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Cross-Linked Hyaluronan Gel Reduces the Acute Rectal Toxicity of Radiotherapy for Prostate Cancer
Purpose: To prospectively analyze whether cross-linked hyaluronan gel reduces the mean rectal dose and acute rectal toxicity of radiotherapy for prostate cancer. Methods and Materials: Between September 2008 and March 2009, we transperitoneally injected 9mL of cross-linked hyaluronan gel (Hylaform; Genzyme Corporation, Cambridge, MA) into the anterior perirectal fat of 10 early-stage prostate cancer patients to increase the separation between
Richard B. Wilder; Greg A. Barme; Ronald F. Gilbert; Richard E. Holevas; Luis I. Kobashi; Richard R. Reed; Ronald S. Solomon; Nancy L. Walter; Lucy Chittenden; Albert V. Mesa; Jeffrey Agustin; Jessica Lizarde; Jorge Macedo; John Ravera; Kenneth M. Tokita
Cross-Linked Hyaluronan Gel Reduces the Acute Rectal Toxicity of Radiotherapy for Prostate Cancer
Purpose: To prospectively analyze whether cross-linked hyaluronan gel reduces the mean rectal dose and acute rectal toxicity of radiotherapy for prostate cancer. Methods and Materials: Between September 2008 and March 2009, we transperitoneally injected 9mL of cross-linked hyaluronan gel (Hylaform; Genzyme Corporation, Cambridge, MA) into the anterior perirectal fat of 10 early-stage prostate cancer patients to increase the separation between the prostate and rectum by 8 to 18mm at the start of radiotherapy. Patients then underwent high-dose rate brachytherapy to 2,200cGy followed by intensity-modulated radiation therapy to 5,040cGy. We assessed acute rectal toxicity using the National Cancer Institute Common Terminology Criteria for Adverse Events v3.0 grading scheme. Results: Median follow-up was 3 months. The anteroposterior dimensions of Hylaform at the start and end of radiotherapy were 13 {+-} 3mm (mean {+-} SD) and 10 {+-} 4mm, respectively. At the start of intensity-modulated radiation therapy, daily mean rectal doses were 73 {+-} 13cGy with Hylaform vs. 106 {+-} 20cGy without Hylaform (p = 0.005). There was a 0% incidence of National Cancer Institute Common Terminology Criteria for Adverse Events v3.0 Grade 1, 2, or 3 acute diarrhea in 10 patients who received Hylaform vs. a 29.7% incidence (n = 71) in 239 historical controls who did not receive Hylaform (p = 0.04). Conclusions: By increasing the separation between the prostate and rectum, Hylaform decreased the mean rectal dose. This led to a significant reduction in the acute rectal toxicity of radiotherapy for prostate cancer.
Wilder, Richard B., E-mail: email@example.com [Cancer Center of Irvine, Irvine, CA (United States); Barme, Greg A.; Gilbert, Ronald F.; Holevas, Richard E.; Kobashi, Luis I.; Reed, Richard R.; Solomon, Ronald S.; Walter, Nancy L.; Chittenden, Lucy; Mesa, Albert V.; Agustin, Jeffrey; Lizarde, Jessica; Macedo, Jorge; Ravera, John; Tokita, Kenneth M. [Cancer Center of Irvine, Irvine, CA (United States)
Rheological Properties of Cross-Linked Hyaluronan-Gelatin Hydrogels for Tissue Engineering
Hydrogels that mimic the natural extracellular matrix (ECM) are used in three-dimensional cell culture, cell therapy, and tissue engineering. A semi-synthetic ECM based on cross-linked hyaluronana offers experimental control of both composition and gel stiffness. The mechanical properties of the ECM in part determine the ultimate cell phenotype. We now describe a rheological study of synthetic ECM hydrogels with storage shear moduli that span three orders of magnitude, from 11 to 3 500 Pa, a range important for engineering of soft tissues. The concentration of the chemically modified HA and the cross-linking density were the main determinants of gel stiffness. Increase in the ratio of thiol-modified gelatin reduced gel stiffness by diluting the effective concentration of the HA component.
Vanderhooft, Janssen L.; Alcoutlabi, Mataz; Magda, Jules J.; Prestwich, Glenn D.
Magnetic Characterization of Iron Oxide Cross Linked Hydro gels
NASA Astrophysics Data System (ADS)
Magnetic hydro gels have potential applications in drug delivery, cells sorting, sensors, and actuating technologies. Iron oxide alginate nanocomposites were synthesized following the method of Kroll et al^1 by cross linking sodium alginate with Fe^2+ and Fe^3+ in methanol: water. The ion-cross linked alginate hydro gels are oxidized in an alkaline solution. The resulting hydro gel consists of iron oxide cross linked alginate. The alginate hydro gels are inert to the reaction conditions and therefore the reaction sequence can be repeated. The multiple loadings result in an increase in the amount of iron oxide and the size of the iron oxide nanoparticles in the cross linked hydro gels. The third and sixth loaded iron oxide alginate hydro gels were dried and characterized by X-ray diffraction (XRD), Transmission Electron Microscopy (TEM), and Superconducting Quantum Interference Device (SQUID) magnetometry. The XRD patterns have characteristic features of ?- Fe_2O3 or Fe_3O4 phases. The average particle size, calculated from the XRD peaks, for third loaded iron oxide alginate was 2 nm. The zero-field-cooled and field-cooled SQUID measurements show the iron oxide nanoparticles are superparamagnetic with blocking temperature (T_B) of approximately 35 K. Above the blocking temperature, the inverse susceptibility versus temperature relationship does not follow the Curie-Weiss law, indicating strong inter-particle interactions. The M vs. H data above the blocking temperature was fitted with a modified Langevin function to obtain additional information about the iron oxide particle size. Details of the relationship between coercive field and temperature as well as the particle size distribution obtained from XRD and TEM measurements will be presented. *Research supported by NSF grant # DGE �980720 **Supported by NSF REU grant # EEC-0097736 ^1E. Kroll, F.M. Winnik, and R.F. Ziolo, Chem. Mater, 8, 1594 (1996).
Senaratne, U.; Powell, N.; Kroll, E.; Tsoi, G.; Naik, R.; Naik, V.; Vaishnava, P. P.; Wenger, L. E.
Inter-?-inhibitor Impairs TSG-6-induced Hyaluronan Cross-linking*
Under inflammatory conditions and in the matrix of the cumulus-oocyte complex, the polysaccharide hyaluronan (HA) becomes decorated covalently with heavy chains (HCs) of the serum glycoprotein inter-?-inhibitor (I?I). This alters the functional properties of the HA as well as its structural role within extracellular matrices. The covalent transfer of HCs from I?I to HA is catalyzed by TSG-6 (tumor necrosis factor-stimulated gene-6), but TSG-6 is also known as a HA cross-linker that induces condensation of the HA matrix. Here, we investigate the interplay of these two distinct functions of TSG-6 by studying the ternary interactions of I?I and TSG-6 with well defined films of end-grafted HA chains. We demonstrate that TSG-6-mediated cross-linking of HA films is impaired in the presence of I?I and that this effect suppresses the TSG-6-mediated enhancement of HA binding to CD44-positive cells. Furthermore, we find that the interaction of TSG-6 and I?I in the presence of HA gives rise to two types of complexes that independently promote the covalent transfer of heavy chains to HA. One type of complex interacts very weakly with HA and is likely to correspond to the previously reported covalent HC�TSG-6 complexes. The other type of complex is novel and binds stably but noncovalently to HA. Prolonged incubation with TSG-6 and I?I leads to HA films that contain, in addition to covalently HA-bound HCs, several tightly but noncovalently bound molecular species. These findings have important implications for understanding how the biological activities of TSG-6 are regulated, such that the presence or absence of I?I will dictate its function.
Baranova, Natalia S.; Foulcer, Simon J.; Briggs, David C.; Tilakaratna, Viranga; Enghild, Jan J.; Milner, Caroline M.; Day, Anthony J.; Richter, Ralf P.
Chitinolytic digestibility of cross-linked chitosan gels by an enzyme complex from Streptomyces kurssanovii.
The action of a microbial chitinolytic enzyme complex from Streptomyces kurssanovii on chitosan gels with various degrees of cross-linking and on the O-derivatives of the gels was examined. The rate of digestion of the gels was shown to depend on the degree of cross-linking as well as on the size of the O-substituent, decreasing greatly with a rise in substitution. PMID:8484909
Tikhonov, V E; Yamskov, I A; Varlamov, V P; Ilyina, A V; Davankov, V A
"Out-gel" tryptic digestion procedure for chemical cross-linking studies with mass spectrometric detection.
SDS-PAGE is one of the most powerful protein separation techniques, and in-gel digestion is the leading method for converting proteins separated by SDS-PAGE into peptides suitable for mass spectrometry-based proteomic studies. In in-gel digestion, proteins are digested within the gel matrix, and the resulting peptides are extracted into an appropriate buffer. Transfer of the digested peptides to the liquid phase for subsequent mass spectrometric analysis, however, may be hampered by peptide-specific characteristics, including size, shape, poor solubility, adsorption to the polyacrylamide, and-in the case of cross-linking applications-by the branched structure of the peptides produced. This can be a limitation in cross-linking studies where efficient recoveries of the cross-linked peptides are critical. To overcome this limitation, we have developed a modification to the standard in-gel digestion procedure for SDS-PAGE-separated cross-linked proteins, based on older passive diffusion methods. By omitting the gel staining and gel fixation steps, intact proteins or cross-linked protein complexes can move through the gel and into the buffer solution. Digestion of the entire protein in the buffer outside the gel increases the probability that most of the proteolytic peptides produced will be present in the final digest solution. The resulting peptide mixture is then freed of SDS and concentrated using SCX (strong cation exchange) zip-tips and analyzed by mass spectrometry. For standard protein identification studies and the recovery of noncross-linked peptides, the in-gel procedure outperformed the out-gel procedure, but for cross-linking studies with enrichable cross-linkers (such as CBDPS), the standard out-gel procedure allowed the recoveries of cross-links not recovered via the in-gel method. Labeling experiments showed that, with an enrichable cross-linker, 93% of the cross-links showed better or equal recoveries with the out-gel procedure, as compared to the in-gel procedure. It should be noted that this method is not designed to replace in-gel digestion for most proteomics applications. However, by using the out-gel method, we were able to detect twice as many interprotein CBDPS cross-links from the histone H2A/H2B complex as were found in the in-gel digested sample. PMID:24354799
Petrotchenko, Evgeniy V; Serpa, Jason J; Cabecinha, Ashley N; Lesperance, Mary; Borchers, Christoph H
Effect of electron beam-cross-linked gels on the rheological properties of raw natural rubber
NASA Astrophysics Data System (ADS)
Electron beam (EB)-cross-linked natural rubber (NR) gels were prepared from latex and characterized by various techniques. The addition of a small amount of these gels to raw NR was found to reduce the apparent shear viscosity and die swell remarkably. This effect was further enhanced with the addition of butyl acrylate as a sensitizer. The apparent shear viscosity first decreased up to 8 phr of gel loading and then increased. However, the percent die swell value decreased steadily upon gel loading. These were explained by calculating principal normal stress difference, the activation energy of melt flow and characteristics of EB-cross-linked gels. These effects were also reflected in the changes of mechanical and dynamic mechanical properties of gel-filled raw NR. Tailoring of the above properties could be done with the help of these gels.
Mitra, Suman; Chattopadhyay, Santanu; Bharadwaj, Y. K.; Sabharwal, S.; Bhowmick, Anil K.
Cross-linking cellulose nanofibrils for potential elastic cryo-structured gels.
Cellulose nanofibrils were produced from P. radiata kraft pulp fibers. The nanofibrillation was facilitated by applying 2,2,6,6-tetramethylpiperidinyl-1-oxyl-mediated oxidation as pretreatment. The oxidized nanofibrils were cross-linked with polyethyleneimine and poly N-isopropylacrylamide-co-allylamine-co-methylenebisacrylamide particles and were frozen to form cryo-structured gels. Samples of the gels were critical-point dried, and the corresponding structures were assessed with scanning electron microscopy. It appears that the aldehyde groups in the oxidized nanofibrils are suitable reaction sites for cross-linking. The cryo-structured materials were spongy, elastic, and thus capable of regaining their shape after a given pressure was released, indicating a successful cross-linking. These novel types of gels are considered potential candidates in biomedical and biotechnological applications. PMID:22152032
Syverud, Kristin; Kirsebom, Harald; Hajizadeh, Solmaz; Chinga-Carrasco, Gary
Cross-linking cellulose nanofibrils for potential elastic cryo-structured gels
NASA Astrophysics Data System (ADS)
Cellulose nanofibrils were produced from P. radiata kraft pulp fibers. The nanofibrillation was facilitated by applying 2,2,6,6-tetramethylpiperidinyl-1-oxyl-mediated oxidation as pretreatment. The oxidized nanofibrils were cross-linked with polyethyleneimine and poly N-isopropylacrylamide- co-allylamine- co-methylenebisacrylamide particles and were frozen to form cryo-structured gels. Samples of the gels were critical-point dried, and the corresponding structures were assessed with scanning electron microscopy. It appears that the aldehyde groups in the oxidized nanofibrils are suitable reaction sites for cross-linking. The cryo-structured materials were spongy, elastic, and thus capable of regaining their shape after a given pressure was released, indicating a successful cross-linking. These novel types of gels are considered potential candidates in biomedical and biotechnological applications.
Syverud, Kristin; Kirsebom, Harald; Hajizadeh, Solmaz; Chinga-Carrasco, Gary
Thermoreversible physical gels of poly(dimethylsiloxane) without cross-links or functionalization.
The preparation of gels of poly(dimethylsiloxane) (PDMS) reported in the literature so far involves catalysts and chemical cross-links (chemical gels) or functionalization with organogelators. We report that thermoreversible physical gels of PDMS, without cross-links or functionalization, can be made with propylamine or hexylamine as a solvent. The gels consist of spherical domains as small as 20 nm. We show that these spherical domains are part of a network. Differential scanning calorimetry (DSC), optical microscopy, and rheology show that the gel is thermoreversible. With the DSC experiments, we have devised a procedure to achieve thermoreversibility with very similar gel-sol transition endotherms in the first and second heating cycles. PMID:23799797
Dahan, Elianne; Sundararajan, Pudupadi R
Robust cross-links in molluscan adhesive gels: Testing for contributions from hydrophobic and electrostatic interactions
The cross-linking interactions that provide cohesive strength to molluscan adhesive gels were investigated. Metal-based interactions have been shown to play an important role in the glue of the slug Arion subfuscus (Draparnaud), but other types of interactions may also contribute to the glue's strength and their role has not been investigated. This study shows that treatments that normally disrupt hydrophobic or electrostatic interactions have little to no effect on the slug glue. High salt concentrations and non-ionic detergent do not affect the solubility of the proteins in the glue or the ability of the glue proteins to stiffen gels. In contrast, metal chelation markedly disrupts the gel. Experiments with gel filtration chromatography identify a 40 kDa protein that is a central component of the cross-links in the glue. This 40 kDa protein forms robust macromolecular aggregations that are stable even in the presence of high concentrations of salt, non-ionic detergent, urea or metal chelators. Metal chelation during glue secretion, however, may block some of these cross-links. Such robust, non-specific interactions in an aqueous environment are highly unusual for hydrogels and reflect an intriguing cross-linking mechanism.
Smith, A.M.; Robinson, T. M.; Salt, M. D.; Hamilton, K. S.; Silvia, B. E.; Blasiak, R.
The microstructure of collagen type I gel cross-linked with gold nanoparticles
Scanning electron microscopy, transmission electron micrsocopy, rheomerty, and electrochemistry were used to provide insight into the microstructure of collagen type I gel (1% w/v) modified with the tiopronin-protected (N-(2-mercaptopropionyl)glycine) gold nanoparticles (TPAu), a multivalent crosslinker. The cross-linking reaction, performed via EDC (1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide) coupling, results in compliant, mechanically stable and continuous gels. The gels contain unusual interconnected collagen-TPAu particles. Electrochemical measurements of 4-hydroxy-(2,2,6,6-tetramethylpiperidine-1-oxyl) (4HT) diffusion within the gel reveal that the gel hindrance is nearly independent of the TPAu concentration. The properties of the collagen-TPAu gel make it suitable for potential biomedical applications, such as delivery of small molecule drugs.
Schuetz, Thomas; Richmond, Nathan; Harmon, Marianne E.; Schuetz, Joseph; Castaneda, Luciano; Slowinska, Katarzyna
The enhanced stability of the cross-linked hylan structure to hydroxyl (OH) radicals compared with the uncross-linked hyaluronan
NASA Astrophysics Data System (ADS)
A comparison has been made of the relative stabilities of hyaluronan and hylan to degradation by OH radicals produced by ?-irradiation of aqueous solutions in N 2O, when G (yield per 100 eV) for OH radicals is 5.6 and H atoms 0.6. Using low angle light scattering and viscometric methods, the change in molecular weight of the polysaccharides was measured with increasing dose. From the yield/dose curves (expressed as breaks per molecule), the initial G value for hyaluronan degradation is � 4. A further slow post-irradiation decrease in molecular weight is observed, which can be brought to completion by incubating the solutions for 1 h at 60�C. Thereafter, the G value for degradation is � 6. A similar post-irradiation degradation was found for hylan. A technique using tetranitromethane (TNM) has been used to distinguish between two types of radicals formed on the hyaluronan backbone. Radicals of the 1-hydroxy-2-alkoxy type (C-2, C-4, C-2 and C3 of the glucuronic acid) would induce strand breakage by alkoxy elimination. For the equivalent alkoxy radical at C6 of the acetamido monosaccharide, ring opening would occur with formation of a hemi-acetal, leading also to strand breakage. The C-2 and C-3 radicals would eliminate water rather than produce breaks by ?-alkoxy elimination. Thus three out of the initially formed radicals would produce breaks by ?-alkoxy formation. These can be stabilised with TNM and distinguished. It is concluded that these are the radicals involved in the post-irradiation thermal degradation process. Comparison of hylan and hyaluronan is, therefore, most valid when this post-irradiation process has been completed. Therefore, all G values for degradation were measured after incubation for 1 h at 60�. This investigation establishes the greater stability of hylan ( Gdegradation = 2) compared to hylan ( Gdegradation = 6). Therefore, in an environment such as supplementation of an inflammed joint where OH radicals are released, hylan is able to retain its integrity as a viscoelastic macromolecule three times better than hyaluronan. Its potential as a viscosupplementation material, or as an inflammatory drug release matrix inserted within the joint is, therefore, greater than non-cross-linked hyaluronan.
Al-Assaf, Saphwan; Phillips, Glyn O.; Deeble, D. J.; Parsons, Barry; Starnes, Hazel; Von Sonntag, C.
Immobilization of Cross-Linked Phenylalanine Ammonia Lyase Aggregates in Microporous Silica Gel
A separable and highly-stable enzyme system was developed by adsorption of phenylalanine ammonia lyase (PAL) from Rhodotorula glutinis in amino-functionalized macroporous silica gel and subsequent enzyme crosslinking. This resulted in the formation of cross-linked enzyme aggregates (PAL-CLEAs) into macroporous silica gel (MSG-CLEAs). The effect of adsorptive conditions, type of aggregating agent, its concentration as well as that of cross-linking agent was studied. MSG-CLEAs production was most effective using ammonium sulfate (40%-saturation), followed by cross-linking for 1 h with 1.5% (v/v) glutaraldehyde. The resulting MSG-CLEAs extended the optimal temperature and pH range compared to free PAL and PAL-CLEAs. Moreover, MSG-CLEAs exhibited the excellent stability of the enzyme against various deactivating conditions such as temperature and denaturants, and showed higher storage stability compared to the free PAL and the conventional PAL-CLEAs. Such as, after 6 h incubation at 60�C, the MSG-CLEAs still retained more than 47% of the initial activity whereas PAL-CLEAs only retained 7% of the initial activity. Especially, the MSG-CLEAs exhibited good reusability due to its suitable size and active properties. These results indicated that PAL-CLEAs on MSG might be used as a feasible and efficient solution for improving properties of immobilized enzyme in industrial application.
Cui, Jian Dong; Li, Lian Lian; Bian, Hong Jie
Preparation of porous, chemically cross-linked, PVdF-based gel polymer electrolytes for rechargeable lithium batteries
This study reports the development of a new system of porous, chemically cross-linked, gel polymer electrolytes based on poly(vinylidene fluoride-co-hexafluoropropylene) (PVdF�HFP) copolymer as a polymer matrix, polyethylene glycol (PEG) as a plasticizer, and polyethylene glycol dimethacrylate (PEGDMA) as a chemical cross-linking oligomer. The electrolytes are prepared by a combination of controlled evaporation and thermal polymerization of PEGDMA. PVdF�HFP\\/PEG\\/PEGDMA gel polymer
C. L Cheng; C. C Wan; Y. Y Wang
Hierarchical cross-linking in physical alginate gels: a rheological and dynamic light scattering investigation
NASA Astrophysics Data System (ADS)
We investigate the dynamics of alginate gels, an important class of biopolymer-based viscoelastic materials, by combining mechanical tests and non-conventional, time-resolved light scattering methods. Two relaxation modes are observed upon applying a compressive or shear stress. Dynamic light scattering and diffusive wave spectroscopy measurements reveal that these modes are associated with discontinuous rearrangement events that restructure the gel network via anomalous, non-diffusive microscopic dynamics. We show that these dynamics are due to both thermal activation and internal stress stored during gelation and propose a scenario where a hierarchy of cross-links with different life times is responsible for the observed complex behavior. Measurements at various temperatures and sample ages are presented to support this scenario.
Larobina, Domenico; Cipelletti, Luca
Using response surface methodology to optimize process parameters and cross-linking agents for production of combined-cross-linked bovine serum albumin gels.
D-optimal design was employed to optimize the mixture of cross-linking agents formulation: microbial transglutaminase (MTGase) and ribose, and the processing parameters (i.e. incubation and heating time) in the mixture in order to obtain combined-cross-linked bovine serum albumin gels that have high gel strength, pH close to neutral and yet medium in browning. Analysis of variance (ANOVA) showed that the contribution of quadratic term to the model over the linear was significant for pH and L* value, whereas linear model was significant for gel strength. Optimization study using response surface methodology (RSM) was performed to the mixture components and process variables and the optimum conditions obtained were: MTGase of 1.34-1.43 g/100 mL, ribose of 1.07-1.16 g/100 mL, incubation time of 5 h at 40 degrees C and heating time of 3 h at 90 degrees C. PMID:19332294
Gan, Chee-Yuen; Alkarkhi, Abbas F M; Easa, Azhar Mat
Citrate cross-linked gels with strain reversibility and viscoelastic behavior accelerate healing of osteochondral defects in a rabbit model.
Most living tissues are viscoelastic in nature. Self-repair due to the dissipation of energy by reversible bonds prevents the rupture of the molecular backbone in these tissues. Recent studies, therefore, have aimed to synthesize biomaterials that approximate the mechanical performance of biological materials with self-recovery properties. We report an environmentally friendly method for the development of ionotropically cross-linked viscoelastic chitosan gels with a modulus comparable to that of living tissues. The strain recovery property was found to be highest for the gels with the lowest cross-linking density. The force-displacement curve showed significant hysteresis due to the presence of reversible bonds in the cross-linked gels. Nanoindentation studies demonstrated the creep phenomenon for the cross-linked chitosan gels. Creep, hysteresis, and plasticity index confirmed the viscoelastic behavior of the cross-linked gels. The viscoelastic gels were implanted at osteochondral defect sites to assess the tissue regeneration ability. In vivo results demonstrated early cartilage formation and woven bone deposition for defects filled with the gels compared to nontreated defects. PMID:24971647
Ghosh, Paulomi; Rameshbabu, Arun Prabhu; Dhara, Santanu
MALDI analysis of proteins after extraction from dissolvable ethylene glycol diacrylate cross-linked polyacrylamide gels.
Although the extraction of intact proteins from polyacrylamide gels followed by mass spectrometric molecular mass determination has been shown to be efficient, there is room for alternative approaches. Our study evaluates ethylene glycol diacrylate, a cleavable cross-linking agent used for a new type of dissolvable gels. It attains an ester linkage that can be hydrolyzed in alkali conditions. The separation performance of the new gel system was tested by 1D and 2D SDS-PAGE using the outer chloroplast envelope of Pisum sativum as well as a soluble protein fraction of human lymphocytes, respectively. Gel spot staining (CBB), dissolving, and extracting were conducted using a custom-developed workflow. It includes protein extraction with an ammonia-SDS buffer followed by methanol treatment to remove acrylamide filaments. Necessary purification for MALDI-TOF analysis was implemented using methanol-chloroform precipitation and perfusion HPLC. Both cleaning procedures were applied to several standard proteins of different molecular weight as well as 'real' biological samples (8-75 kDa). The protein amounts, which had to be loaded on the gel to detect a peak in MALDI-TOF MS, were in the range of 0.1 to 5 ?g, and the required amount increased with increasing mass. PMID:23775326
Papasotiriou, Dimitrios G; Markoutsa, Stavroula; Gorka, Jan; Schleiff, Enrico; Karas, Michael; Meyer, Bjoern
Generalized structural description of calcium-sodium aluminosilicate hydrate gels: the cross-linked substituted tobermorite model.
Structural models for the primary strength and durability-giving reaction product in modern cements, a calcium (alumino)silicate hydrate gel, have previously been based solely on non-cross-linked tobermorite structures. However, recent experimental studies of laboratory-synthesized and alkali-activated slag (AAS) binders have indicated that the calcium-sodium aluminosilicate hydrate [C-(N)-A-S-H] gel formed in these systems can be significantly cross-linked. Here, we propose a model that describes the C-(N)-A-S-H gel as a mixture of cross-linked and non-cross-linked tobermorite-based structures (the cross-linked substituted tobermorite model, CSTM), which can more appropriately describe the spectroscopic and density information available for this material. Analysis of the phase assemblage and Al coordination environments of AAS binders shows that it is not possible to fully account for the chemistry of AAS by use of the assumption that all of the tetrahedral Al is present in a tobermorite-type C-(N)-A-S-H gel, due to the structural constraints of the gel. Application of the CSTM can for the first time reconcile this information, indicating the presence of an additional activation product that contains highly connected four-coordinated silicate and aluminate species. The CSTM therefore provides a more advanced description of the chemistry and structure of calcium-sodium aluminosilicate gel structures than that previously established in the literature. PMID:23534827
Myers, Rupert J; Bernal, Susan A; San Nicolas, Rackel; Provis, John L
Cross-linking by protein oxidation in the rapidly setting gel-based glues of slugs
SUMMARY The terrestrial slug Arion subfuscus secretes a glue that is a dilute gel with remarkable adhesive and cohesive strength. The function of this glue depends on metals, raising the possibility that metal-catalyzed oxidation plays a role. The extent and time course of protein oxidation was measured by immunoblotting to detect the resulting carbonyl groups. Several proteins, particularly one with a relative molecular mass (Mr) of 165�103, were heavily oxidized. Of the proteins known to distinguish the glue from non-adhesive mucus, only specific size variants were oxidized. The oxidation appears to occur within the first few seconds of secretion. Although carbonyls were detected by 2,4-dinitrophenylhydrazine (DNPH) in denatured proteins, they were not easily detected in the native state. The presence of reversible cross-links derived from carbonyls was tested for by treatment with sodium borohydride, which would reduce uncross-linked carbonyls to alcohols, but stabilize imine bonds formed by carbonyls and thus lead to less soluble complexes. Consistent with imine bond formation, sodium borohydride led to a 20�35% decrease in the amount of soluble protein with a Mr of 40�165 (�103) without changing the carbonyl content per protein. In contrast, the nucleophile hydroxylamine, which would competitively disrupt imine bonds, increased protein solubility in the glue. Finally, the primary amine groups on a protein with a Mr of 15�103 were not accessible to acid anhydrides. The results suggest that cross-links between aldehydes and primary amines contribute to the cohesive strength of the glue.
Bradshaw, Andrew; Salt, Michael; Bell, Ashley; Zeitler, Matt; Litra, Noelle; Smith, Andrew M.
Cross-linking by protein oxidation in the rapidly setting gel-based glues of slugs.
The terrestrial slug Arion subfuscus secretes a glue that is a dilute gel with remarkable adhesive and cohesive strength. The function of this glue depends on metals, raising the possibility that metal-catalyzed oxidation plays a role. The extent and time course of protein oxidation was measured by immunoblotting to detect the resulting carbonyl groups. Several proteins, particularly one with a relative molecular mass (M(r)) of 165 x 10�, were heavily oxidized. Of the proteins known to distinguish the glue from non-adhesive mucus, only specific size variants were oxidized. The oxidation appears to occur within the first few seconds of secretion. Although carbonyls were detected by 2,4-dinitrophenylhydrazine (DNPH) in denatured proteins, they were not easily detected in the native state. The presence of reversible cross-links derived from carbonyls was tested for by treatment with sodium borohydride, which would reduce uncross-linked carbonyls to alcohols, but stabilize imine bonds formed by carbonyls and thus lead to less soluble complexes. Consistent with imine bond formation, sodium borohydride led to a 20-35% decrease in the amount of soluble protein with a M(r) of 40-165 (x 10�) without changing the carbonyl content per protein. In contrast, the nucleophile hydroxylamine, which would competitively disrupt imine bonds, increased protein solubility in the glue. Finally, the primary amine groups on a protein with a M(r) of 15 x 10� were not accessible to acid anhydrides. The results suggest that cross-links between aldehydes and primary amines contribute to the cohesive strength of the glue. PMID:21525316
Bradshaw, Andrew; Salt, Michael; Bell, Ashley; Zeitler, Matt; Litra, Noelle; Smith, Andrew M
Transglutaminase cross-linking of milk proteins and impact on yoghurt gel properties
A novel yoghurt process was investigated in which milk proteins were covalently cross-linked by a microbial transglutaminase (TG) preparation containing glutathione (TG+GSH). As unheated milk is normally less reactive towards TG, TG+GSH was applied to enable non-inhibited cross-linking without requiring a pre-heat treatment beyond pasteurisation conditions. After the TG+GSH incubation phase, the enzyme was inactivated by heat treatment of the
Martin P. B�nisch; Manfred Huss; Kerstin Weitl; Ulrich Kulozik
Temperature-induced sol-gel transition and microgel formation in ?-actinin cross-linked actin networks: A rheological study
NASA Astrophysics Data System (ADS)
We have studied the sol-gel transition, the viscoelastic and the structural properties of networks constituted of semiflexible actin filaments cross-linked by ?-actinin. Cross-linking was regulated in a reversible way by varying the temperature through the association-dissociation equilibrium of the actin-?-actinin system. Viscoelastic parameters [shear storage modulus G'(?), phase shift tan(?)(?), creep compliance J(t)] were measured as a function of temperature and actin-to-cross-linker ratio by a magnetically driven rotating disc rheometer. G'(?) and tan(?)(?) were studied at a frequency ? corresponding to the elastic plateau regime of the G'(?) versus ? spectrum of the purely entangled solution. The microstructure of the networks was viewed by negative staining electron microscopy (EM). The phase shift tan(?) (or equivalently the viscosity ?) diverges and reaches a maximum when approaching the apparent gel point from lower and higher temperatures, and the maximum defines the gel point (temperature Tg). The elastic plateau modulus G'N diverges at temperatures beyond this gel point T
Tempel, M.; Isenberg, G.; Sackmann, E.
A physical cross-linking process of cellulose nanofibril gels with shear-controlled fibril orientation
Cellulose nanofibrils constitute the smallest fibrous components of wood, with a width of approximately 4\\\\,nm and a length in the micrometer range. They consist of aligned linear cellulose chains with crystallinity exceeding 60\\\\,\\\\%, rendering stiff, high-aspect-ratio rods. These properties are advantageous in the reinforcement components of composites. Cross-linked networks of fibrils can be used as templates into which a polymer
A. B. Fall; S. B. Lindstr�m; J. Sprakel; L. W�gberg
Preparation of porous, chemically cross-linked, PVdF-based gel polymer electrolytes for rechargeable lithium batteries
NASA Astrophysics Data System (ADS)
This study reports the development of a new system of porous, chemically cross-linked, gel polymer electrolytes based on poly(vinylidene fluoride-co-hexafluoropropylene) (PVdF-HFP) copolymer as a polymer matrix, polyethylene glycol (PEG) as a plasticizer, and polyethylene glycol dimethacrylate (PEGDMA) as a chemical cross-linking oligomer. The electrolytes are prepared by a combination of controlled evaporation and thermal polymerization of PEGDMA. PVdF-HFP/PEG/PEGDMA gel polymer electrolytes with a composition of 5/3/2 exhibit both high ambient ionic conductivity, viz., >1 mS cm -1, and a high tensile modulus of 52 MPa, because of their porous and network structures. All the blends of electrolytes are electrochemically stable up to 5 V versus Li/Li + in the presence of 1 M LiPF 6/ethylene carbonate-diethyl carbonate (EC-DEC). With these polymer electrolytes, rechargeable lithium batteries composed of carbon anode and LiCoO 2 cathode have acceptable cycleability and a good rate capability.
Cheng, C. L.; Wan, C. C.; Wang, Y. Y.
Local application of hyaluronan gel in conjunction with periodontal surgery: a randomized controlled trial.
Hyaluronic acid application has been proven to be beneficial in a number of medical disciplines. The aim of the current study was to clinically evaluate the effect of local application of hyaluronan gel in conjunction with periodontal surgery. Fourteen patients with chronic periodontitis having four interproximal intrabony defects (?3 mm) with probing depth values >5 mm were included in this split-mouth study. Following initial nonsurgical periodontal therapy and re-evaluation, defects were randomly assigned to be treated with modified Widman flap (MWF) surgery in conjunction with either 0.8% hyaluronan gel (test) or placebo gel (control) application. Clinical attachment level (CAL), probing depth (PD), gingival recession (GR), plaque index (PI), and bleeding on probing (BOP) values were taken at baseline and 3 and 6 months. Differences between test and control sites were evaluated using a Wilcoxon signed-rank and a McNemar test. A Friedman and a Cochran test were used to test equal ranks over time. Statistically significant differences were noted for CAL and GR (P < 0.05) in favor of the test sites. No significant differences were found regarding PD, BOP, or PI values (P > 0.05). Hyaluronan gel application in conjunction with periodontal surgery appears to result in significant improvement of CAL and in a reduction in GR. Hyaluronan gel application appears to improve the clinical outcome of MWF surgery. PMID:22012469
Fawzy El-Sayed, Karim M; Dahaba, Moushira A; Aboul-Ela, Shadw; Darhous, Mona S
Topical 3% diclofenac in 2.5% hyaluronan gel for the treatment of actinic keratoses.
Actinic Keratoses (AKs) are epidermal skin lesions that have the potential to develop into squamous cell carcinoma. Many of the treatment options available can cause discomfort, pain or skin irritation. Topical 3% diclofenac in 2.5% hyaluronan gel (Solaraze, Bioglan Pharma) is a relatively new treatment that has been shown to be effective and well tolerated for the treatment of AKs. PMID:14716439
Rivers, J K
A study of cross-linked PEO gel polymer electrolytes using bisphenol A ethoxylate diacrylate: ionic conductivity and mechanical properties
NASA Astrophysics Data System (ADS)
Ionic conductivity, mechanical stability and electrochemical property were examined on the poly ethyleneoxide (PEO) based cross-linked gel polymer electrolyte. Bisphenol A ethoxylate diacrylates (BPAEDA) with different numbers of ethylene oxide units ( n=4, 15) were used as cross-linkers. The curing kinetics of the cross-linker was examined by an FT-IR study with two types of initiators and at different curing temperatures. Higher conductivity was observed for the longer ethylene oxide unit. Conductivity of the polymer electrolyte prepared from BPAEDA ( n=15) and liquid electrolyte (70 wt.%) was 3.47�10 -3 S/cm at 30 �C. Improved tensile strength and elongation at break were realized from the polymer electrolyte using BPAEDA as a cross-linker. Electrolyte having higher cross-linking density showed higher tensile strength, but showed decreased flexibility. The stability of the polymer electrolyte against electrochemical oxidation could be extended to larger than 4.5 V (versus Li/Li +). Lithium cycling efficiency of the polymer electrolyte was about 80%.
Kang, Yongku; Cheong, Kwangjo; Noh, Kun-Ae; Lee, Changjin; Seung, Do-Young
Effectiveness of auto-cross-linked hyaluronic acid gel in the prevention of intrauterine adhesions after hysteroscopic adhesiolysis: a prospective, randomized, controlled study
BACKGROUND: A prospective, randomized, controlled study was performed to assess the efficacy of auto-cross- linked hyaluronic acid (ACP) gel in preventing the development of intrauterine adhesions following hysteroscopic adhesiolysis. METHODS: Ninety-two patients with irregular menses and intrauterine adhesions referred to the Hysteroscopic Unit of the University of Naples ''Federico II''. Patients were randomized to two different groups. Group A were
Giuseppe Acunzo; Maurizio Guida; Massimiliano Pellicano; Giovanni Antonio Tommaselli; Attilio Di; Spiezio Sardo; Giuseppe Bifulco; Domenico Cirillo; Alex Taylor; Carmine Nappi
Conformational and dynamical behaviors of cross-linked poly(?-methyl l-glutamate) gel as studied by high-resolution solid-state 13C NMR spectroscopy
Cross-linked poly(?-methyl l-glutamate) (PMLG) was prepared by the ester-amide exchange reaction using diaminododecane as a cross-linker and was swollen by chloroform (CHCl3), trifluoracetic acid (TFA) and a mixture of TFA and CHCl3. The structural and dynamical analyses of the PMLG gel were carried out by means of high-resolution solid-state 13C NMR spectroscopy. From the experimental results on 13C chemical shifts
Chenhua Zhao; Shingo Matsukawa; Masatoshi Kobayashi; Isao Ando
The efficacy of 3% diclofenac in 2.5% hyaluronan gel base for treatment of recurrent aphthous stomatitis (RAS): A double blind study
Purpose: To evaluate the effects of 3% Diclofenac in 2.5% hyaluronan base versus 2.5% gel base alone on the pain duration and healing time of the minor aphthous ulcers. Materials and Methods: 44 patients with minor RAS were selected randomly and treated with 3% Diclofenac in 2.5% hyaluronan or the 2.5% hyaluronan base alone. Pain duration and healing duration of
Iraji Fariba; Ghafgasi Taghi; Samani Mazdak; Tashakori Maryam; Siadat Amir Hossein; Enshaieh Shahla; Shahmoradi Zabiholah
Transformation of metal-organic framework to polymer gel by cross-linking the organic ligands preorganized in metal-organic framework.
Until now, seamless fusion of metal-organic frameworks (MOFs) and covalently cross-linked polymer gels (PG) at molecular level has been extremely rare, since these two matters have been regarded as opposite, that is, hard versus soft. In this report, we demonstrate transformation of cubic MOF crystals to PG via inner cross-linking of the organic linkers in the void space of MOF, followed by decomposition of the metal coordination. The obtained PG behaved as a polyelectrolyte gel, indicating the high content of ionic groups inside. Metal ions were well adsorbed in the PG due to its densely packed carboxylate groups. A chimera-type hybrid material consisting of MOF and PG was obtained by partial hydrolysis of resulting cross-linked MOF. The shape of resulting PG network well reflected the crystal structure of MOF employed as a template. Our results will connect the two different network materials that have been ever studied in the two different fields to provide new soft and hard hybrid materials, and the unique copolymerization in the large void space of the MOF will open a new horizon toward "ideal network polymers" never prepared before now. PMID:23472763
Ishiwata, Takumi; Furukawa, Yuki; Sugikawa, Kouta; Kokado, Kenta; Sada, Kazuki
Influence of titanium oxide and titanium peroxy gel on the breakdown of hyaluronan by reactive oxygen species
The molecular events occuring at the interface between titanium and connective tissue were investigated in order to help explain the unique biocompatible properties of titanium implants and their successful osseointegration into bone tissue. In this study the influence of commercially pure titanium and titanium peroxy gels on the breakdown of the connective tissue component and serum derived factor, hyaluronan, by
G. C. Taylor; R. J. Waddington; R. Moseley; K. R. Williams; G. Embery
A Technique for High-Throughput Protein Crystallization in Ionically Cross-Linked Polysaccharide Gel Beads for X-Ray Diffraction Experiments
A simple technique for high-throughput protein crystallization in ionically cross-linked polysaccharide gel beads has been developed for contactless handling of crystals in X-ray crystallography. The method is designed to reduce mechanical damage to crystals caused by physical contact between crystal and mount tool and by osmotic shock during various manipulations including cryoprotection, heavy-atom derivatization, ligand soaking, and diffraction experiments. For this study, protein crystallization in alginate and ?-carrageenan gel beads was performed using six test proteins, demonstrating that proteins could be successfully crystallized in gel beads. Two complete diffraction data sets from lysozyme and ID70067 protein crystals in gel beads were collected at 100 K without removing the crystals; the results showed that the crystals had low mosaicities. In addition, crystallization of glucose isomerase was carried out in alginate gel beads in the presence of synthetic zeolite molecular sieves (MS), a hetero-epitaxic nucleant; the results demonstrated that MS can reduce excess nucleation of this protein in beads. To demonstrate heavy-atom derivatization, lysozyme crystals were successfully derivatized with K2PtBr6 within alginate gel beads. These results suggest that gel beads prevent serious damage to protein crystals during such experiments.
Sugahara, Michihiro
A technique for high-throughput protein crystallization in ionically cross-linked polysaccharide gel beads for x-ray diffraction experiments.
A simple technique for high-throughput protein crystallization in ionically cross-linked polysaccharide gel beads has been developed for contactless handling of crystals in X-ray crystallography. The method is designed to reduce mechanical damage to crystals caused by physical contact between crystal and mount tool and by osmotic shock during various manipulations including cryoprotection, heavy-atom derivatization, ligand soaking, and diffraction experiments. For this study, protein crystallization in alginate and ?-carrageenan gel beads was performed using six test proteins, demonstrating that proteins could be successfully crystallized in gel beads. Two complete diffraction data sets from lysozyme and ID70067 protein crystals in gel beads were collected at 100 K without removing the crystals; the results showed that the crystals had low mosaicities. In addition, crystallization of glucose isomerase was carried out in alginate gel beads in the presence of synthetic zeolite molecular sieves (MS), a hetero-epitaxic nucleant; the results demonstrated that MS can reduce excess nucleation of this protein in beads. To demonstrate heavy-atom derivatization, lysozyme crystals were successfully derivatized with K2PtBr6 within alginate gel beads. These results suggest that gel beads prevent serious damage to protein crystals during such experiments. PMID:24740192
Sugahara, Michihiro
Synthesis of nanocomposites from Pd(0) and a hyper-cross-linked functional resin obtained from a conventional gel-type precursor.
Hyper-cross-linked resins stemming from a gel-type poly-chloromethylated poly(styrene-co-divinylbenzene) resin (GT) have been investigated by a multi-methodological approach based on elemental analysis, scanning electron microscopy, X-ray microanalysis, and solvent absorption. The hyper-cross-linking of the parent resin was accomplished by Friedel-Crafts alkylation of the phenyl rings of the resins with the chloromethyl groups. This produced a permanent pore system comprising both micropores (<2.0 nm in diameter) and mesopores (2.2 nm). The chloromethyl groups that did not react in the hyper-cross-linking step were transformed into methylmercaptan groups and the latter were then converted into sulfonic groups by oxidation with hydrogen peroxide. By this procedure the extensive permanent porosity of the parent unsulfonated hyper-cross-linked polymer (HGT) was retained by the sulfonated polymer (HGTS). The final exchange capacity of HGTS was determined to be 0.36 mmol g(-1). HGTS was easily metalated with Pd(II) and the subsequent reduction of the metal centers with either aqueous sodium borohydride, formaldehyde, or dihydrogen produced three Pd(0)/HGTS nanocomposites. The metal nanoparticles had diameters in the 1-6 nm range for all the nanocomposites, as determined by TEM, but with somewhat different distributions. When formaldehyde was used, more than 90% of the nanoparticles were less than 3 nm and their radial distribution throughout the polymer beads was quite homogeneous. These findings show that with this reducing agent the metal nanoparticles are generated within the pore system of the polymer matrix, hence their size is controlled by the dimensions of the pores of the polymeric support. PMID:23728964
Je?�bek, Karel; Zecca, Marco; Centomo, Paolo; Marchionda, Federico; Peruzzo, Luca; Canton, Patrizia; Negro, Enrico; Di Noto, Vito; Corain, Benedetto
Improved agarose gel electrophoresis method and molecular mass calculation for high molecular mass hyaluronan.
The molecular mass of the polysaccharide hyaluronan (HA) is an important determinant of its biological activity and physicochemical properties. One method currently used for the analysis of the molecular mass distribution of an HA sample is gel electrophoresis. In the current work, an improved agarose gel electrophoresis method for analysis of high molecular mass HA is presented and validated. HA mobility in 0.5% agarose minigels was found to be linearly related to the logarithm of molecular mass in the range from approximately 200 to 6000 kDa. A sample load of 2.5 ?g for polydisperse HA samples was employed. Densitometric scanning of stained gels allowed analysis of the range of molecular masses present in the sample as well as calculation of weight-average and number-average values. The method was validated for a polydisperse HA sample with a weight-average molecular mass of approximately 2000 kDa. Excellent agreement was found between the weight-average molecular mass determined by electrophoresis and that determined by rheological measurement of the solution viscosity. The revised method was then used to show that heating solutions of HA at 100�C, followed by various cooling procedures, had no effect on the HA molecular mass distribution. PMID:21683677
Cowman, Mary K; Chen, Cherry C; Pandya, Monika; Yuan, Han; Ramkishun, Dianne; LoBello, Jaclyn; Bhilocha, Shardul; Russell-Puleri, Sparkle; Skendaj, Eraldi; Mijovic, Jovan; Jing, Wei
Cross-Linked Supramolecular Polymer Gels Constructed from Discrete Multi-pillar[5]arene Metallacycles and Their Multiple Stimuli-Responsive Behavior.
A new family of discrete hexakis-pillar[5]arene metallacycles with different sizes have been successfully prepared via coordination-driven self-assembly, which presented very few successful examples of preparation of discrete multiple pillar[n]arene derivatives. These newly designed hexakis-pillar[5]arene metallacycles were well characterized with one-dimensional (1-D) multinuclear NMR ((1)H and (31) P NMR), two-dimensional (2-D) (1)H-(1)H COSY and NOESY, ESI-TOF-MS, elemental analysis, and PM6 semiempirical molecular orbital methods. Furthermore, the host-guest complexation of such hexakis-pillar[5]arene hosts with a series of different neutral ditopic guests G1-6 were well investigated. Through host-guest interactions of hexakis-pillar[5]arene metallacycles H2 or H3 with the neutral dinitrile guest G5, the cross-linked supramolecular polymers H2?(G5)3 or H3?(G5)3 were successfully constructed at the high-concentration region, respectively. Interestingly, these cross-linked supramolecular polymers transformed into the stable supramolecular gels upon increasing the concentrations to a relatively high level. More importantly, by taking advantage of the dynamic nature of metal-ligand bonds and host-guest interactions, the reversible multiple stimuli-responsive gel-sol phase transitions of such polymer gels were successfully realized under different stimuli, such as temperature, halide, and competitive guest, etc. The mechanism of such multiple stimuli-responsive processes was well illustrated by in situ multinuclear NMR investigation. This research not only provides a highly efficient approach to the preparation of discrete multiple pillar[n]arene derivatives but also presents a new family of multiple stimuli-responsive "smart" soft matters. PMID:24571308
Li, Zhong-Yu; Zhang, Yanyan; Zhang, Chang-Wei; Chen, Li-Jun; Wang, Chao; Tan, Hongwei; Yu, Yihua; Li, Xiaopeng; Yang, Hai-Bo
Noncovalently and covalently cross-linked polyurethane gels as alignment media and the suppression of residual polymer signals using diffusion-filtered spectroscopy.
With polyurethane (PU), a novel alignment medium for organic solvents is introduced and characterized, which is very robust and easy to produce on a large scale. Linear PU already constitutes an elastomer gel with several solvents based on its ability to form hydrogen bonds. Covalent cross-linking of the polymer with accelerated electrons provides an alignment medium with different properties. However, PU exhibits a number of undesired polymer signals in corresponding spectra, which ideally have to be removed spectroscopically. Within this context, we demonstrate the applicability of diffusion-filtered experiments for removal of the polymer signals. Example spectra for the usefulness of PU alignment media are provided for the common test molecules strychnine and norcamphor. PMID:23280657
Kaden, Peter; Freudenberger, J Christoph; Luy, Burkhard
The effect of F127 addition on the properties of PEGDA\\/PVdF cross-linked gel polymer electrolytes
The ternary polymer membranes consisting of poly(ethylene glycol) diacrylate (PEGDA), poly(vinylidene fluoride) (PVdF), and polyethylene oxide-co-polypropylene oxide-co-polyethylene oxide (PEO-PPO-PEO, F127) were prepared as the matrix of gel polymer electrolytes (GPEs) by a solvent controlled evaporation procedure. In the ternary system, the unraveled PVdF polymer chains are randomly mixed with the liquid PEGDA oligomer, which are entangled with themselves and the
Yan-Jie Wang; Dukjoon Kim
An agarose gel electrophoretic method for analysis of hyaluronan molecular weight distribution.
An electrophoretic method is described for determining the molecular weight distribution of hyaluronan (HA). The method involves separation of HA by electrophoresis on a 0.5% agarose gel, followed by detection of HA using the cationic dye Stains-All (3,3'-dimethyl-9-methyl-4,5,4'5'-dibenzothiacarbocyanine). The recommended sample load is 7 micrograms. Calibration of the method with HA standards of known molecular weight has established a linear relationship between electrophoretic mobility and the logarithm of the weight-average molecular weight over the range of approximately 0.2-6 x 10(6). The separated HA pattern may also be visualized after electrotransfer of HA from the agarose gel to a nylon membrane. The membrane may be stained with the dye alcian blue. Alternatively, specific detection of HA from impure samples can be achieved by probing the nylon membrane with biotin-labeled HA-binding protein and subsequent interaction with a streptavidin-linked gold reagent and silver staining for amplification. The electrophoretic method was used to analyze HA in two different liquid connective tissues. Normal human knee joint synovial fluid showed a narrow HA molecular weight distribution, with a peak at 6-7 x 10(6). Owl monkey vitreous HA also showed a narrow molecular weight distribution, with a peak at 5-6 x 10(6). These results agree well with available published data and indicate the applicability of the method to the analysis of impure HA samples which may be available in limited amounts. PMID:8080084
Lee, H G; Cowman, M K
Agarose and Polyacrylamide Gel Electrophoresis Methods for Molecular Mass Analysis of 5-500 kDa Hyaluronan
Agarose and polyacrylamide gel electrophoresis systems for the molecular mass-dependent separation of hyaluronan (HA) in the size range of approximately 5�500 kDa have been investigated. For agarose-based systems, the suitability of different agarose types, agarose concentrations, and buffers systems were determined. Using chemoenzymatically synthesized HA standards of low polydispersity, the molecular mass range was determined for each gel composition, over which the relationship between HA mobility and logarithm of the molecular mass was linear. Excellent linear calibration was obtained for HA molecular mass as low as approximately 9 kDa in agarose gels. For higher resolution separation, and for extension to molecular masses as low as approximately 5 kDa, gradient polyacrylamide gels were superior. Densitometric scanning of stained gels allowed analysis of the range of molecular masses present in a sample, and calculation of weight-average and number-average values. The methods were validated for polydisperse HA samples with viscosity-average molecular masses of 112, 59, 37, and 22 kDa, at sample loads of 0.5 �g (for polyacrylamide) to 2.5 �g (for agarose). Use of the methods for electrophoretic mobility shift assays was demonstrated for binding of the HA-binding region of aggrecan (recombinant human aggrecan G1-IGD-G2 domains) to a 150 kDa HA standard.
Bhilocha, Shardul; Amin, Ripal; Pandya, Monika; Yuan, Han; Tank, Mihir; LoBello, Jaclyn; Shytuhina, Anastasia; Wang, Wenlan; Wisniewski, Hans-Georg; de la Motte, Carol; Cowman, Mary K.
Functional Properties of Hydroxypropylated, Cross-Linked, and Hydroxypropylated Cross-Linked Tuber and Root Starches
Cereal Chem. 84(1):30-37 Functional properties of some underexploited tuber and root starches (true yam, gourd yam, taro, lotus, and sweet potato) were investigated before and after hydroxypropylation, cross-linking, and hydroxypropylation and cross-linking using potato starch as the reference. Low swelling ability, poor viscosity development but high shear stability, gel hardness, and resistance to enzyme hydrolysis was observed in starches from
Anil Gunaratne; Harold Corke
Application of NMR spectroscopy and multidimensional imaging to the gelcasting process and in-situ real-time monitoring of cross-linking polyacrylamide gels
In the gelcasting process, a slurry of ceramic powder in a solution of organic monomers is cast in a mold. The process is different from injection molding in that it separates mold-filling from setting during conversion of the ceramic slurry to a formed green part. In this work, NMR spectroscopy and imaging have been conducted for in-situ monitoring of the gelation process and for mapping the polymerization. {sup 1}H nuclear magnetic resonance spectra have been obtained during polymerization of a premix of soluble reactive methacrylamide (monomer) and N, N`-methylene bisacrylamide (cross-linking molecules). The premix was polymerized by adding ammonium persulfate (initiator) and tetramethyl-ethylene-diamine (accelerator) to form long-chain, cross-linked polymers. The time-varying spin-lattice relaxation times T{sub 1} during polymerization have been studied at 25 and 35{degrees}C, and the variation of spectra and T{sub 1} with respect to extent of polymerization has been determined. To verify homogeneous polymerization, multidimensional NMR imaging was utilized for in-situ monitoring of the process. The intensities from the images are modeled and the correspondence shows a direct extraction of T{sub 1} data from the images.
Ahuja, S.; Dieckman, S.L.; Gopalsami, N. [and others
DNA Interstrand Cross-Linking by Epichlorohydrin
Epichlorohydrin (ECH), an important industrial chemical, is a bifunctional alkylating agent with the potential to form DNA cross-links. Occupational exposure to this suspect carcinogen leads to chromosomal aberrations, and ECH has been shown previously to undergo reaction with DNA in vivo and in vitro.We used denaturing polyacrylamide gel electrophoresis to monitor the possible formation of interstrand cross-links within DNA oligomers by ECH and the related compound, epibromohydrin (EBH). Although both compounds did indeed form cross-links between deoxyguanosine residues, EBH was a more efficient cross-linker than ECH. The optimal pH for cross-linking also varied, with ECH more efficient at pH 5.0 and EBH more efficient at pH 7.0. Both agents were relatively flexible in the sequences targeted, with comparable efficiencies for 5?-GGC and 5?GC sites. Furthermore, interstrand cross-linking by the two optical isomers of ECH correlated with their relative cytotoxicities, with R-ECH about twice as potent as S-ECH.
Romano, Keith P.; Newman, Adam G.; Zahran, Rami W.; Millard, Julie T.
Kinetics of the swelling of a gel of cross-linked polyvinyl alcohol during the synthesis of copper-containing composite based on it
NASA Astrophysics Data System (ADS)
The kinetics of processes that occur during the chemical reduction of copper in a gel of polyvinyl alcohol (PVA) are described. It is shown that we can assess the processes occurring in the polymer from changes in the degree of swelling. The effects the nature of the precursor (precursor compound) has on the conditions of reduction are analyzed. It is found that crystals of reduced copper do not change the volume of PVA gel, which has a positive effect on the mechanical stability of the resulting composite.
Khudyakova, S. N.; Tokmachev, M. G.; Ferapontov, N. B.
Hyaluronan: from extracellular glue to pericellular cue
Hyaluronan is an extracellular and cell-surface-associated polysaccharide that is traditionally regarded as a biological 'goo' that participates in lubricating joints or holding together gel-like connective tissues. Although these are common physiological roles of hyaluronan in adult organisms, hyaluronan also functions as a microenvironmental cue that co-regulates cell behaviour during embryonic development, healing processes, inflammation and tumour development. Recent work highlights
Bryan P. Toole
Flexible all-solid-state supercapacitors based on graphene/carbon black nanoparticle film electrodes and cross-linked poly(vinyl alcohol)-H2SO4 porous gel electrolytes
NASA Astrophysics Data System (ADS)
Flexible all-solid-state supercapacitors (SCs) are fabricated using graphene/carbon black nanoparticle (GCB) film electrodes and cross-linked poly(vinyl alcohol)-H2SO4 porous gel electrolytes (gPVAP-H2SO4). The GCB composite films, with carbon black (CB) nanoparticles uniformly distributed in the graphene nanosheets, greatly improve the active surface areas and ion transportation of pristine graphene film. The porous structure of as-prepared gPVAP-H2SO4 membrane improves the equilibrium swelling ratio in electrolyte and provides interconnected ion transport channels. The chemical crosslinking solves the fluidity problem of PVA-H2SO4 gel electrolyte at high temperature. As-fabricated GCB//gPVAP(20)-H2SO4//GCB flexible SC displays an increased specific capacitance (144.5�F�g-1 at 0.5�A�g-1) and a higher specific capacitance retention (67.9% from 0.2 to 4�A�g-1). More importantly, the flexible SC possesses good electrochemical performance at high temperature (capacitance retention of 78.3% after 1000 cycles at 70��C).
Fei, Haojie; Yang, Chongyang; Bao, Hua; Wang, Gengchao
Sealing effects of cross-linked gelatin.
Surgical sealants form gel when applied to tissues. Currently, fibrin sealant has been successfully used in many surgical fields, but it has several disadvantages, including possible virus transmission, low adhesive strength, and high cost. In this study, gelatin and glutaraldehyde (GA) solutions were chosen to demonstrate the effectiveness of cross-linked gelatin gel as sealant and barrier, both of which have long been used in medical applications. It was found that the gelatin gel prepared from 26?wt% gelatin and 1?wt% GA solutions exhibited bonding strength almost three times higher than that of fibrin glue. The bonding strength increased with the increasing gelatin and GA concentrations. When a needle hole on PTFE vascular grafts was sealed with the gelatin gel, the water-resistant pressure significantly increased upon rubbing and was twice higher than that of fibrin glue. The cytotoxicity of gelatin gel was found to be much lower than that of albumin glue prepared at the same composition as commercially available BioGlue�. The gelatin gel was found to be also effective as barrier to prevent adhesion in a rat cecum abrasion model. PMID:22274878
Suzuki, S; Ikada, Y
Conjugation of hyaluronan to proteins.
Polymer conjugation has been widely exploited to prolong half-life and reduce immunogenicity of therapeutic proteins. Here, the potentials of hyaluronic acid (HA) have been investigated by studying the conjugates with two model enzymes, trypsin and RNase A, and with insulin. As the direct coupling of proteins to the HA's carboxylic groups can cause cross-linking problems, a hyaluronan-aldehyde derivative has been synthesized for N-terminal site-selective conjugation. HA conjugation, termed HAylation, preserved the activities of enzymes and their thermal stabilities. Insulin HAylation was studied by preparing two conjugates with different peptide loadings (32% and 17%, w/w). Noticeably, the conjugate with the lower loading showed the greater effect on blood glucose level. The 17% HA-insulin conjugate showed a lowering effect on blood glucose level for up to 6h, while free insulin exhausted its action after 1h. This study highlights the potentials of hyaluronan-aldehyde for protein delivery. PMID:23399272
Mero, Anna; Pasqualin, Matteo; Campisi, Monica; Renier, Davide; Pasut, Gianfranco
Photoreactivities and thermal properties of psoralen cross-links
The authors have studied the photoreaction of 8-methoxypsoralen (8-MOP), 4,5',8-trimethylpsoralen (TMP), and 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) with a pair of 18-base-long oligonucleotides in which a 14-base region is complementary. Only one 5'TpA site, favored for both monoadduct and cross-link formation with psoralen, is present in this oligonucleotide pair. They have used this model system to demonstrate, for the first time, strand specificity in the photoreaction of psoralen with DNA. They found that the two types of cross-links which form at this site have large differences in thermal stabilities. In addition, the denaturation of each cross-links isomer duplex occurred in at least three stages, which can be visualized as three bands in thermal equilibrium under the conditions of a denaturing polyacrylamide gel. This novel observation suggests that there are several domains differing in thermal stability in a psoralen cross-link.
Yeung, A.T.; Jones, B.K.; Chu, C.T.
Collagen cross linking: Current perspectives
Keratoconus is a common ectatic disorder occurring in more than 1 in 1,000 individuals. The condition typically starts in adolescence and early adulthood. It is a disease with an uncertain cause and its progression is unpredictable, but in extreme cases, vision deteriorates and can require corneal transplant surgery. Corneal collagen cross-linking (CCL) with riboflavin (C3R) is a recent treatment option that can enhance the rigidity of the cornea and prevent disease progression. Since its inception, the procedure has evolved with newer instrumentation, surgical techniques, and is also now performed for expanded indications other than keratoconus. With increasing experience, newer guidelines regarding optimization of patient selection, the spectrum of complications and their management, and combination procedures are being described. This article in conjunction with the others in this issue, will try and explore the uses of collagen cross-linking (CXL) in its current form.
Rao, Srinivas K
In vitro hemocompatibility testing of UV-modified hyaluronan hydrogels.
Hydrogels (hylans) based on cross-linked hyaluronan (HA) are potentially good biomaterials for vascular tissue engineering applications because they are highly non-antigenic and -immunogenic. To facilitate surface endothelialization, vital to vascular deployment, we irradiated the gel surface with low wavelength UV light. This process micro-textures the smooth gel surface to provide sites for cell anchorage and causes limited scission of native long-chain HA yielding smaller fragments that elicit an enhanced cell response. In the current in vitro study, we assessed the effects of UV irradiation on the short-term (<45 min) interaction between hylan gels and human blood cells (RBCs, platelets) and coagulation proteins at physiologic temperature. Although the lowered hydrophilicity of irradiated (UV) hylans elicited greater vascular cell response relative to unmodified (U) hylans, platelet deposition was unaffected and much lower compared to collagen-coated glass controls. The adhered platelets were rounded or mildly pseudopodic and did not express p-selectin, an activation marker. Both gel types induced identical, and minimal platelet release as measured using an platelet factor 4 ELISA, and identically deferred the intrinsic and extrinsic coagulation pathways. Both gel types induced elevated levels of contact activation of bound, but not plasma-phase factor XII relative to controls. Hemolysis rates were also identical and within accepted standards. We conclude that UV-treatment of hylans, useful to improve surface endothelialization, does not compromise their short-term hemocompatibility, vital to their use as vascular implant materials. PMID:16143386
Amarnath, Leena Pravina; Srinivas, Arvind; Ramamurthi, Anand
Hyaluronan in human malignancies
Hyaluronan, a major macropolysaccharide in the extracellular matrix of connective tissues, is intimately involved in the biology of cancer. Hyaluronan accumulates into the stroma of various human tumors and modulates intracellular signaling pathways, cell proliferation, motility and invasive properties of malignant cells. Experimental and clinicopathological evidence highlights the importance of hyaluronan in tumor growth and metastasis. A high stromal hyaluronan content is associated with poorly differentiated tumors and aggressive clinical behavior in human adenocarcinomas. Instead, the squamous cell carcinomas and malignant melanomas tend to have a reduced hyaluronan content. In addition to the stroma-cancer cell interaction, hyaluronan can influence stromal cell recruitment, tumor angiogenesis and epithelial-mesenchymal transition. Hyaluronan receptors, hyaluronan synthases and hyaluronan degrading enzymes, hyaluronidases, are involved in the modulation of cancer progression, depending on the tumor type. Furthermore, intracellular signaling and angiogenesis are affected by the degradation products of hyaluronan. Hyaluronan has also therapeutic implications since it is involved in multidrug resistance.
Sironen, R.K. [Institute of Clinical Medicine, Pathology and Forensic Medicine, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio (Finland) [Institute of Clinical Medicine, Pathology and Forensic Medicine, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio (Finland); Department of Pathology, Kuopio University Hospital, P.O. Box 1777, FI-70211 Kuopio (Finland); Tammi, M.; Tammi, R. [Institute of Biomedicine, Anatomy, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio (Finland)] [Institute of Biomedicine, Anatomy, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio (Finland); Auvinen, P.K. [Department of Oncology, Kuopio University Hospital, P.O. Box 1777, FI-70211 Kuopio (Finland)] [Department of Oncology, Kuopio University Hospital, P.O. Box 1777, FI-70211 Kuopio (Finland); Anttila, M. [Institute of Clinical Medicine, Pathology and Forensic Medicine, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio (Finland) [Institute of Clinical Medicine, Pathology and Forensic Medicine, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio (Finland); Department of Gynecology and Obstetrics, Kuopio University Hospital, P.O. Box 1777, FI-70211 Kuopio (Finland); Kosma, V-M., E-mail: firstname.lastname@example.com [Institute of Clinical Medicine, Pathology and Forensic Medicine, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio (Finland); Department of Pathology, Kuopio University Hospital, P.O. Box 1777, FI-70211 Kuopio (Finland)
Extreme dryness and DNA-protein cross-links
NASA Astrophysics Data System (ADS)
Exposure of fungal conidia (Aspergillus ochraceus) or spores of Bacillus subtilis to extreme dryness or vacuum induces DNA lesions, including strand breaks and the formation of DNA-protein cross-links. In wet cells only a small amount of protein is bound to DNA, but exposure to conditions of lowered water activity results in an increasing number of cross-links between DNA and proteins. In fungal conidia these cross-links are detected after selective iodination (125J) of the DNA-bound proteins followed by gel electrophoresis and subsequent autoradiography. Another approach is the labelling of DNA with 32p by means of nick translation and the detection of differences in the electrophoretic mobility of DNA before and after digestion with proteinase K of proteins bound to DNA.
Bieger-Dose, A.; Dose, K.; Meffert, R.; Mehler, M.; Risi, S.
Cross-linking of dark-adapted frog photoreceptor disk membranes. Evidence for monomeric rhodopsin.
A model for random cross-linking of identical monomers diffusing in a membrane was formulated to test whether rhodopsin's cross-linking behavior was quantitatively consistent with a monomeric structure. Cross-linking was performed on rhodopsin both in intact retinas and in isolated rod outer segment (ROS) membranes using the reagent glutaraldehyde. The distribution of covalent oligomers formed was analyzed by SDS-polyacrylamide gel electrophoresis and compared to predictions for the random model. A similar analysis was made for ROS membranes cross-linked by diisocyanatohexane and retinas cross-linked by cupric ion complexed with o-phenanthroline. Patterns of cross-linking produced by these three reagents are reasonably consistent with the monomer model. Glutaraldehyde was also used to cross-link the tetrameric protein aldolase in order to verify that cross-linking of a stable oligomer, under conditions comparable to those used for ROS, yielded the pattern predicted for a tetrameric protein having D2 symmetry. This pattern is markedly different from the one for a random-collision model. Moreover, a comparison of rates showed that aldolase cross-linking with glutaraldehyde is significantly faster than cross-linking of membrane-bound rhodopsin. It is concluded that rhodopsin is monomeric in dark-adapted photoreceptor membranes and that the observed cross-linking results from collisions between diffusing rhodopsin molecules.
Downer, N W
Corneal collagen cross-linking.
Corneal collagen cross-linking (CXL) with riboflavin and ultraviolet-A (UVA) is a new technique of corneal tissue strengthening by using riboflavin as a photosensitizer and UVA to increase the formation of intra and interfibrillar covalent bonds by photosensitized oxidation.Keratocyte apoptosis in the anterior segment of the corneal stroma all the way down to a depth of about 300 microns has been described and a demarcation line between the treated and untreated cornea has been clearly shown. It is important to ensure that the cytotoxic threshold for the endothelium has not been exceeded by strictly respecting the minimal corneal thickness. Confocal microscopy studies show that repopulation of keratocytes is already visible 1 month after the treatment, reaching its pre-operative quantity and quality in terms of functional morphology within 6 months after the treatment. The major indication for the use of CXL is to inhibit the progression of corneal ectasias, such as keratoconus and pellucid marginal degeneration. CXL may also be effective in the treatment and prophylaxis of iatrogenic keratectasia, resulting from excessively aggressive photoablation. This treatment has also been used to treat infectious corneal ulcers with apparent favorable results. Combination with other treatments, such as intracorneal ring segment implantation, limited topography-guided photoablation and conductive keratoplasty have been used with different levels of success. PMID:20543933
Jankov Ii, Mirko R; Jovanovic, Vesna; Nikolic, Ljubisa; Lake, Jonathan C; Kymionis, Georgos; Coskunseven, Efekan
Corneal Collagen Cross-Linking
Corneal collagen cross-linking (CXL) with riboflavin and ultraviolet-A (UVA) is a new technique of corneal tissue strengthening by using riboflavin as a photosensitizer and UVA to increase the formation of intra and interfibrillar covalent bonds by photosensitized oxidation. Keratocyte apoptosis in the anterior segment of the corneal stroma all the way down to a depth of about 300 microns has been described and a demarcation line between the treated and untreated cornea has been clearly shown. It is important to ensure that the cytotoxic threshold for the endothelium has not been exceeded by strictly respecting the minimal corneal thickness. Confocal microscopy studies show that repopulation of keratocytes is already visible 1 month after the treatment, reaching its pre-operative quantity and quality in terms of functional morphology within 6 months after the treatment. The major indication for the use of CXL is to inhibit the progression of corneal ectasias, such as keratoconus and pellucid marginal degeneration. CXL may also be effective in the treatment and prophylaxis of iatrogenic keratectasia, resulting from excessively aggressive photoablation. This treatment has also been used to treat infectious corneal ulcers with apparent favorable results. Combination with other treatments, such as intracorneal ring segment implantation, limited topography-guided photoablation and conductive keratoplasty have been used with different levels of success.
Jankov II, Mirko R.; Jovanovic, Vesna; Nikolic, Ljubisa; Lake, Jonathan C.; Kymionis, Georgos; Coskunseven, Efekan
Controlled degradation of hydrogels using multi-functional cross-linking molecules.
Hydrogels, chemically cross-linked or physically entangled, have found a number of applications as novel delivery vehicles of drugs and cells. However, the narrow ranges of degradation rates and mechanical strength currently available from many hydrogels limits their applications. We have hypothesized that utilization of multi-functional cross-linking molecules to form hydrogels could provide a wider range and tighter control over the degradation rates and mechanical stiffness of gels than bi-functional cross-linking molecules. To address the possibility, we isolated alpha-L-guluronate residues of sodium alginate, and oxidized them to prepare poly(aldehyde guluronate) (PAG). Hydrogels were formed with either poly(acrylamide-co-hydrazide) (PAH) as a multi-functional cross-linking molecule or adipic acid dihydrazide (AAD) as a bi-functional cross-linking molecule. The initial properties and degradation behavior of both PAG gel types were monitored. PAG/PAH hydrogels showed higher mechanical stiffness before degradation and degraded more slowly than PAG/AAD gels, at the same concentration of cross-linking functional groups. The enhanced mechanical stiffness and prolonged degradation behavior could be attributed to the multiple attachment points of PAH in the gel at the same concentration of functional groups. This approach to regulating gel properties with multifunctional cross-linking molecules could be broadly used in hydrogels. PMID:14751730
Lee, Kuen Yong; Bouhadir, Kamal H; Mooney, David J
Controlled Compaction with Ruthenium-catalyzed Photochemical Cross-linking of Fibrin-based Engineered Connective Tissue
Tissue engineering utilizing fibrin gel as a scaffold has the advantage of creating a completely biological replacement. Cells seeded in a fibrin gel can induce fibril alignment by traction forces when subjected to appropriate mechanical constraints. While gel compaction is key to successful tissue fabrication, excessive compaction can result due to low gel stiffness. This study investigated using ruthenium-catalyzed photo-cross-linking as a method to increase gel stiffness in order to minimize over-compaction. Cross-links between the abundant tyrosine molecules that comprise fibrin were created upon exposure to blue light. Cross-linking was effective in increasing the stiffness of the fibrin gel by 93% with no adverse effects on cell viability. Long-term culture of cross-linked tubular constructs revealed no detrimental effects on cell proliferation or collagen deposition due to cross-linking. After 4 weeks of cyclic distension, the cross-linked samples were more than twice as long as non-cross-linked controls, with similar cell and collagen contents. However, the cross-linked samples required a longer incubation period to achieve a UTS and modulus comparable to controls. This study shows that photo-cross-linking is an attractive option to stiffen the initial fibrin gel and thereby reduce cell-induced compaction, which can allow for longer incubation periods and thus more tissue growth without compaction below a useful size.
Syedain, Zeeshan H.; Bjork, Jason; Sando, Lillian; Tranquillo, Robert T.
Thermo-cross-linked elastomeric opal films.
An efficient and convenient thermal cross-linking protocol in elastomeric opal films leading to fully reversible and stretch-tunable optical materials is reported. In this study, functional monodisperse core-shell particles were arranged in a face-centered cubic (fcc) lattice structure by a melt flow process. A problem up to now was that un-cross-linked films could not be drawn fully reversibly and hence lost their optical and mechanical performance. After thermal cross-linking reaction, the obtained films can be drawn like rubbers and the color of their Bragg reflection changes because of controlled lattice deformation, which makes the cross-linked films mechanochromic sensors. Different techniques were developed for the cross-linking of the films a posteriori, after their preparation in the melt flow process. A photo-cross-linking approach was reported earlier. This study now deals with a very efficient thermo-cross-linking approach based on the chemistry of hydroxyl- and isocyanate-functionalities that form urethane bridges. The focus of the present work is the mechanism and efficiency of this cross-linking process for elastomeric opal films with excellent mechanical and optical properties. PMID:24134322
Sch�fer, Christian G; Viel, Benjamin; Hellmann, Goetz P; Rehahn, Matthias; Gallei, Markus
Cross-Linked Antioxidant Nanozymes for Improved Delivery to CNS
Formulations of antioxidant enzymes, superoxide dismutase 1 (SOD1, also known as Cu/Zn SOD) and catalase were prepared by electrostatic coupling of enzymes with cationic block copolymers, polyethyleneimine-poly(ethylene glycol) or poly(L-lysine)-poly(ethylene glycol), followed by covalent cross-linking to stabilize nanoparticles. Different cross-linking strategies (using glutaraldehyde, bis-(sulfosuccinimidyl)suberate sodium salt or 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride with N-hydroxysulfosuccinimide) and reaction conditions (pH and polycation/protein charge ratio) were investigated that allowed immobilizing active enzymes in cross-linked nanoparticles, termed �nanozymes�. Bi-enzyme nanoparticles, containing both SOD1 and catalase were also formulated. Formation of complexes was confirmed using denaturing gel electrophoresis and western blotting and physicochemical characterization was conducted using dynamic light scattering and atomic force microscopy. In vivo studies of 125I-labeled SOD1-containing nanozymes in mice demonstrated its increased stability in both blood and brain and increased accumulation in brain tissues, compared to non-cross-linked complexes and native SOD1. Future studies will evaluate potential of these formulations for delivery of antioxidant enzymes to central nervous system to attenuate oxidative stress associated with neurological diseases.
Klyachko, Natalia L.; Manickam, Devika S.; Brynskikh, Anna M.; Uglanova, Svetlana V.; Li, Shu; Higginbotham, Sheila M.; Bronich, Tatiana K.; Batrakova, Elena V.; Kabanov, Alexander V.
The effect of cross-link distributions in axially-ordered, cross-linked networks
Cross-linking between the constituent chains of biopolymers has a marked effect on their materials properties. In certain of these materials, such as fibrillar collagen, increases in cross-linking lead to an increase in the melting temperature. Fibrillar collagen is an axially-ordered network of cross-linked polymer chains exhibiting a broadened denaturation transition, which has been explained in terms of the successive denaturation with temperature of multiple species. We model axially-ordered cross-linked materials as stiff chains with distinct arrangements of cross-link-forming sites. Simulations suggest that systems composed of chains with identical arrangements of cross-link-forming sites exhibit critical behavior. In contrast, systems composed of non-identical chains undergo a crossover. This model suggests that the arrangement of cross-link-forming sites may contribute to the broadening of the denaturation transition in fibrillar collagen.
Bennett, C. Brad; Kruczek, James; Rabson, D. A.; Matthews, W. Garrett; Pandit, Sagar A.
Highly cross-linked nanoporous polymers
Condensation polymerization followed by a supercritical extraction step can be used to obtain highly cross-linked nanoporous polymers with high surface area, controlled pore sizes and rigid structural integrity. The invention polymers are useful for applications requiring separation membranes.
Steckle, Jr., Warren P. (Los Alamos, NM); Apen, Paul G. (Los Alamos, NM) [Los Alamos, NM; Mitchell, Michael A. (Los Alamos, NM) [Los Alamos, NM
Highly cross-linked nanoporous polymers
Condensation polymerization followed by a supercritical extraction step can be used to obtain highly cross-linked nanoporous polymers with high surface area, controlled pore sizes and rigid structural integrity. The invention polymers are useful for applications requiring separation membranes.
Steckle, Jr., Warren P. (Los Alamos, NM); Apen, Paul G. (Los Alamos, NM) [Los Alamos, NM; Mitchell, Michael A. (Los Alamos, NM) [Los Alamos, NM
Intramolecular cross-linking in the native JHBP molecule.
Juvenile hormone binding protein (JHBP) acts as a shuttle, carrying one of the most crucial hormones for insect development to target tissues. We have found that although the JHBP molecule does not contain tryptophan residues, it exhibits a weak fluorescence maximum near 420nm upon excitation at 315nm. Gel filtration experiments performed in denaturing conditions and ESI-MS analyses excluded the possibility that some low molecular ligand was bound to the protein molecules. Further UV and CD spectroscopy studies, as well as immunoblotting, showed that the unusual JHBP optical properties were due to dityrosine intramolecular cross-linking. These bridges were detected both in native and recombinant protein molecules. We believe that in Galleria mellonella hemolymph the DT generation occurs via ROS-mediated oxidation leading to the formation of cross-linked JHBP monomers. MS analyses of peptides generated after JHBP proteolysis indicated, that the dityrosine bridge occurs between the Y128 and Y130 residues. PMID:22086120
Bystranowska, Dominika; Szewczuk, Zbigniew; Lisowski, Marek; Sitkiewicz, Ewa; Dobryszycki, Piotr; O?yhar, Andrzej; Kochman, Marian
Cross-linked biopolymer bundles: Cross-link reversibility leads to cooperative binding/unbinding phenomena
NASA Astrophysics Data System (ADS)
We consider a biopolymer bundle consisting of filaments that are cross-linked together. The cross-links are reversible: they can dynamically bind and unbind adjacent filament pairs as controlled by a binding enthalpy. The bundle is subjected to a bending deformation and the corresponding distribution of cross-links is measured. For a bundle consisting of two filaments, upon increasing the bending amplitude, a first-order transition is observed. The transition is from a state where the filaments are tightly coupled by many bound cross-links, to a state of nearly independent filaments with only a few bound cross-links. For a bundle consisting of more than two filaments, a series of first-order transitions is observed. The transitions are connected with the formation of an interface between regions of low and high cross-link densities. Combining umbrella sampling Monte Carlo simulations with analytical calculations, we present a detailed picture of how the competition between cross-link shearing and filament stretching drives the transitions. We also find that, when the cross-links become soft, collective behavior is not observed: the cross-links then unbind one after the other leading to a smooth decrease of the average cross-link density.
Vink, Richard L. C.; Heussinger, Claus
Physicochemical properties of collagen solutions cross-linked by glutaraldehyde.
The physicochemical properties of collagen solutions (5 mg/ml) cross-linked by various amounts of glutaraldehyde (GTA) [GTA/collagen (w/w)?= 0-0.5] under acidic condition (pH 4.00) were examined. Based on the results of the determination of residual amino group content, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, dynamic rheological measurements, differential scanning calorimetry and atomic force microscopy (AFM), it was proved that the collagen solutions possessed strikingly different physicochemical properties depending on the amount of GTA. At low GTA amounts [GTA/collagen (w/w)?? 0.1], the residual amino group contents of the cross-linked collagens decreased largely from 100% to 32.76%, accompanied by an increase in the molecular weight. Additionally, increases of the fiber diameter and the values of G', G? and ?* were measured, while the thermal denaturation temperature (Td) did not change visibly and the fluidity of collagen samples was still retained with increasing the GTA amount. When the ratio of GTA to collagen exceeded 0.1, although the residual amino group content only decreased by ~8.2%, the cross-linked collagen solution [GTA/collagen (w/w)?= 0.3] displayed a clear loss of flow and a sudden rise (~2.0 �C) of the Td value compared to the uncross-linked collagen solution, probably illustrating that the collagen solution was converted into a gel with mature network structure-containing nuclei observed in AFM image. It was conjectured that the physicochemical properties of the collagen solutions might be in connection with the cross-linking between collagen molecules from the same aggregate or different aggregates. PMID:24564765
Tian, Zhenhua; Li, Conghu; Duan, Lian; Li, Guoying
Comparative analysis of hyaluronan gel and xanthan-based chlorhexidine gel, as adjunct to scaling and root planing with scaling and root planing alone in the treatment of chronic periodontitis: A preliminary study
Aim: The aim of this study was to evaluate the effects of hyaluronan (HA) and chlorhexidine (CHX) gels as adjunct to scaling and root planing (SRP) in the treatment of chronic periodontitis. Materials and Methods: Sixty patients within the age group of 30-65 years recruited to participate in the study were randomly equally divided into three groups. Complete SRP and subgingival debridement were performed within 6 h in all the patients. For control (Group I) patients, SRP was the only treatment modality given; for Group II and III patients, at least 8 teeth with 4-8 mm probing pocket depth (PPD) were selected for subgingival application of HA gel and CHX gel, respectively. Clinical periodontal parameters such as gingival index, PPD, and clinical attachment level (CAL) were recorded at baseline and 3 months, whereas plaque index was recorded at baseline, 1 month, and 3 months. For measuring systemic/hematological parameters, blood samples for laboratory tests for total leucocyte count (TLC), differential leucocyte count (DLC), and C-reactive protein (CRP) were obtained using standard 2-mL syringe from each subject in all the three groups at baseline, 24 h, and on the 1 month and 3 months post-baseline. Results: In all the three groups, a significant reduction in PPD and gain in CAL were observed between baseline and 3 months follow-up (P< 0.05); however, at 3 months, change in PPD and CAL was more in Group II than Group III, but the difference was non-significant, and Group I (control) showed less changes in PPD and CAL than both experimental groups. Only one patient revealed positive value for CRP at baseline only, and hence could not be statistically analyzed. In all the three groups, the peak values for TLC count were observed at 24 h. At 1-month and 3-month intervals, a significant improvement in TLC and DLC counts was observed among the experimental (HA gel/SRP and Xan-CHX gel) groups as compared to control group (SRP alone).
Chauhan, Avinash Singh; Bains, Vivek K.; Gupta, Vivek; Singh, G. P.; Patil, Sudhir S.
Tuning chemical and physical cross-links in silk electrogels for morphological analysis and mechanical reinforcement.
Electrochemically controlled, reversible assembly of biopolymers into hydrogel structures is a promising technique for on-demand cell or drug encapsulation and release systems. An electrochemically sol-gel transition has been demonstrated in regenerated Bombyx mori silk fibroin, offering a controllable way to generate biocompatible and reversible adhesives and other biomedical materials. Despite the involvement of an electrochemically triggered electrophoretic migration of the silk molecules, the mechanism of the reversible electrogelation remains unclear. It is, however, known that the freshly prepared silk electrogels (e-gels) adopt a predominantly random coil conformation, indicating a lack of cross-linking as well as thermal, mechanical, and morphological stabilities. In the present work, the tuning of covalent and physical ?-sheet cross-links in silk hydrogels was studied for programming the structural properties. Scanning electron microscopy (SEM) revealed delicate morphology, including locally aligned fibrillar structures, in silk e-gels, preserved by combining glutaraldehyde-cross-linking and ethanol dehydration. Fourier transform infrared (FTIR) spectroscopic analysis of either electrogelled, vortex-induced or spontaneously formed silk hydrogels showed that the secondary structure of silk e-gels was tunable between non-?-sheet-dominated and ?-sheet-dominated states. Dynamic oscillatory rheology confirmed the mechanical reinforcement of silk e-gels provided by controlled chemical and physical cross-links. The selective incorporation of either chemical or physical or both cross-links into the electrochemically responsive, originally unstructured silk e-gel should help in the design for electrochemically responsive protein polymers. PMID:23859710
Lin, Yinan; Xia, Xiaoxia; Shang, Ke; Elia, Roberto; Huang, Wenwen; Cebe, Peggy; Leisk, Gary; Omenetto, Fiorenzo; Kaplan, David L
Structural and functional studies of cross-linked Go protein subunits.
The guanine nucleotide binding proteins (G proteins) that couple hormone and other receptors to a variety of intracellular effector enzymes and ion channels are heterotrimers of alpha, beta, and gamma subunits. One way to study the interfaces between subunits is to analyze the consequences of chemically cross-linking them. We have used 1,6-bismaleimidohexane (BMH), a homobifunctional cross-linking reagent that reacts with sulfhydryl groups, to cross-link alpha to beta subunits of Go and Gi-1. Two cross-linked products are formed from each G protein with apparent molecular masses of 140 and 122 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both bands formed from Go reacted with anti-alpha o and anti-beta antibody. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is anomalous since the undenatured, cross-linked proteins have the same Stokes radius as the native, uncross-linked alpha beta gamma heterotrimer. Therefore, each cross-linked product contains one alpha and one beta subunit. Activation of Go by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) does not prevent cross-linking of alpha to beta gamma, consistent with an equilibrium between associated and dissociated subunits even in the presence of GTP gamma S. The same cross-linked products of Go are formed in brain membranes reacted with BMH as are formed in solution, indicating that the residues cross-linked by BMH in the pure protein are accessible when Go is membrane bound. Analysis of tryptic peptides formed from the cross-linked products indicates that the alpha subunit is cross-linked to the 26-kDa carboxyl-terminal portion of the beta subunit. The cross-linked G protein is functional, and its alpha subunit can change conformation upon binding GTP gamma S. GTP gamma S stabilizes alpha o to digestion by trypsin (Winslow, J.W., Van Amsterdam, J.R., and Neer, E.J. (1986) J. Biol. Chem. 261, 7571-7579) and also stabilizes the alpha subunit in the cross-linked product. Cross-linked G o can be ADP-ribosylated by pertussis toxin. This ADP-ribosylation is inhibited by GTP gamma S with a concentration dependence that is indistinguishable from that of the control, uncross-linked G o. These two kinds of experiments indicate that alpha o is able to change its conformation even though it cannot separate completely from beta gamma. Thus, although dissociation of the subunits accompanies activation of G o in solution, it is not obligatory for a conformational change to occur in the alpha subunit. PMID:1899868
Yi, F; Denker, B M; Neer, E J
Composition of cross-linked 125I-follitropin-receptor complexes
Both of the alpha and beta subunits of intact human follitropin (FSH) were radioiodinated with SVI-sodium iodide and chloramine-T and could be resolved on sodium dodecyl sulfate-polyacrylamide gels. Radioiodinated FSH was affinity-cross-linked with a cleavable (nondisulfide) homobifunctional reagent to its membrane receptor on the porcine granulosa cell surface as well as to a Triton X-100-solubilized form of the receptor. Cross-linked samples revealed three additional bands of slower electrophoretic mobility, corresponding to 65, 83, and 117 kDa, in addition to the hormone bands. The hormone alpha beta dimer band corresponded to 43 kDa. Formation of the three bands requires the SVI-hormone to bind specifically to the receptor with subsequent cross-linking. Binding was prevented by an excess of the native hormone but not by other hormones. A monofunctional analog of the cross-linking reagent failed to produce the three bands. Reagent concentration-dependent cross-linking revealed that their formation was sequential; smaller complexes formed first and then larger ones. When gels of cross-linked complexes were treated to cleave covalent cross-links and then electrophoresed in a second dimension, 18-, 22-, and 34-kDa components were released, in addition to the alpha and beta subunits of the hormone.
Shin, J.; Ji, T.H.
Phased psoralen cross-links do not bend the DNA double helix
Although the chemical reaction of psoralens with nucleic acids is well understood, the structure of psoralen-DNA cross-linked products is still not clear. Model building studies based on the crystal structure of the psoralen-thymine monoadduct suggest that each cross-link bends the DNA double helix by 46.5/sup 0/. Here the authors use gel electrophoresis to test the validity of the current models. They have synthesized a series of DNA fragments (21-24 base pairs in length), each containing one unique T-A site for 4'-(hydroxymethyl)-4,5'8-trimethylpsoralen (HMT) cross-linking. Because of an estimated 28/sup 0/ unwinding of the helix by HMT, one expects that the 22-bp cross-linked fragment will be repeated nearly in phase with the average helical screw when multimerized. In that sequence ligation will maximally amplify any deformation to the double helix. They find that the ligated multimers of cross-linked DNA migrate close to the multimers of non-cross-linked DNA on polyacrylamide gels. These observations place an upper limit of 10/sup 0/ on DNA bending induced by psoralen cross-linking and indicate unwinding by about 1 bp, as well as stiffening of the double helix. These properties are not unexpected for classical intercalators.
Haran, T.E.; Crothers, D.M.
Cross-linking Chemistry of Squid Beak*
In stark contrast to most aggressive predators, Dosidicus gigas (jumbo squids) do not use minerals in their powerful mouthparts known as beaks. Their beaks instead consist of a highly sclerotized chitinous composite with incremental hydration from the tip to the base. We previously reported l-3,4-dihydroxyphenylalanine (dopa)-histidine (dopa-His) as an important covalent cross-link providing mechanical strengthening to the beak material. Here, we present a more complete characterization of the sclerotization chemistry and describe additional cross-links from D. gigas beak. All cross-links presented in this report share common building blocks, a family of di-, tri-, and tetra-histidine-catecholic adducts, that were separated by affinity chromatography and high performance liquid chromatography (HPLC) and identified by tandem mass spectroscopy and proton nuclear magnetic resonance (1H NMR). The data provide additional insights into the unusually high cross-link density found in mature beaks. Furthermore, we propose both a low molecular weight catechol, and peptidyl-dopa, to be sclerotization agents of squid beak. This appears to represent a new strategy for forming hard tissue in animals. The interplay between covalent cross-linking and dehydration on the graded properties of the beaks is discussed.
Miserez, Ali; Rubin, Daniel; Waite, J. Herbert
Cross-linking chemistry of squid beak.
In stark contrast to most aggressive predators, Dosidicus gigas (jumbo squids) do not use minerals in their powerful mouthparts known as beaks. Their beaks instead consist of a highly sclerotized chitinous composite with incremental hydration from the tip to the base. We previously reported l-3,4-dihydroxyphenylalanine (dopa)-histidine (dopa-His) as an important covalent cross-link providing mechanical strengthening to the beak material. Here, we present a more complete characterization of the sclerotization chemistry and describe additional cross-links from D. gigas beak. All cross-links presented in this report share common building blocks, a family of di-, tri-, and tetra-histidine-catecholic adducts, that were separated by affinity chromatography and high performance liquid chromatography (HPLC) and identified by tandem mass spectroscopy and proton nuclear magnetic resonance ((1)H NMR). The data provide additional insights into the unusually high cross-link density found in mature beaks. Furthermore, we propose both a low molecular weight catechol, and peptidyl-dopa, to be sclerotization agents of squid beak. This appears to represent a new strategy for forming hard tissue in animals. The interplay between covalent cross-linking and dehydration on the graded properties of the beaks is discussed. PMID:20870720
Miserez, Ali; Rubin, Daniel; Waite, J Herbert
Polyimide Aerogels with Three-Dimensional Cross-Linked Structure
NASA Technical Reports Server (NTRS)
Polyimide aerogels with three-dimensional cross-linked structure are made using linear oligomeric segments of polyimide, and linked with one of the following into a 3D structure: trifunctional aliphatic or aromatic amines, latent reactive end caps such as nadic anhydride or phenylethynylphenyl amine, and silica or silsesquioxane cage structures decorated with amine. Drying the gels supercritically maintains the solid structure of the gel, creating a polyimide aerogel with improved mechanical properties over linear polyimide aerogels. Lightweight, low-density structures are desired for acoustic and thermal insulation for aerospace structures, habitats, astronaut equipment, and aeronautic applications. Aerogels are a unique material for providing such properties because of their extremely low density and small pore sizes. However, plain silica aerogels are brittle. Reinforcing the aerogel structure with a polymer (X-Aerogel) provides vast improvements in strength while maintaining low density and pore structure. However, degradation of polymers used in cross-linking tends to limit use temperatures to below 150 C. Organic aerogels made from linear polyimide have been demonstrated, but gels shrink substantially during supercritical fluid extraction and may have lower use temperature due to lower glass transition temperatures. The purpose of this innovation is to raise the glass transition temperature of all organic polyimide aerogel by use of tri-, tetra-, or poly-functional units in the structure to create a 3D covalently bonded network. Such cross-linked polyimides typically have higher glass transition temperatures in excess of 300 400 C. In addition, the reinforcement provided by a 3D network should improve mechanical stability, and prevent shrinkage on supercritical fluid extraction. The use of tri-functional aromatic or aliphatic amine groups in the polyimide backbone will provide such a 3D structure.
Panek, John
Firming of Bread Crumb with Cross-Linked Waxy Barley Starch Substituted for Wheat Starch
Cereal Chem. 69(3):321-325 White pan bread was baked from flour that had been fractionated a higher enthalpy of melting than that of control bread crumb, except and reconstituted using cross-linked waxy barley starch (5.9% amylose at 6 hr after baking. Furthermore, a 50% gel of cross-linked waxy barley content) in place of prime wheat starch (28.3% amylose content). starch in
Actin Filament Length Tunes Elasticity of Flexibly Cross-Linked Actin Networks
Networks of the cytoskeletal biopolymer actin cross-linked by the compliant protein filamin form soft gels that stiffen dramatically under shear stress. We demonstrate that the elasticity of these networks shows a strong dependence on the mean length of the actin polymers, unlike networks with small, rigid cross-links. This behavior is in agreement with a model of rigid filaments connected by multiple flexible linkers.
Kasza, K.E.; Broedersz, C.P.; Koenderink, G.H.; Lin, Y.C.; Messner, W.; Millman, E.A.; Nakamura, F.; Stossel, T.P.; MacKintosh, F.C.; Weitz, D.A.
Cross-linked silicon based polymer nano-wire formation by high energy charged particles
High energy charged particles penetrating into silicon based polymer materials cause non-homogeneous cross-linking in the nanospace along the particle trajectories, giving nano-gels (nano-wires) of polymers with cylindrical structures of regular length and thickness. Polycarbosilanes with a variety of molecular weights were used as the target polymer materials in the present study, and the preparation of nano-wires based on cross-linked polycarbosilanes
S. Seki; S. Tsukuda; S. Tagawa; M. Sugimoto
The influence of Hofmeister series ions on hyaluronan swelling and viscosity.
The dissolution of hyaluronan in water leads to its degradation, and as a result its molecular weight decreases. The degradation of hyaluronan is mainly influenced by temperature, solution composition, and also its pH. This study describes the influence of Hofmeister series ions on hyaluronan behaviour and hyaluronan film swelling by solutions of these ions. It was found that Hofmeister ions show lyotropic effects influencing the entanglement of hyaluronan coils and their expansion from solid polymer films into swollen gel state. The hydrophobic and hydrophilic interactions in the structure of hyaluronan macromolecules are represented by the mutual diffusion coefficient D(c), the mean mutual diffusion coefficient D(s), the expansion work of coil swelling RA(delta,s), the activation enthalpy of diffusion connected with swelling H(D,s) and kinematic viscosity of hyaluronan-ions solutions nu. PMID:18560327
Mr�cek, Ales; Varhan�kov�, J�lia; Lehock�, Mari�n; Grundelov�, Lenka; Pokopcov�, Alena; Velebn�, Vladim�r
Electrophoretic mobility of duplex DNA cross-linked by mechlorethamine at a cytosine-cytosine mismatch pair.
The common nitrogen mustard, mechlorethamine, can form a covalent cross-link between the two bases of a cytosine-cytosine mismatch pair within a DNA duplex. The cross-linked species can be readily separated from DNA monoadducts and unreacted strands using denaturing polyacrylamide gel electrophoresis. Here, using DNA 19 mer duplexes that are mechlorethamine cross-linked at a C(4)-C(35), C(7)-C(32), C(10)-C(29), or C(13)-C(26) mismatch pair, we show that the denaturing polyacrylamide gel electrophoresis mobility of the cross-linked species is particularly sensitive to the proximity of the C-C cross-link to the duplex end. Species that are cross-linked at a C(4)-C(35) mismatch have greater mobilities than those cross-linked at C(7)-C(32) or C(13)-C(26), and the species with a central C(10)-C(29) cross-link have the lowest mobility. The mobility is also dependent on the proximity of the cross-link to a 5'-(32)P-phosphate or a 5'-fluorescein label. We interpret these results in terms of the conformational properties of the cross-linked species in the denaturing gel. The results are consistent with the retention of partial duplex character at the end proximal to the cross-link, with an influence on the mobility of the GC/AT ratio proximal to the cross-link and at the duplex end, and a small but discernible effect of the label. PMID:23334930
Romero, Rebecca M; Rojsittisak, Pornchai; Haworth, Ian S
Covalent cross-linking of poly(A) to Escherichia coli ribosomes, and localization of the cross-link site within the 16S RNA.
Poly(A) can be cross-linked to E. coli 70S ribosomes in the presence of tRNALys by mild ultraviolet irradiation. The cross-linking reaction is exclusively with the 30S subunit, and involves primarily the RNA moiety. Following a partial nuclease digestion, cross-linked complexes containing poly(A) and fragments of the 16S RNA were isolated by affinity chromatography on oligo(dT)-cellulose. The complexes were purified by gel electrophoresis and subjected to oligonucleotide analysis, which revealed a single cross-link site within positions 1394-1399 of the 16S RNA. The same pattern of cross-linking, at about one-fifth of the intensity, was observed in the absence of tRNALys. The cross-link site to poly(A), together with other sites in the 16S RNA that have been implicated in ribosomal function, is discussed in the framework of our recent model for the three-dimensional structure of 16S RNA; all of the functional sites are clustered together in two distinct groups in the model. Images
Stiege, W; Stade, K; Schuler, D; Brimacombe, R
Cross-linked structure of network evolution.
We study the temporal co-variation of network co-evolution via the cross-link structure of networks, for which we take advantage of the formalism of hypergraphs to map cross-link structures back to network nodes. We investigate two sets of temporal network data in detail. In a network of coupled nonlinear oscillators, hyperedges that consist of network edges with temporally co-varying weights uncover the driving co-evolution patterns of edge weight dynamics both within and between oscillator communities. In the human brain, networks that represent temporal changes in brain activity during learning exhibit early co-evolution that then settles down with practice. Subsequent decreases in hyperedge size are consistent with emergence of an autonomous subgraph whose dynamics no longer depends on other parts of the network. Our results on real and synthetic networks give a poignant demonstration of the ability of cross-link structure to uncover unexpected co-evolution attributes in both real and synthetic dynamical systems. This, in turn, illustrates the utility of analyzing cross-links for investigating the structure of temporal networks. PMID:24697374
Bassett, Danielle S; Wymbs, Nicholas F; Porter, Mason A; Mucha, Peter J; Grafton, Scott T
Electrospinning covalently cross-linking biocompatible hydrogelators
Many hydrogel materials of interest are homogeneous on the micrometer scale. Electrospinning, the formation of sub-micrometer to micrometer diameter fibers by a jet of fluid formed under an electric field, is one process being explored to create rich microstructures. However, electrospinning a hydrogel system as it reacts requires an understanding of the gelation kinetics and corresponding rheology near the liquid-solid transition. In this study, we correlate the structure of electrospun fibers of a covalently cross-linked hydrogelator with the corresponding gelation transition and kinetics. Polyethylene oxide (PEO) is used as a carrier polymer in a chemically cross-linking poly(ethylene glycol)-high molecular weight heparin (PEG-HMWH) hydrogel. Using measurements of gelation kinetics from multiple particle tracking microrheology (MPT), we correlate the material rheology with the the formation of stable fibers. An equilibrated, cross-linked hydrogel is then spun and the PEO is dissolved. In both cases, microstructural features of the electrospun fibers are retained, confirming the covalent nature of the network. The ability to spin fibers of a cross-linking hydrogel system ultimately enables the engineering of materials and microstructural length scales suitable for biological applications.
Schultz, Kelly M.; Campo-Deano, Laura; Baldwin, Aaron D.; Kiick, Kristi L.; Clasen, Christian; Furst, Eric M.
Highly cross-linked nanoporous polymers
Condensation polymerization followed by a supercritical extraction step can be used to obtain highly cross-linked nanoporous polymers with high surface area, controlled pore sizes and rigid structural integrity. The invention polymers are useful for applications requiring separation membranes. 1 fig.
Steckle, W.P. Jr.; Apen, P.G.; Mitchell, M.A.
Cross-linking in type IV collagen.
Type IV collagen could not be extracted from human placenta using 6M-urea containing 10mM-dithiothreitol, indicating that the type IV molecule is stabilized within the basement membrane by covalent cross-links. However, various forms of type IV collagen molecule were extractable by means of increasingly severe proteolytic conditions. Type IV collagen tetramers ('spiders') lacking only the C-terminal globular region (NC1) were further digested to the 'long-form' 7S fragment and an assortment of helical rod-like molecules derived from the 'leg' region. These molecules were separated by salt fractionation and examined by rotary-shadowing electron microscopy. Isolation of these fractions from placenta which had been reduced with NaB3H4 revealed that both the 7S (N-terminal) and C-terminal regions contained significant proportions of reducible lysine-derived cross-links. These cross-links were shown to be exclusively the stable oxo-imine hydroxylysino-5-oxonorleucine. The number of the cross-links per molecule was significantly lower than might be expected in order to fully stabilize the molecule. These results suggest that the keto-imine cross-links in type IV collagen have been stabilized in part by conversion into an unknown non-reducible form, although a sensitive immunoassay failed to show the presence of any pyridinoline. Comparison with the fibrous collagen from placenta suggested that the rate of this conversion is much greater in basement-membrane collagen. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5.
Bailey, A J; Sims, T J; Light, N
Cross-Linking Studies of Lysozyme Nucleation
NASA Technical Reports Server (NTRS)
Tetragonal chicken egg white crystals consist of 4(sub 3) helices running in alternating directions, the helix rows having a two fold symmetry with each other. The unit cell consists of one complete tetrameric turn from each of two adjacent helices (an octamer). PBC analysis indicates that the helix intermolecular bonds are the strongest in the crystal, therefore likely formed first. AFM analysis of the (110) surface shows only complete helices, no half steps or bisected helices being found, while AFM line scans to measure the growth step increments show that they are multiples of the 4(sub 3) helix tetramer dimensions. This supports our thesis that the growth units are in fact multiples of the four molecule 4(sub 3) helix unit, the "average" growth unit size for the (110) face being an octamer (two turns about the helix) and the (101) growth unit averaging about the size of a hexamer. In an effort to better understand the species involved in the crystal nucleation and growth process, we have initiated an experimental program to study the species formed in solution compared to what is found in the crystal through covalent cross-linking studies. These experiments use the heterobifunctional cross-linking agent aminoethyl-4-azidonitroanaline (AEANA). An aliphatic amine at one end is covalently attached to the protein by a carbodiimide-mediated reaction, and a photo reactive group at the other can be used to initiate crosslinking. Modifications to the parent structure can be used to alter the distance between the two reactive groups and thus the cross-linking agents "reach". In practice, the cross-linking agent is first coupled to the asp101 side chain through the amine group. Asp101 lies within the active site cleft, and previous work with fluorescent probes had shown that derivatives at this site still crystallize in the tetragonal space group. This was also found to be the case with the AEANA derivative, which gave red tetragonal crystals. The protein now has a reactive group that can be photoactivated at a specific point in the nucleation or crystal growth process to "capture" protein molecules bound within reach of the crosslinking agent. If those bound protein molecules have a defined geometric relationship with the capturing molecule, such as would be found in a crystal, then the photoreacted cross-linking site should be consistent. Random protein interactions, typical of an amorphous precipitate or interaction, would show a random cross-linking reaction. The results of these and other experiments will be presented.
Forsythe, Elizabeth; Pusey, Marc
Plasticizer migration from cross-linked flexible PVC. 1. Effects on tribology
NASA Astrophysics Data System (ADS)
Utilization of soft PVC is restricted by plasticizer migration that can affect material properties, as well as its toxicity. Modifying the chemical structure of PVC is one of the most effective tool to reduce the diffusion of plasticizer. In this work, a soft cross-linked PVC was obtained using a difunctional amine, namely isophoron diamine (IPDA) as the cross-linking agent. The gel content (wt %) was evaluated by weighting the insoluble portion obtained through solvent extraction technique. Thermogravimetric analysis (TGA) revealed that cross-linking reactions promote thermal degradation phenomena in the polymer matrix. Tribological properties of soft uncross-linked, cross-linked and rigid PVC were determined. Soft formulations were held in contact for 32 days with rigid PVC sheets. Plasticizer migration towards the interface causes an increase of dynamic friction compared to that of the reference rigid PVC.
Pannico, M.; Persico, P.; Ambrogi, V.; Carfagna, C.
Complications of Corneal Collagen Cross-Linking
Cross-linking of corneal collagen (CXL) is a promising approach for the treatment of keratoconus and secondary ectasia. Several long-term and short-term complications of CXL have been studied and documented. The possibility of a secondary infection after the procedure exists because the patient is subjected to epithelial debridement and the application of a soft contact lens. Formation of temporary corneal haze, permanent scars, endothelial damage, treatment failure, sterile infiltrates, and herpes reactivation are the other reported complications of this procedure. Cross-linking is a low-invasive procedure with low complication and failure rate but it may have direct or primary complications due to incorrect technique application or incorrect patient's inclusion and indirect or secondary complications related to therapeutic soft contact lens, patient's poor hygiene, and undiagnosed concomitant ocular surface diseases.
Dhawan, Shikha; Rao, Kavita; Natrajan, Sundaram
Collagen cross-linking in thin corneas
Collagen cross-linking (CXL) has become the standard of care for progressive keratoconus, after numerous clinical studies have established its efficacy and safety in suitably selected eyes. The standard protocol is applicable in eyes which have a minimum corneal thickness of 400 ?m after epithelial debridement. This prerequisite was stipulated to protect the corneal endothelium and intraocular tissues from the deleterious effect of ultraviolet-A (UVA) radiation. However, patients with keratoconus often present with corneal thickness of less than 400 ?m and could have otherwise benefited from this procedure. A few modifications of the standard procedure have been suggested to benefit these patients without a compromise in safety. Transepithelial cross-linking, pachymetry-guided epithelial debridement before cross-linking, and the use of hypoosmolar riboflavin are some of the techniques that have been attempted. Although clinical data is limited at the present time, these techniques are worth considering in patients with thin corneas. Further studies are needed to scientifically establish their efficacy and safety.
Padmanabhan, Prema; Dave, Abhishek
Bone Fragility and Collagen Cross-Links
Infrared imaging analysis of iliac crest biopsy specimens from patients with osteoporotic and multiple spontaneous fractures shows significant differences in the spatial variation of the nonreducible: reducible collagen cross-links at bone-forming trabecular surfaces compared with normal bone. Introduction Although the role of BMC and bone mineral quality in determining fracture risk has been extensively studied, considerably less attention has been paid to the quality of collagen in fragile bone. Materials and Methods In this study, the technique of Fourier transform infrared imaging (FTIRI) was used to determine the ratio of nonreducible:reducible cross-links, in 2- to 4-?m-thick sections, from human iliac crest biopsy specimens (N = 27) at bone-forming trabecular surfaces. The biopsy specimens were obtained from patients that had been diagnosed as high- or low-turnover osteoporosis, as well as premenopausal women <40 years of age, with normal BMD and biochemistry, who suffered multiple spontaneous fractures. The obtained values were compared with previously published analyses of trabecular bone from normal non-osteoporotic subjects (N = 14, 6 males and 8 females; age range, 51�70 years). Results and Conclusions Collagen cross-links distribution within the first 50 ?m at forming trabecular surfaces in patients with fragile bone was markedly different compared with normal bone.
Paschalis, Eleftherios P; Shane, Elizabeth; Lyritis, George; Skarantavos, Grigoris; Mendelsohn, Richard; Boskey, Adele L
Positive tone cross-linked resists based on photoacid inhibition of cross linking
NASA Astrophysics Data System (ADS)
A resist imaging design that utilizes photoacid inhibition of cationic polymerization and cross-linking during a postexposure bake step has been studied. The key to the design approach is the use of two different polymerization catalysts/initiators: (1) a photoacid produced from a photoacid generator (PAG) upon exposure of the resist that can result in polymerization and cross-linking of the resist matrix and (2) a thermal cross-linking catalyst (TCC) designed to thermally catalyze epoxide-phenol cross-linking. The TCC can be chosen from a variety of compounds such as triphenylphosphine (TPP) or imidazole. When only one of these catalysts (e.g TPP or photoacid) is present in an epoxide and phenol containing resist matrix, it will individually catalyze cross-linking. When they are present together, they effectively quench one another and little to no cross-linking occurs. This approach can be used to switch the tone of a resist from negative (photoacid catalyzed) to positive (TCC catalyzed and photoacid inhibited). The effect of the ratio of TCC:PAG was examined and the optimal ratio for positive tone behavior was determined. Resist contrast can be modified by optimization of epoxide:phenol ratio in the formulation. Dual tone behavior with positive tone at low dose and negative tone at higher doses can be observed in certain formulation conditions. Initial EUV patterning shows poor results, but the source of the poor imaging is not yet understood.
Lawson, Richard A.; Chun, Jun Sung; Neisser, Mark; Tolbert, Laren M.; Henderson, Clifford L.
Enzymatically cross-linked alginic-hyaluronic acid composite hydrogels as cell delivery vehicles.
An injectable composite gel was developed from alginic and hyaluronic acid. The enzymatically cross-linked injectable gels were prepared via the oxidative coupling of tyramine modified sodium algiante and sodium hyaluronate in the presence of horse radish peroxidase (HRP) and hydrogen peroxide (H2O2). The composite gels were prepared by mixing equal parts of the two tyraminated polymer solutions in 10U HRP and treating with 1.0% H2O2. The properties of the alginate gels were significantly affected by the addition of hyaluronic acid. The percentage water absorption and storage modulus of the composite gels were found to be lower than the alginate gels. The alginate and composite gels showed lower protein release compared to hyaluronate gels in the absence of hyaluronidase. Even hyaluronate gels showed only approximately 10% protein release after 14 days incubation in phosphate buffer solution. ATDC-5 cells encapsulated in the injectable gels showed high cell viability. The composite gels showed the presence of enlarged spherical cells with significantly higher metabolic activity compared to cells in hyaluronic and alginic acid gels. The results suggest the potential of the composite approach to develop covalently cross-linked hydrogels with tuneable physical, mechanical, and biological properties. PMID:23357799
Ganesh, Nitya; Hanna, Craig; Nair, Shantikumar V; Nair, Lakshmi S
Enzymatically Cross-linked Alginic-Hyaluronic acid Composite Hydrogels As Cell Delivery Vehicles
An injectable composite gel was developed from alginic and hyaluronic acid. The ezymatically cross-linked injectable gels were prepared via the oxidative coupling of tyramine modified sodium algiante and sodium hyaluronate in the presence of horse radish peroxidase (HRP) and hydrogen peroxide (H2O2). The composite gels were prepared by mixing equal parts of the two tryaminated polymer solutions in 10U HRP and treating with 1.0% H2O2. The properties of the alginate gels were significanly affected by the addition of hyaluronic acid. The percentage water absorption and storage modulus of the composite gels were found to be lower than the alginate gels. The alginate and composite gels showed lower protein release compared to hyaluronate gels in the absence of hyaluronidase. Even hyaluronate gels showed only approximately 10% protein release after 14 days incubation in phosphate buffer solution. ATDC-5 cells encapsulated in the injectable gels showed high cell viability. The composite gels showed the presence of enlarged spherical cells with significantly higher metabolic activity compared to cells in hyaluronic and alginic acid gels. The results suggest the potential of the composite approach to develop covalently cross-linked hydrogels with tuneable physical, mechanical, and biological properties.
Ganesh, Nitya; Hanna, Craig; Nair, Shantikumar V.; Nair, Lakshmi S.
Characterization of receptors for VIP on pancreatic acinar cell plasma membranes using covalent cross-linking
Vasoactive intestinal peptide (VIP) receptors on guinea pig pancreatic acini differ from those on all other tissues in containing a high-affinity VIP receptor and a low-affinity VIP receptor that has a high affinity for secretin. To characterize the molecular components of these receptors, /sup 125/I-VIP was covalently cross-linked to these receptors by four different cross-linking agents: disuccinimidyl suberate, ethylene glycol bis (succinimidyl succinate), dithiobis (succinimidylpropionate), and m-maleimidobenzoyl N-hydroxysuccinimide ester. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated a single major polypeptide band of M/sub r/ 45,000 and a minor polypeptide band of M/sub r/ 30,000 were cross-linked to /sup 125/I-VIP. Covalent cross-linking only occurred when a cross-linking agent was added, was inhibited by GTP, was inhibited by VIP receptor agonist or antagonists that interact with VIP receptors, and not by other pancreatic secretagogues that interact with difference receptors. Thus the high-affinity VIP receptor on pancreatic acinar cell membranes consists of a single major polypeptide of M/sub r/ 45,000, and this polypeptide is not a subunit of a larger disulfide-linked structure. Furthermore, either the low-affinity VIP/secretin-preferring receptor was not covalently cross-linked under the experimental conditions or it consist of a major polypeptide with the same molecular weight as the high-affinity VIP receptor.
McArthur, K.E.; Wood, C.L.; O'Dorisio, M.S.; Zhou, Z.C.; Gardner, J.D.; Jensen, R.T.
Triple shape memory effects of cross-linked polyethylene/polypropylene blends with cocontinuous architecture.
In this paper, the triple shape memory effects (SMEs) observed in chemically cross-linked polyethylene (PE)/polypropylene (PP) blends with cocontinuous architecture are systematically investigated. The cocontinuous window of typical immiscible PE/PP blends is the volume fraction of PE (v(PE)) of ca. 30-70 vol %. This architecture can be stabilized by chemical cross-linking. Different initiators, 2,5-dimethyl-2,5-di(tert-butylperoxy)-hexane (DHBP), dicumylperoxide (DCP) coupled with divinylbenzene (DVB) (DCP-DVB), and their mixture (DHBP/DCP-DVB), are used for the cross-linking. According to the differential scanning calorimetry (DSC) measurements and gel fraction calculations, DHBP produces the best cross-linking and DCP-DVB the worst, and the mixture, DHBP/DCP-DVB, is in between. The chemical cross-linking causes lower melting temperature (Tm) and smaller melting enthalpy (?Hm). The prepared triple shape memory polymers (SMPs) by cocontinuous immiscible PE/PP blends with v(PE) of 50 vol % show pronounced triple SMEs in the dynamic mechanical thermal analysis (DMTA) and visual observation. This new strategy of chemically cross-linked immiscible blends with cocontinuous architecture can be used to design and prepare new SMPs with triple SMEs. PMID:23713446
Zhao, Jun; Chen, Min; Wang, Xiaoyan; Zhao, Xiaodong; Wang, Zhenwen; Dang, Zhi-Min; Ma, Lan; Hu, Guo-Hua; Chen, Fenghua
Cross-linking for microbial keratitis
The success of collagen cross-linking as a clinical modality to modify the clinical course in keratoconus seems to have fueled the search for alternative applications for this treatment. Current clinical data on its efficacy is limited and laboratory data seems to indicate that it performs poorly against resistant strains of bacteria and against slow growing organisms. However, the biological plausibility of crosslinking and the lack of effective strategies in managing infections with these organisms continue to focus attention on this potential treatment. Well-conducted experimental and clinical studies with controls are required to answer the questions of its efficacy in future.
Vazirani, Jayesh; Vaddavalli, Pravin K
Characterization of receptors for VIP on pancreatic acinar cell plasma membranes using covalent cross-linking.
Vasoactive intestinal peptide (VIP) receptors on guinea pig pancreatic acini differ from those on all other tissues in containing a high-affinity VIP receptor and a low-affinity VIP receptor that has a high affinity for secretin. To characterize the molecular components of these receptors, 125I-VIP was covalently cross-linked to these receptors by four different cross-linking agents: disuccinimidyl suberate, ethylene glycol bis (succinimidyl succinate), dithiobis (succinimidylpropionate), and m-maleimidobenzoyl N-hydroxysuccinimide ester. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated a single major polypeptide band of Mr 45,000 and a minor polypeptide band of Mr 30,000 were cross-linked to 125I-VIP. Covalent cross-linking only occurred when a cross-linking agent was added, was inhibited by GTP, was inhibited by VIP receptor agonists or antagonists that interact with VIP receptors, and not by other pancreatic secretagogues that interact with different receptors. For inhibiting both cross-linking and binding of 125I-VIP to the major polypeptide Mr 45,000 and the minor polypeptide Mr 30,000 components, the relative potencies were VIP greater than helodermin greater than rat growth hormone releasing factor greater than peptide histidine isoleucine greater than secretin. The apparent molecular weight of the cross-linked polypeptides were unchanged by dithiothreitol. Thus the high-affinity VIP receptor on pancreatic acinar cell membranes consists of a single major polypeptide of Mr 45,000, and this polypeptide is not a subunit of a larger disulfide-linked structure. Furthermore, either the low-affinity VIP/secretin-preferring receptor was not covalently cross-linked under the experimental conditions or it consists of a major polypeptide with the same molecular weight as the high-affinity VIP receptor. PMID:2435170
McArthur, K E; Wood, C L; O'Dorisio, M S; Zhou, Z C; Gardner, J D; Jensen, R T
Stretching and bending in cross-linked biopolymer networks
NASA Astrophysics Data System (ADS)
The elastic response of cross-linked biopolymer networks is usually interpreted in terms of affine stretching models, adopted from the theory of rubber-elasticity valid for flexible polymer gels. Unlike flexible polymers, however, stiff polymers have a highly anisotropic elastic response, where the low-energy elastic excitations are actually of bending nature. As a consequence, similar to springs connected in series, one would expect the softer bending mode to dominate the elastic energy rather than the stiff stretching mode. We propose a theory that, unlike recent affine models, properly accounts for the soft bending response of stiff polymers. It allows calculating the macroscopic elastic moduli starting from a microscopic characterization of the (non-affine) deformation field. The calculated scaling properties for the shear modulus are in excellent agreement with the results of recent simulations obtained in simple two-dimensional model networks, and can also be applied to rationalize bulk rheological data in reconstituted actin networks.
Heussinger, Claus; Frey, Erwin
Effects of alginate hydrogel cross-linking density on mechanical and biological behaviors for tissue engineering.
An effective cross-linking of alginate gel was made through reaction with calcium carbonate (CaCO3). We used human chondrocytes as a model cell to study the effects of cross-linking density. Three different pore size ranges of cross-linked alginate hydrogels were fabricated. The morphological, mechanical, and rheological properties of various alginate hydrogels were characterized and responses of biosynthesis of cells encapsulated in each gel to the variation in cross-linking density were investigated. Desired outer shape of structure was maintained when the alginate solution was cross-linked with the applied method. The properties of alginate hydrogel could be tailored through applying various concentrations of CaCO3. The rate of synthesized GAGs and collagens was significantly higher in human chondrocytes encapsulated in the smaller pore structure than that in the larger pore structure. The expression of chondrogenic markers, including collagen type II and aggrecan, was enhanced in the smaller pore structure. It was found that proper structural morphology is a critical factor to enhance the performance and tissue regeneration. PMID:24880568
Jang, Jinah; Seol, Young-Joon; Kim, Hyeon Ji; Kundu, Joydip; Kim, Sung Won; Cho, Dong-Woo
Improving the mechanical and thermal properties of gelatin hydrogels cross-linked by cellulose nanowhiskers.
This study demonstrates the preparation of a renewable and biocompatible hydrogel with superior mechanical properties consisting of a gelatin matrix cross-linked with oxidized cellulose nanowhiskers. We found an increased degree of chemical cross-linking (0.14-17%) between gelatin and nanowhiskers with the increased amount of aldehyde contents (0.062-0.230mmolg(-1)). (1)H nuclear magnetic resonance (NMR) T(2) relaxation experiments on D(2)O swollen hydrogels demonstrated systems consisting of both gelatin and cellulose nanowhiskers displayed a higher percentage of "ridge" protons, attributed in part to increasing chemical cross-linking junction points between gelatin and nanowhiskers. This increase in hydrogel rigidity not only modified local chain dynamics but also influenced gel swelling, showing relatively reduced water uptake ability than that of the neat gelatin. Rheological measurements confirmed a 150% improvement in storage modulus (G') of the cross-linked hydrogels compared to neat gelatin. Chemical cross-linking also increased the resistance of the gels towards thermal degradation above the melting temperature of gelatin as observed by thermal scanning experiments. PMID:23121958
Dash, Rajalaxmi; Foston, Marcus; Ragauskas, Arthur J
Corneal collagen cross-linking: A review
The aim was to review the published literature on corneal collagen cross-linking. The emphasis was on the seminal publications, systemic reviews, meta-analyses and randomized controlled trials. Where such an evidence did not exist, selective large series cohort studies, case controlled studies and case series with follow-up preferably greater than 12 months were included. Riboflavin/Ultraviolet A (UVA) corneal collagen cross-linking appears to be the first treatment modality to halt the progression of keratoconus and other corneal ectatic disorders with improvement in visual, keratometric and topographic parameters documented by most investigators. Its precise mechanism of action at a molecular level is as yet not fully determined. Follow-up is limited to 4�6 years at present but suggests continued stability and improvement in corneal shape with time. Most published data are with epithelium-off techniques. Epithelium-on studies suggest some efficacy but less than with the epithelium-off procedures and long-term data are not currently available. The use of Riboflavin/UVA CXL for the management of infectious and non-infectious keratitis appears very promising. Its use in the management of bullous keratopathy is equivocal. Investigation of other methodologies for CXL are under investigation.
O'Brart, David P.S.
Corneal Collagen Cross-Linking Outcomes: Review
Keratoconus is a condition characterized by biomechanical instability of the cornea, presenting in a progressive, asymmetric and bilateral way. Corneal collagen cross-linking with riboflavin and UVA (CXL) is a new technique of corneal tissue strengthening that combines the use of riboflavin as a photo sensitizer and UVA irradiation. The studies showed that CXL was effective in halting the progression of keratoconus over a period of up to four years. The published studies also revealed a reduction of max K readings by more than 2 D, while the postoperative SEQ was reduced by an average of more than 1 D, and refractive cylinder decreased by about 1 D. No eyes lost any line of BCDVA. Moreover, there was no significant decrease in endothelial cell density. It was also found that CXL treatment was effective with reducing corneal and total wavefront aberrations. Corneal cross-linking has also led to an arrest and/or even a partial reversal of keratectasia in the treatment of iatrogenic ectasia after excimer laser ablation. A primary intervention such as CXL should be considered to potentially increase the biomechanical stability of the corneal tissue and postpone the need of lamellar or penetrating keratoplasty.
Jankov II, Mirko R; Jovanovic, Vesna; Delevic, Sladjana; Coskunseven, Efekan
21 CFR 177.1211 - Cross-linked polyacrylate copolymers.
Code of Federal Regulations, 2010 CFR
...section, the cross-linked polyacrylate copolymers consist of: (1...grafted copolymer of cross-linked sodium polyacrylate identified as 2-propenoic acid...hydrolyzed polyvinyl acetate, sodium salts, graft (CAS Reg....
21 CFR 177.1211 - Cross-linked polyacrylate copolymers.
Code of Federal Regulations, 2010 CFR
...section, the cross-linked polyacrylate copolymers consist of: (1...grafted copolymer of cross-linked sodium polyacrylate identified as 2-propenoic acid...hydrolyzed polyvinyl acetate, sodium salts, graft (CAS Reg....
A novel hydrogel crosslinked hyaluronan with glycol chitosan.
A novel hydrogel was prepared by crosslinking hyaluronan with glycol chitosan in aqueous solution using water soluble carbodiimide at nearly neutral pH and room temperature. The products can be easily formulated into injectable gels, various films, membranes and sponges for soft tissue augmentation, viscosupplementation, drug delivery, preventing adhesion of post operation, wound dressing and tissue engineering scaffolds. The said hydrogel has high water adsorption property and biostability. Rheololgical results of the gel showed a soft and viscoelastic structure. FTIR further confirmed the formation of amide bonds between carboxyl groups of hyaluronan and amine groups of glycol chitosan and no N-acylurea and other derivatives were identified. PMID:17143757
Wang, Wei
Nanoscopic replication of cross-linked hydrogel in high-porosity nanoporous silica
Nanoscopic network structure of N-isopropylacrylamide (NIPAM) gel cross-linked with N,N?-methelenbisacrylamide (BIS) was replicated in sol�gel derived silica matrix. As a result of that, the silica became a nanoporous solid with a high-porosity, which reaches 60�80%. The porosity was totally due to mesopores whose size was within 10nm. Electron microscope observation revealed that the nanoscale network structure were replicated in the
Ken-ichi Kurumada; Takanori Nakamura; Atsushi Suzuki; Naoki Umeda; Naoki Kishimoto; Masahiko Hiro
21 CFR 177.2420 - Polyester resins, cross-linked.
Code of Federal Regulations, 2011 CFR
...2011-04-01 2011-04-01 false Polyester resins, cross-linked. 177.2420 Section...for Repeated Use � 177.2420 Polyester resins, cross-linked. Cross-linked polyester resins may be safely used as articles or...
21 CFR 177.2420 - Polyester resins, cross-linked.
Code of Federal Regulations, 2012 CFR
...2012-04-01 2012-04-01 false Polyester resins, cross-linked. 177.2420 Section...for Repeated Use � 177.2420 Polyester resins, cross-linked. Cross-linked polyester resins may be safely used as articles or...
Additional Applications of Corneal Cross Linking
Corneal collagen cross-linking (CxL) is a prevalent surgical method for the management of keratoconus. However, literature suggests that, further to keratoconus, CxL has a beneficial impact on a series of corneal related diseases and states. This article attempts to provide a contemporary review for all additional applications of CxL. Specifically, it outlines the most recent studies that demonstrate the beneficial impact of CxL for iatrogenic ectasias, pellucid marginal degeneration, infectious keratitis, bullous keratitopathy, and for ulcerative keratitis. The outcome of this review indicates that CxL could serve as a primary or adjuvant therapeutic modality for all aforementioned corneal-related pathologic states.
Kozobolis, Vassilios; Labiris, Georgios; Gkika, Maria; Sideroudi, Haris
Additional applications of corneal cross linking.
Corneal collagen cross-linking (CxL) is a prevalent surgical method for the management of keratoconus. However, literature suggests that, further to keratoconus, CxL has a beneficial impact on a series of corneal related diseases and states. This article attempts to provide a contemporary review for all additional applications of CxL. Specifically, it outlines the most recent studies that demonstrate the beneficial impact of CxL for iatrogenic ectasias, pellucid marginal degeneration, infectious keratitis, bullous keratitopathy, and for ulcerative keratitis. The outcome of this review indicates that CxL could serve as a primary or adjuvant therapeutic modality for all aforementioned corneal-related pathologic states. PMID:21448300
Kozobolis, Vassilios; Labiris, Georgios; Gkika, Maria; Sideroudi, Haris
Microtubule Actin Cross-Linking Factor (Macf)
We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends�PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH2 terminus. However, unlike dystonin, mACF7 does not contain a coiled�coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest�specific protein, Gas2. In this paper, we demonstrate that the NH2-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.
Leung, Conrad L.; Sun, Dongming; Zheng, Min; Knowles, David R.; Liem, Ronald K.H.
Influence of Chemical Cross-Linking on the Over Voltage Positive Temperature Coefficient of Linear Low Density Polyethylene\\/carbon Black\\/aluminum Hydroxide Nano Composites
The effect of cross-linking of blends on the stability of positive temperature coefficient (PTC) and the elimination of negative temperature coefficient (NTC) were investigated. Linear low density polyethylene (LLDPE) was chemically cross-linking with various amounts of dicumyl peroxide (DCP). The resulting of cross-linking structure of composites was analyzed by gel content, differential scanning calorimetry (DSC), and dynamic mechanical analysis (DMA).
C. Y. Huang; C. S. Tsai; K. Y. Tsao
Synthesis of acrylic and allylic bifunctional cross-linking monomers derived from PET waste
NASA Astrophysics Data System (ADS)
An acrylic and two novel allylic monomers synthesized from bis (hydroxyethyl) terephthalate, BHET, are reported. This was obtained by glycolysis of post-consumer PET with boiling ethylene glycol. The bifunctional monomer bis(2-(acryloyloxy)ethyl) terephthalate was obtained from acryloyl chloride, while the allylic monomers 2-(((allyloxi)carbonyl)oxy) ethyl (2-hydroxyethyl) terephthalate and bis(2-(((allyloxi)carbonyl)oxy)ethyl) terephthalate, from allyl chloroformate. Cross-linking was studied in bulk polymerization using two different thermal initiators. Monomers were analyzed by means of 1H NMR and the cross-linked polymers by infrared spectroscopy. Gel content higher than 90% was obtained for the acrylic monomer. In the case of the mixture of the allylic monomers, the cross-linked polymer was 80 % using BPO initiator, being this mixture 24 times less reactive than the acrylic monomer.
Cruz-Aguilar, A.; Herrera-Gonz�lez, A. M.; V�zquez-Garc�a, R. A.; Navarro-Rodr�guez, D.; Core�o, J.
Control of dehydrodiferulate cross-linking in pectins from sugar-beet tissues.
Pectins were extracted from roots, petioles and leaves of sugar beet, and cross-linked using hydrogen peroxide and peroxidase. The effects on dehydrodiferulate formation were monitored by HPLC and TLC. Dehydrodimers were formed in different proportions to those found in vivo. There was a net loss of around 50% of the phenolic groups (monomers plus dimers) during dimerisation. Gel filtration showed that root and petiole pectin, but not leaf pectin, increased in molecular weight during cross-linking. The effects of varying the cross-linking conditions were investigated, and it was found that hydrogen peroxide concentration was the most important factor in controlling both the type and amount of dehydrodiferulate formed. PMID:15110691
Baydoun, Elias A -H; Pavlencheva, Natalie; Cumming, Carol M; Waldron, Keith W; Brett, Christopher T
Covalent cross-linking of vasoactive intestinal polypeptide to its receptors on intact human lymphoblasts.
125I-labeled vasoactive intestinal polypeptide (125I-VIP) was covalently cross-linked with its binding sites on intact cultured human lymphoblasts by each of three bifunctional reagents: disuccinimidyl suberate (DSS), ethylene glycol bis(succinimidyl succinate) (EGS), and N-succinimidyl 6-(4'-azido-2'-nitrophenylamino) hexanoate (SANAH). A fourth cross-linking agent with a shorter chain length, N-hydroxysuccinimidyl 4-azidobenzoate (HSAB), was much less effective in cross-linking 125I-VIP to the site. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography demonstrated a band of Mr approximately equal to 50,000 +/- 3,000, regardless of which cross-linker was used. The labeling of this band was specific in that it was prevented by 10(-6) M unlabeled VIP and was partially blocked by the homologous hormones secretin and glucagon. The relative potencies of these peptides in blocking the cross-linking of 125I-VIP to the Mr approximately equal to 50,000 band of the lymphoblasts (VIP greater than secretin greater than or equal to glucagon) were similar to those previously found for competitive inhibition of 125I-VIP binding to its putative high-affinity receptor on these cells. The covalent cross-linking required a bifunctional reagent; it was dependent on both the number of Molt cells and the concentration of 125I-VIP. The apparent molecular weight of the cross-linked species was unchanged by treatment with dithiothreitol. These observations suggest that the Mr = 50,000 species represents 125I-VIP cross-linked to a specific plasma membrane receptor and that the receptor does not contain interchain disulfide bonds. PMID:2981838
Wood, C L; O'Dorisio, M S
Gelation of Covalently Cross-Linked PEG-Heparin Hydrogels
We study PEG�heparin hydrogels to identify compositions that lead to gel formation and measure the corresponding gelation kinetics. The material consists of a maleimide-functionalized high molecular weight heparin (HMWH) backbone covalently cross-linked with bis-thiol poly(ethylene glycol) (PEG). Using multiple particle tracking microrheology, we investigate a broad composition space, defined by the number of maleimide functional sites per HMWH (f = 3.9�11.8), the molecular weight of the PEG cross-linker (Mn = 2000, 5000, and 10 000), and the concentrations of the heparin and PEG polymers. Gelation kinetics are characterized by time�cure superposition, yielding the gel time, tc, and the critical relaxation exponent, n. Gelation times range from 5 < tc ? 45 min, with the fastest kinetics occurring for the highest HMWH maleimide functionalities. tc depends nonmonotonically on the PEG cross-linker molecular weight, suggesting that gelation is affected by the length of the cross-linker relative to intermolecular interactions between heparin molecules. The critical relaxation exponent decreases from n = 0.52 for PEG 2000 to n = 0.39 for PEG 10 000. Finally, 219 equilibrated samples taken over the entire composition space are identified as liquid or solid, defining the �gelation envelope�. The boundaries of this empirical gelation envelope are in good agreement with Flory�Stockmayer theory. In all, microrheological measurements enable characterization over a large parameter space and provide crucial insight into the gelation of complex, multifunctional hydrogelators used in therapeutic applications.
Schultz, Kelly M.; Baldwin, Aaron D.; Kiick, Kristi L.; Furst, Eric M.
Organization of photosystem I polypeptides examined by chemical cross-linking
NASA Technical Reports Server (NTRS)
Photosystem I from the cyanobacterium Synechocystis sp. PCC 6803 was examined using the chemical cross-linkers glutaraldehyde and N-ethyl-1-3-[3-(dimethylamino)propyl]carbodiimide to investigate the organization of the polypeptide subunits. Thylakoid membranes and photosystem I, which was isolated by Triton X-100 fractionation, were treated with cross-linking reagents and were resolved using a Tricine/urea low-molecular-weight resolution gel system. Subunit-specific antibodies and western blotting analysis were used to identify the components of cross-linked species. These analyses identified glutaraldehyde-dependent cross-linking products composed of small amounts of PsaD and PsaC, PsaC and PsaE, and PsaE and PsaF. The novel cross-link between PsaE and PsaF was also observed following treatment with N-ethyl-1-3-[3-(dimethylamino)propyl]carbodiimide. These cross-linking results suggest a structural interaction between PsaE and PsaF and predict a transmembrane topology for PsaF.
Armbrust, T. S.; Chitnis, P. R.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)
Cross-Link Guided Molecular Modeling with ROSETTA
Chemical cross-links identified by mass spectrometry generate distance restraints that reveal low-resolution structural information on proteins and protein complexes. The technology to reliably generate such data has become mature and robust enough to shift the focus to the question of how these distance restraints can be best integrated into molecular modeling calculations. Here, we introduce three workflows for incorporating distance restraints generated by chemical cross-linking and mass spectrometry into ROSETTA protocols for comparative and de novo modeling and protein-protein docking. We demonstrate that the cross-link validation and visualization software Xwalk facilitates successful cross-link data integration. Besides the protocols we introduce XLdb, a database of chemical cross-links from 14 different publications with 506 intra-protein and 62 inter-protein cross-links, where each cross-link can be mapped on an experimental structure from the Protein Data Bank. Finally, we demonstrate on a protein-protein docking reference data set the impact of virtual cross-links on protein docking calculations and show that an inter-protein cross-link can reduce on average the RMSD of a docking prediction by 5.0 �. The methods and results presented here provide guidelines for the effective integration of chemical cross-link data in molecular modeling calculations and should advance the structural analysis of particularly large and transient protein complexes via hybrid structural biology methods.
Leitner, Alexander; Rosenberger, George; Aebersold, Ruedi; Malmstrom, Lars
NASA Astrophysics Data System (ADS)
Kidney stones cannot be formed as long as crystals are passed in the urine. However, when crystals are retained it becomes possible for them to aggregate and form a stone. Crystals are expected to be formed not earlier than the distal tubules and collecting ducts. Studies both in vitro and in vivo demonstrate that calcium oxalate monohydrate crystals do not adhere to intact distal epithelium, but only when the epithelium is proliferating or regenerating, so that it possesses dedifferentiated cells expressing hyaluronan, osteopontin (OPN) and their mutual receptor CD44 at the apical cell membrane. The polysaccharide hyaluronan is an excellent crystal binding molecule because of its negative ionic charge. We hypothesized that the risk for crystal retention in the human kidney would be increased when tubular cells express hyaluronan at their apical cell membrane. Two different patient categories in which nephrocalcinosis frequently occurs were studied to test this hypothesis (preterm neonates and kidney transplant patients). Hyaluronan (and OPN) expression at the luminal membrane of tubular cells indeed was observed, which preceded subsequent retention of crystals in the distal tubules. Tubular nephrocalcinosis has been reported to be associated with decline of renal function and thus further studies to extend our knowledge of the mechanisms of retention and accumulation of crystals in the kidney are warranted. Ultimately, this may allow the design of new strategies for the prevention and treatment of both nephrocalcinosis and nephrolithiasis in patients.
Asselman, Marino
Corneal Cross-Linking and Safety Issues
Purpose: To compile the safety aspects of the corneal collagen cross-linking (CXL) by means of the riboflavin/UVA (370 nm) approach. Materials and Methodology: Analysis of the current treatment protocol with respect to safety during CXL. Results: The currently used UVA dose density of 5.4 J/cm2 and the corresponding irradiance of 3 mW/cm2 are below the known damage thresholds of UVA for the corneal endothelium, lens, and retina. Regarding the photochemical damages due to the free radicals the damage threshold for endothelial cells is 0.35 mW/cm2. In a 400?m thick corneal stroma saturated with riboflavin, the irradiance at the endothelial level is about 0.18 mW/cm2, which is a factor of 2 smaller than the damage threshold. Conclusion: As long as the corneal stroma treated has a minimal thickness of 400 microns (as recommended), neither corneal endothelium nor deeper structures such as lens and retina will suffer any damages. The light source should provide a homogenous irradiance avoiding hot spots.
Spoerl, Eberhard; Hoyer, Anne; Pillunat, Lutz E; Raiskup, Frederik
Nonlinear elasticity of cross-linked networks
NASA Astrophysics Data System (ADS)
Cross-linked semiflexible polymer networks are omnipresent in living cells. Typical examples are actin networks in the cytoplasm of eukaryotic cells, which play an essential role in cell motility, and the spectrin network, a key element in maintaining the integrity of erythrocytes in the blood circulatory system. We introduce a simple mechanical network model at the length scale of the typical mesh size and derive a continuous constitutive law relating the stress to deformation. The continuous constitutive law is found to be generically nonlinear even if the microscopic law at the scale of the mesh size is linear. The nonlinear bulk mechanical properties are in good agreement with the experimental data for semiflexible polymer networks, i.e., the network stiffens and exhibits a negative normal stress in response to a volume-conserving shear deformation, whereby the normal stress is of the same order as the shear stress. Furthermore, it shows a strain localization behavior in response to an uniaxial compression. Within the same model we find a hierarchy of constitutive laws depending on the degree of nonlinearities retained in the final equation. The presented theory provides a basis for the continuum description of polymer networks such as actin or spectrin in complex geometries and it can be easily coupled to growth problems, as they occur, for example, in modeling actin-driven motility.
John, Karin; Caillerie, Denis; Peyla, Philippe; Raoult, Annie; Misbah, Chaouqi
Intra-articular hyaluronans in knee osteoarthritis: rationale and practical considerations.
Intra-articular hyaluronans are used to treat pain associated with osteoarthritis of the knee. Many controlled clinical studies have demonstrated their efficacy for this indication. The rationale for the use of hyaluronans therapeutically is based on observations that hyaluronic acid is an important component of the synovial fluid acting as a cushion and lubricant for the joint and also serving as a major component of the extracellular matrix of the cartilage, helping to enhance the ability of cartilage to resist shear and maintain a resiliency to compression. While intra-articular hyaluronans are indicated at this time only for the treatment of pain in osteoarthritis of the knee, there are data to suggest that they may also be useful in treating degenerative disease of other articular joints, as well as have an impact on disease progression. The mechanisms by which hyaluronans mediate their clinical benefit seem to be multifactorial and biologically related, in contrast to the notion that they provide only viscous fluid replacement. The safety profile of intra-articular hyaluronans is very favorable and, because they are used as a local therapy, there are no known drug interactions-an advantage for patients receiving treatment for comorbid conditions. Some adverse effects, such as pseudosepsis, have been associated with cross-linked hyaluronan agents and do not appear to be class related. PMID:15005296
Kelly, Michael A; Kurzweil, Peter R; Moskowitz, Roland W
Vitamin C hinders radiation cross-linking in aqueous poly(vinyl alcohol) solutions
NASA Astrophysics Data System (ADS)
Poly(vinyl alcohol) (PVA) is a promising semi-crystalline material for biomedical applications. It is soluble in water and can be formed into hydrogels by freezing and thawing or crystallizing from an aqueous theta solution such as that of polyethylene glycol (PEG). Radiation cross-linking caused by sterilization or high dose irradiation of concentrated PVA solutions could compromise some properties of these hydrogels. Therefore, we hypothesized that radiation cross-linking of PVA solutions and PVA-PEG theta gels could be prevented by using the antioxidant vitamin C as an anticross-linking agent. Our hypothesis tested positive. Vitamin C concentrations of 0.75 and 4.5 mol/mol of PVA repeating unit could prevent cross-linking in 17.5 wt/v% PVA solutions made with PVA molecular weight of 115,000 g/mol irradiated to 25 and 100 kGy, respectively. Vitamin C also prevented cross-linking in 25 kGy irradiated PVA-PEG theta gels containing up to 5 wt% PEG and decreased the viscosity of those up to 39 wt%.
Oral, Ebru; Bodugoz-Senturk, Hatice; Macias, Celia; Muratoglu, Orhun K.
Gelatin hydrogels cross-linked with bisvinyl sulfonemethyl. 2. The physical and chemical networks.
This paper deals with the physical and the chemical gelation of gelatin in the presence of a reactant, bisvinyl sulfonemethyl (BVSM). The strategy of this investigation is to separate the contributions of the two types of cross-links in order to deduce the resultant elasticity of the network. In addition, the question raised by several authors concerning an increase of the thermal stability of the triple helices in the presence of cross-links was examined by using several techniques. In this study, the concentration of gelatin and BVSM were kept constant, while the influence of the thermal protocols was put in evidence. The gel formation was followed by rheological, thermodynamic (microcalorimetry), and optical spectroscopy (optical rotation) measurements. The results demonstrate the large differences which arise on the storage moduli by changing the thermal protocols. Cross-linking of the networks in the presence of the triple helices induce a heterogeneous repartition of the bonds, which can form along the triple helices and at the end of the sequences. Consequently, the rubber like network obtained by denaturation of the triple helices is still reminiscent of the initial twist of the chains, and a large modulus is observed, as if rigid segments were still present (storage modulus 10 times larger than for random cross-linking). The hydrogels have an elastic modulus which is larger that the addition of the physical and chemical contributions. The interpretation of the network elasticity is based on the predominant role of the rigid rods of triple helices, where the BVSM cross-links can either modify the ratio between the apparent length and distance between rods, l/d, and/or increase the rigidity of the interchain connections, which are loose coils for the physical gels. The hydrogels investigated have a network which is still close to the percolation threshold of the physical gel, and therefore, the statistical models known for well developed networks cannot be directly validated in these experimental conditions. PMID:16981771
Hellio-Serughetti, Dominique; Djabourov, Madeleine
Hyaluronan (HA) Interacting Proteins RHAMM and Hyaluronidase Impact Prostate Cancer Cell Behavior and Invadopodia Formation in 3D HA-Based Hydrogels
To study the individual functions of hyaluronan interacting proteins in prostate cancer (PCa) motility through connective tissues, we developed a novel three-dimensional (3D) hyaluronic acid (HA) hydrogel assay that provides a flexible, quantifiable, and physiologically relevant alternative to current methods. Invasion in this system reflects the prevalence of HA in connective tissues and its role in the promotion of cancer cell motility and tissue invasion, making the system ideal to study invasion through bone marrow or other HA-rich connective tissues. The bio-compatible cross-linking process we used allows for direct encapsulation of cancer cells within the gel where they adopt a distinct, cluster-like morphology. Metastatic PCa cells in these hydrogels develop fingerlike structures, �invadopodia�, consistent with their invasive properties. The number of invadopodia, as well as cluster size, shape, and convergence, can provide a quantifiable measure of invasive potential. Among candidate hyaluronan interacting proteins that could be responsible for the behavior we observed, we found that culture in the HA hydrogel triggers invasive PCa cells to differentially express and localize receptor for hyaluronan mediated motility (RHAMM)/CD168 which, in the absence of CD44, appears to contribute to PCa motility and invasion by interacting with the HA hydrogel components. PCa cell invasion through the HA hydrogel also was found to depend on the activity of hyaluronidases. Studies shown here reveal that while hyaluronidase activity is necessary for invadopodia and inter-connecting cluster formation, activity alone is not sufficient for acquisition of invasiveness to occur. We therefore suggest that development of invasive behavior in 3D HA-based systems requires development of additional cellular features, such as activation of motility associated pathways that regulate formation of invadopodia. Thus, we report development of a 3D system amenable to dissection of biological processes associated with cancer cell motility through HA-rich connective tissues.
Gurski, Lisa A.; Nguyen, Ngoc T.; Xiao, Longxi; van Golen, Kenneth L.; Jia, Xinqiao; Farach-Carson, Mary C.
The Role of Nonenzymatic Glycation and Carbonyls in Collagen Cross-Linking for the Treatment of Keratoconus
Purpose. Corneal cross-linking (CXL) is a treatment for keratoconus that eliminates the need for keratoplasty in most patients. However, its molecular mechanisms remain under study. Advanced glycation end products (AGEs) have been suggested by many studies as the causative strengthening agent during CXL, though no studies to date have directly tested this hypothesis. Methods. Corneas of young rabbits and sharks were pretreated with pyridoxal hydrochloride and copper ions before CXL. Two known inhibitors of AGE formation, aminoguanidine and rifampicin, were applied during CXL in the treatment solution. Tensile strength tests were conducted after these experiments to detect diminished or accentuated corneal stiffening after CXL. SDS-PAGE was performed on type I collagen cross-linked in the absence and presence of AGE inhibitors. Results. Pretreatment with pyridoxal hydrochloride resulted in significantly higher corneal stiffening after CXL. AGE inhibitors significantly diminished cross-linking as detected by both tensile strength measurements using whole corneas and gel electrophoresis of in vitro cross-linking of type I collagen in solution, in the presence and absence of the inhibitors. Rifampicin inhibited CXL more significantly than aminoguanidine in gel electrophoresis and tensile strength tests, confirming recent findings on its efficacy as an AGE inhibitor. Conclusions. Data presented here suggest that CXL is carbonyl dependent and involves the formation of AGE cross-links. Six possible cross-linking mechanisms are discussed.
Littlechild, Stacy; McCall, Scott; Zhang, Yuntao; Conrad, Gary W.
Trametes sp. as a source of biopolymer cross-linking agents: laccase induced gelation of ferulated arabinoxylans.
This study examined the feasibility of using Trametes sp. grown under drought conditions to catalyze the covalent cross-linking of ferulated arabinoxylans. The rate of polymerization of ferulated arabinoxylans solutions at 0.5% (w/v) was dose dependent on the laccase concentration in the system. Above 1.2 nkat laccase/mg ferulated arabinoxylan, the rate of cross-linking reached a plateau, suggesting that the reaction reached saturation. For 2% (w/v) ferulated arabinoxylans treated with laccase (1.6 nkat/mg ferulated arabinoxylan), stable gels were formed after 4 h at 25 degrees C, with cross-linking contents (diferulic and triferulic acid) contents of 0.03 and 0.015 microg/mg ferulated arabinoxylan, respectively. This study demonstrated that Trametes sp. can be a source of biopolymer cross-linking enzymes like laccase. PMID:19924054
Castillo, Alva; Rasc�n-Chu, Agust�n; Vargas, Georgina; Carvajal-Mill�n, Elizabeth; Valenzuela-Soto, Elisa; Sotelo-Mundo, Rogerio R; Mart�nez, Ana Luisa
Cross-linking of two antiparallel vortex tubes
The detailed mechanisms in vortex cross-linking are unveiled by adequately resolved, direct numerical simulation of two viscous vortex tubes. There are three characteristic phases: (1) inviscid induction followed by core flattening and stretching; (2) bridging of the two vortices by accumulation of annihilated and then cross-linked vortex lines; and (3) threading of the remnants of the initial vortex pair in
M. V. Melander; F. Hussain
Structure of Glutaraldehyde Cross-Linked Ryanodine Receptor
The ryanodine receptor (RyR) family of calcium release channels plays a vital role in excitation-contraction coupling (ECC). Along with the dihydropyridine receptor (DHPR), calsequestrin, and several other smaller regulatory and adaptor proteins, RyRs form a large dynamic complex referred to as ECC machinery. Here we describe a simple cross-linking procedure that can be used to stabilize fragile components of the ECC machinery, for the purpose of structural elucidation by single particle cryo-electron microscopy (cryo-EM). As a model system, the complex of the FK506-binding protein (FKBP12) and RyR1 was used to test the cross-linking protocol. Glutaraldehyde fixation led to complete cross-linking of receptor-bound FKBP12 to RyR1, and also to extensive cross-linking of the four subunits comprising RyR to one another without compromising the RyR1 ultrastructure. FKBP12 cross-linked with RyR1 was visualized in 2D averages by single particle cryo-EM. Comparison of control RyR1 and cross-linked RyR1 3D reconstructions revealed minor conformational changes at the transmembrane assembly and at the cytoplasmic region. Intersubunit cross-linking enhanced [3H]ryanodine binding to RyR1. Based on our findings we propose that intersubunit cross-linking of RyR1 by glutaraldehyde induced RyR1 to adopt an open like conformation.
Strauss, Joshua D.; Wagenknecht, Terence
Multi-Scale Modeling of Cross-Linked Nanotube Materials
NASA Technical Reports Server (NTRS)
The effect of cross-linking single-walled carbon nanotubes on the Young's modulus of a nanotube-reinforced composite is modeled with a multi-scale method. The Young's modulus is predicted as a function of nanotube volume fraction and cross-link density. In this method, the constitutive properties of molecular representative volume elements are determined using molecular dynamics simulation and equivalent-continuum modeling. The Young's modulus is subsequently calculated for cross-linked nanotubes in a matrix which consists of the unreacted cross-linking agent. Two different cross-linking agents are used in this study, one that is short and rigid (Molecule A), and one that is long and flexible (Molecule B). Direct comparisons between the predicted elastic constants are made for the models in which the nanotubes are either covalently bonded or not chemically bonded to the cross-linking agent. At a nanotube volume fraction of 10%, the Young's modulus of Material A is not affected by nanotube crosslinking, while the Young's modulus of Material B is reduced by 64% when the nanotubes are cross-linked relative to the non-cross-linked material with the same matrix.
Frankland, S. J. V.; Odegard, G. M.; Herzog, M. N.; Gates, T. S.; Fay, C. C.
Structural Analysis and Mechanical Characterization of Hyaluronic Acid-Based Doubly Cross-Linked Networks
We have created a new class of hyaluronic acid (HA)-based hydrogel materials with HA hydrogel particles (HGPs) embedded in and covalently cross-linked to a secondary network. HA HGPs with an average diameter of ?900 nm and narrow particle size distribution were synthesized using a refined reverse micelle polymerization technique. The average mesh size of the HGPs was estimated to be approximately 5.5 to 7.0 nm by a protein uptake experiment. Sodium periodate oxidation not only introduced aldehyde groups to the particles but also reduced the average particle size. The aldehyde groups generated were used as reactive handles for subsequent cross-linking with an HA derivative containing hydrazide groups. The resulting macroscopic gels contain two distinct hierarchical networks (doubly cross-linked networks, DXNs): one within individual particles and another among different particles. Bulk gels (BGs) formed by direct mixing of HA derivatives with mutually reactive groups were included for comparison. The hydrogel microstructures were collectively characterized by microscopy and neutron scattering techniques. Their viscoelasticity was quantified at low frequencies (0.1?10 Hz) using a controlled stress rheometer and at high frequencies (up to 200 Hz) with a home-built torsional wave apparatus. Both BGs and DXNs are stable elastic gels that become stiffer at higher frequencies. The HA-based DXN offers unique structural hierarchy and mechanical properties that are suitable for soft tissue regeneration.
Jha, Amit K.; Hule, Rohan A.; Jiao, Tong; Teller, Sean S.; Clifton, Rodney J.; Duncan, Randall L.; Pochan, Darrin J.; Jia, Xinqiao
Elasticity, biodegradability and cell adhesive properties of chitosan/hyaluronan multilayer films
NASA Astrophysics Data System (ADS)
In the bioengineering field, a recent and promising approach to modifying biomaterial surfaces is the layer-by-layer (LbL) technique used to build thin polyelectrolyte multilayer films. In this work, we focused on polyelectrolyte multilayer films made of two polysaccharides, chitosan (CHI) and hyaluronan (HA), and on the control of their physico-chemical and cell adhesive properties by chemical cross-linking. CHI/HA films were cross-linked using a water soluble carbodiimide and observed by confocal laser scanning microscopy (CLSM) with a fluorescently labeled CHI. Film thicknesses were similar for native and cross-linked films. The film nanometer roughness was measured by atomic force microscopy and was found to be higher for cross-linked films. Cross-linking the films also leads to a drastic change in film stiffness. The elastic modulus of the films (Young's modulus) as measured by AFM nano-indentation was about tenfold increased for cross-linked films as compared to native ones. From a biological point of view, cross-liked films are more resistant to enzymatic degradation by hyaluronidase. Furthermore, the increase in film stiffness has a favorable effect on the adhesion and spreading of chondrosarcoma cells. Thus, the CHI/HA cross-linked films could be used for various applications due to their adhesive properties and to their mechanical properties (including stability in enzymatic media).
Schneider, Aurore; Richert, Ludovic; Francius, Gregory; Voegel, Jean-Claude; Picart, Catherine
Collagen cross-link synthesis in cultured vascular endothelium.
Cultured vascular endothelium secretes the enzyme lysyl oxidase which cross-links both collagen and elastin. The major reducible cross-link synthesized by cultured human umbilical arterial and venous endothelium is dihydroxylysinonorleucine (di-OH-LNL). Treatment of the cultures with the lathyrogen beta-aminopropionitrile (BAPN), which inhibits lysyl oxidase, inhibited synthesis of this cross-link. Cultured porcine aortic endothelium synthesized three major reducible lysine-derived cross-links: dihydroxylysinonorleucine (di-OH-LNL), hydroxylysinonorleucine (OH-LNL) and lysinonorleucine (LNL); BAPN also inhibited synthesis of these three cross-links. Earlier in-vivo observations on BAPN-treated chick embryos had shown a 20% increase in the hydration of cartilage and other tissues; the likeliest explanation was that cross-link disruption permitted the proteoglycans in cartilage to express their hydrophilic nature when freed of their collagenous network. Capillary basement membrane contains laminin, proteoglycan and type IV collagen. Following the finding of oedema in lathyritic cartilage, we would propose that agents which disrupt collagen cross-links in cultured vascular endothelium, damaging capillary basement membrane, be considered as one possible mechanism in the pathogenesis of oedema.
Levene, C. I.; Heale, G.; Robins, S. P.
Redox-Responsive, Core Cross-Linked Polyester Micelles
Monomethoxy poly(ethylene glycol)-b-poly(Tyr(alkynyl)-OCA), a biodegradable amphiphilic block copolymer, was synthesized by means of ring-opening polymerization of 5-(4-(prop-2-yn-1-yloxy)benzyl)-1,3-dioxolane-2,4-dione (Tyr(alkynyl)-OCA) and used to prepare core cross-linked polyester micelles via click chemistry. Core cross-linking not only improved the structural stability of the micelles but also allowed controlled release of cargo molecules in response to the reducing reagent. This new class of core cross-linked micelles can potentially be used in controlled release and drug delivery applications.
Zhang, Zhonghai; Yin, Lichen; Tu, Chunlai; Song, Ziyuan; Zhang, Yanfeng; Xu, Yunxiang; Tong, Rong; Zhou, Qin; Ren, Jie; Cheng, Jianjun
Cross-Link-Governed Dynamics of Biopolymer Networks
NASA Astrophysics Data System (ADS)
Recent experiments show that networks of stiff biopolymers cross-linked by transient linker proteins exhibit complex stress relaxation, enabling network flow at long times. We present a model for the dynamics controlled by cross-links in such networks. We show that a single microscopic time scale for cross-linker unbinding leads to a broad spectrum of macroscopic relaxation times and a shear modulus G�?1/2 for low frequencies ?. This model quantitatively describes the measured rheology of actin networks cross-linked with ?-actinin-4 over more than four decades in frequency.
Broedersz, Chase P.; Depken, Martin; Yao, Norman Y.; Pollak, Martin R.; Weitz, David A.; Mackintosh, Frederick C.
Exploration of chemically cross-linked metal-organic frameworks.
A series of geometrically constrained, cross-linked benzene dicarboxylic acid (bdc) derivatives have been synthesized and incorporated into the canonical isoreticular metal-organic framework (IRMOF) lattice. Only certain cross-links, which allow for the proper relative orientation of the bdc subunits, form the desired IRMOF. Design criteria from these cross-linked ligands allowed for the rational design of two oligomeric ligands composed of three bdc monomers tethered together. These oligomeric ligands were also readily incorporated into an IRMOF lattice with a high degree of crystallinity and porosity, providing a new dimension to rational ligand design for metal-organic frameworks. PMID:24943327
Allen, C A; Cohen, S M
Rearrangement of interstrand cross-links into intrastrand cross-links in cis-diamminedichloroplatinum(II)-modified DNA.
In the reaction of the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP) with DNA, bifunctional intrastrand and interstrand cross-links are formed. In this work, we show that at 37 degrees C interstrand cross-links (ICL) are labile and rearrange into intrastrand cross-links. The ICL instability was first studied with a 10 base pairs (bp) double-stranded oligonucleotide containing a unique site-specific ICL resulting from chelation of the N7 position of two guanine residues on the opposite strands of DNA at the d(GC/GC) site by a cis-diammineplatinum(II) residue. The bonds between the platinum and the N7 of guanine residues within the interstrand adduct are cleaved. In 50 mM NaCl or NaClO4, this cleavage results in the formation of monofunctional adducts which subsequently form intrastrand cross-links. One cleavage reaction takes place per cross-linked duplex in either of both DNA strands. Whereas the starting cross-linked 10 bp duplex is hydrogen bonded, the two complementary DNA strands separate after the cleavage of the ICL. Under these conditions, the cleavage reaction is irreversible allowing its rate measurement (t1/2= 29+/-2 h) and closure of monofunctional adducts to intrastrand cross-links occurs within single-stranded DNA. Within a longer cross-linked oligonucleotide (20 bp), ICL are apparently more stable (t1/2= 120+/-12 h) as a consequense of monofunctional adducts closure back to ICL. We propose that the ICL cleavage is reversible in DNA and that these adducts rearrange finally into intrastrand cross-links. Our results could explain an 'ICL unhooking' in previously reported in vivo repair studies [Zhenet al. (1993)Carcinogenesis14, 919-924].
Perez, C; Leng, M; Malinge, J M
Quantitative affinity electrophoresis of RNA-small molecule interactions by cross-linking the ligand to acrylamide.
We show that the affinity electrophoresis analysis of RNA-small molecule interactions can be made quantifiable by cross-linking the ligand to the gel matrix. Using an RNA-aminoglycoside model system to verify our method, we attached an acryloyl chloride molecule to the aminoglycosides paromomycin and neomycin B to synthesize an acrylamide-aminoglycoside monomer. This molecule was then used as a component in gel polymerization for affinity electrophoresis, covalently attaching an aminoglycoside molecule to the gel matrix. To test RNA binding to the cross-linked aminoglycosides, we used the aminoglycoside binding RNA molecule derived from thymidylate synthase messenger RNA (mRNA) that contains a C-C mismatch. Binding is indicated by the difference in RNA mobility between gels with cross-linked ligand, with ligand embedded during polymerization, and with no ligand present. Critically, the predicted straight line relationship between the reciprocal of the relative migration of the RNA and the ligand concentration is obtained when using cross-linked aminoglycosides, whereas a straight line is not obtained using embedded aminoglycosides. Average apparent dissociation constants are determined from the slope of the line from these plots. This method allows an easy quantitative comparison between different nucleic acid molecules for a small molecule ligand. PMID:23928050
Boodram, Sherry N; McCann, Lucas C; Organ, Michael G; Johnson, Philip E
Remarkable swelling capability of amino Acid based cross-linked polymer networks in organic and aqueous medium.
This work reports design and synthesis of side chain amino acid based cross-linked polymeric gels, able to switch over from organogel to hydrogel by a simple deprotection reaction and showing superabsorbancy in water. Amino acid based methacrylate monomers, tert-butoxycarbonyl (Boc)-l/d-alanine methacryloyloxyethyl ester (Boc-l/d-Ala-HEMA), have been polymerized in the presence of a cross-linker via conventional free radical polymerization (FRP) and the reversible addition-fragmentation chain transfer (RAFT) technique for the synthesis of cross-linked polymer gels. The swelling behaviors of these organogels are investigated in organic solvents, and they behave as superabsorbent materials for organic solvents such as dichloromethane, acetone, tetrahydrofuran, etc. Swollen cross-linked polymer gels release the absorbed organic solvent rapidly. After Boc group deprotection from the pendant alanine moiety, the organogels transform to the hydrogels due to the formation of side chain ammonium (-NH3(+)) groups, and these hydrogels showed a significantly high swelling ratio (?560 times than their dry volumes) in water. The morphology of organogels and hydrogels is studied by field emission scanning electron microscopy (FE-SEM). Amino acid based cross-linked gels could find applications as absorbents for oil spilled on water as well as superabsorbent hydrogels. PMID:24556036
Roy, Saswati Ghosh; Haldar, Ujjal; De, Priyadarsi
21 CFR 177.2420 - Polyester resins, cross-linked.
Code of Federal Regulations, 2010 CFR
...exceed 0.2 percent of the polyester resin. 4. Solvents for... Asbestos Glass fiber Polyester fiber produced by the condensation...glycol 6000 Silicon dioxide Wax, petroleum Complying with... (c) The cross-linked polyester resins, with or...
21 CFR 177.2420 - Polyester resins, cross-linked.
Code of Federal Regulations, 2010 CFR
...exceed 0.2 percent of the polyester resin. 4. Solvents for... Asbestos Glass fiber Polyester fiber produced by the condensation...glycol 6000 Silicon dioxide Wax, petroleum Complying with... (c) The cross-linked polyester resins, with or...
Effectiveness of trimethylopropane trimethacrylate for the electron-beam-irradiation-induced cross-linking of polylactic acid
NASA Astrophysics Data System (ADS)
The purpose of this research was to investigate the effects of various loading levels of trimethylopropane trimethacrylate (TMPTMA) on the properties of polylactic acid (PLA) cross-linked via electron-beam irradiation. PLA was compounded with 3-5 wt.% of TMPTMA to induce cross-linking upon subjection to electron-beam irradiation doses of 25-250 kGy. The physical properties of the PLA samples were characterised by means of X-ray diffraction, gel fraction and scanning electron microscopy analyses on fractured surfaces after tensile tests. The presence of TMPTMA in PLA was found to effectively increase the crystallite size and gel fraction. However, higher loading levels of TMPTMA could compromise the properties of the PLA/TMPTMA samples, indicating that a larger amount of monomer free radicals might promote degradation within the substantially cross-linked amorphous phase. Irradiation-induced cross-linking in the samples could improve the cross-linking density while decreasing the elongation and interfering with the crystallisation. These effects are caused by the intensive irradiation-induced chain scission that is responsible for the deterioration of the mechanical and crystalline properties of the samples.
Ng, Hon-Meng; Bee, Soo-Tueen; Ratnam, C. T.; Sin, Lee Tin; Phang, Yee-Yao; Tee, Tiam-Ting; Rahmat, A. R.
Identification of cross-linked peptides from large sequence databases
We describe a method to identify cross-linked peptides from complex samples and large protein sequence databases. The advance was achieved by combining isotopically tagged cross-linkers, chromatographic enrichment, targeted proteomics, and a novel search engine called xQuest. This software reduces the search space by an upstream candidatepeptide search before the recombination step; we show that xQuest can identify cross-linked peptides from a total E. coli lysate with an unrestricted database search.
Rinner, Oliver; Seebacher, Jan; Walzthoeni, Thomas; Mueller, Lukas; Beck, Martin; Schmidt, Alexander; Mueller, Markus; Aebersold, Ruedi
Thermomechanical Behavior of Collagen-Cross-Linked Porcine Cornea
Purpose: Collagen cross-linking using combined riboflavin\\/UVA treatment has been shown to increase the biomechanical rigidity of the cornea and has been used successfully for the treatment of progressive keratoconus. From morphological and biochemical investigations, a different degree of cross-linking for the anterior and posterior stroma by the treatment is suggested. The present study was undertaken to better evaluate this effect
Eberhard Spoerl; Gregor Wollensak; Dag-Daniel Dittert; Theo Seiler
Large Scale Chemical Cross-linking Mass Spectrometry Perspectives
The spectacular heterogeneity of a complex protein mixture from biological samples becomes even more difficult to tackle when one�s attention is shifted towards different protein complex topologies, transient interactions, or localization of PPIs. Meticulous protein-by-protein affinity pull-downs and yeast-two-hybrid screens are the two approaches currently used to decipher proteome-wide interaction networks. Another method is to employ chemical cross-linking, which gives not only identities of interactors, but could also provide information on the sites of interactions and interaction interfaces. Despite significant advances in mass spectrometry instrumentation over the last decade, mapping Protein-Protein Interactions (PPIs) using chemical cross-linking remains time consuming and requires substantial expertise, even in the simplest of systems. While robust methodologies and software exist for the analysis of binary PPIs and also for the single protein structure refinement using cross-linking-derived constraints, undertaking a proteome-wide cross-linking study is highly complex. Difficulties include i) identifying cross-linkers of the right length and selectivity that could capture interactions of interest; ii) enrichment of the cross-linked species; iii) identification and validation of the cross-linked peptides and cross-linked sites. In this review we examine existing literature aimed at the large-scale protein cross-linking and discuss possible paths for improvement. We also discuss short-length cross-linkers of broad specificity such as formaldehyde and diazirine-based photo-cross-linkers. These cross-linkers could potentially capture many types of interactions, without strict requirement for a particular amino-acid to be present at a given protein-protein interface. How these shortlength, broad specificity cross-linkers be applied to proteome-wide studies? We will suggest specific advances in methodology, instrumentation and software that are needed to make such a leap.
Zybailov, Boris L.; Glazko, Galina V.; Jaiswal, Mihir; Raney, Kevin D.
Structure of glutaraldehyde cross-linked ryanodine receptor.
The ryanodine receptor (RyR) family of calcium release channels plays a vital role in excitation-contraction coupling (ECC). Along with the dihydropyridine receptor (DHPR), calsequestrin, and several other smaller regulatory and adaptor proteins, RyRs form a large dynamic complex referred to as ECC machinery. Here we describe a simple cross-linking procedure that can be used to stabilize fragile components of the ECC machinery, for the purpose of structural elucidation by single particle cryo-electron microscopy (cryo-EM). As a model system, the complex of the FK506-binding protein (FKBP12) and RyR1 was used to test the cross-linking protocol. Glutaraldehyde fixation led to complete cross-linking of receptor-bound FKBP12 to RyR1, and also to extensive cross-linking of the four subunits comprising RyR to one another without compromising the RyR1 ultrastructure. FKBP12 cross-linked with RyR1 was visualized in 2D averages by single particle cryo-EM. Comparison of control RyR1 and cross-linked RyR1 3D reconstructions revealed minor conformational changes at the transmembrane assembly and at the cytoplasmic region. Intersubunit cross-linking enhanced [(3)H]ryanodine binding to RyR1. Based on our findings we propose that intersubunit cross-linking of RyR1 by glutaraldehyde induced RyR1 to adopt an open like conformation. PMID:23333333
Strauss, Joshua D; Wagenknecht, Terence
Cross-linking and the molecular packing of corneal collagen
NASA Technical Reports Server (NTRS)
We have quantitatively characterized, for the first time, the cross-linking in bovine cornea collagen as a function of age. The major iminium reducible cross-links were dehydro-hydroxylysinonorleucine (deH-HLNL) and dehydro-histidinohydroxymerodesmosine (deH-HHMD). The former rapidly diminished after birth; however, the latter persisted in mature animals at a level of 0.3 - 0.4 moles/mole of collagen. A nonreducible cross-link, histidinohydroxylysinonorleucine (HHL), previously found only in skin, was also found to be a major mature cross-link in cornea. The presence of HHL indicates that cornea fibrils have a molecular packing similar to skin collagen. However, like deH-HHMD, the HHL content in corneal fibrils only reaches a maximum value with time about half that of skin. These data suggest that the corneal fibrils are comprised of discrete filaments that are internally stabilized by HHL and deH-HHMD cross-links. This pattern of intermolecular cross-linking would facilitate the special collagen swelling property required for corneal transparency.
Yamauchi, M.; Chandler, G. S.; Tanzawa, H.; Katz, E. P.
Pericellular Hyaluronan and Prostate Cancer.
National Technical Information Service (NTIS)
The working hypothesis of the present project is that hyaluronan (HA) is responsible for maintaining pericellular spaces between nests of prostate tumor cells that are vital for the diffusion of nutrients required for tumor growth. To test this hypothesis...
C. B. Underhill
Synthesis and use of bifunctional chloromethylalkanedione derivatives of variable chain length for cross-linking thiol groups in oligomeric proteins. Specific cross-linking in glyceraldehyde 3-phosphate dehydrogenase.
Bischloromethylpentanedione, bischloromethylhexanedione, bischloromethyloctanedione and bischloromethyldecanedione were synthesized from their corresponding dicarboxylic acids via the bis-acyl chloride and the bisdiazomethylketone derivatives. These compounds proved to be highly specific cross-linking reagents for rabbit skeletal-muscle glyceraldehyde 3-phosphate dehydrogenase. Incubation of the enzyme with cross-linking reagents resulted in both a time- and concentration-dependent formation of covalently linked oligomeric structures. The major cross-linked product detected by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was the dimer (mol. wt. 72000). Sepharose 6B chromatography of the cross-linked enzyme showed that it still existed as the tetramer. Cross-linking was dependent on the native structure of the enzyme, since it was abolished on denaturation of the enzyme. The actual covalently linked product depends on the conditions of modification and the chain length of the reagent. The maximum yield of dimer (70-80%) was obtained with bischloromethylhexanedione, and the yield decreased with either shorter- or longer-chain compounds. The calculated distance between the two reactive points in bischloromethylhexanedione is 1.21-1.45nm. Bischloromethylhexanedione modified at least two thiol groups per monomer. Modification of the active-site thiol, cysteine-149, was not essential for cross-linking, since glyceraldehyde 3-phosphate dehydrogenase carboxymethylated on cysteine-149 still reacted to form the dimer. The rate of chemical cross-linking was markedly decreased by increasing the NAD(+) occupancy of the enzyme active sites. These experiments are discussed in terms of the asymmetry of the enzyme structure in solution. PMID:588252
Bloxham, D P
Random networks of cross-linked directed polymers.
We explore the effect of random permanent cross-links on a system of directed polymers confined between two planes with their end points free to slide on them. We treat the cross-links as quenched disorder and we use a semimicroscopic replica field theory to study the structure and elasticity of this system. Upon increasing the cross-link density, we get a continuous gelation transition signaled by the emergence of a finite in-plane localization length. The distribution of localization length turns out to depend on the height along the preferred direction of the directed polymers. The gelation transition also gives rise to a finite in-plane shear modulus which we calculate and turns out to be universal, i.e., independent of the energy and length scales of the polymers and the cross-links. Using a symmetry argument, we show that cross-links of negligible extent along the preferred axis of the directed polymers do not cause any renormalization to the tilt modulus of the uncross-linked system. PMID:20365585
Ulrich, Stephan; Zippelius, Annette; Benetatos, Panayotis
Synthesis and enzymatic degradation of epichlorohydrin cross-linked pectins.
The water solubility of pectin was successfully decreased by cross-linking with increasing amounts of epichlorohydrin in the reaction media. The initial molar ratios of epichlorohydrin/ galacturonic acid monomer in the reaction mixtures were 0, 0.37, 0.56, 0.74, 1.00, 1.47, and 2.44. The resulting epichlorohydrin cross-linked pectins were thus referred to as C-LP0, C-LP37, C-LP56, C-LP75, C-LP100, C-LP150, and C-LP250, respectively. Methoxylation degrees ranged from 60.5 +/- 0.9% to 68.0 +/- 0.6%, and the effective cross-linking degrees, determined by quantification of the hydroxyl anions consumed during the reaction, were 0, 17.8, 26.0, 38.3, 46.5, 53.5, and 58.7%. respectively. After incubating the different cross-linked pectins (0.5% w/v) in 25 mL of 0.05 M acetate-phosphate buffer (pH 4.5), containing 50 microL of Pectinex Ultra SP-L (pectinolytic enzymes), between 60 and 80% of the pectin osidic bounds were broken in less than 1 hr. Moreover, increasing the cross-linking degree only resulted in a weak slowing on the enzymatic degradation velocity. PMID:12648017
Semd�, Rasman�; Mo�s, Andr� J; Devleeschouwer, Michel J; Amighi, Karim
Spectroscopic characterization of collagen cross-links in bone
NASA Technical Reports Server (NTRS)
Collagen is the most abundant protein of the organic matrix in mineralizing tissues. One of its most critical properties is its cross-linking pattern. The intermolecular cross-linking provides the fibrillar matrices with mechanical properties such as tensile strength and viscoelasticity. In this study, Fourier transform infrared (FTIR) spectroscopy and FTIR imaging (FTIRI) analyses were performed in a series of biochemically characterized samples including purified collagen cross-linked peptides, demineralized bovine bone collagen from animals of different ages, collagen from vitamin B6-deficient chick homogenized bone and their age- and sex-matched controls, and histologically stained thin sections from normal human iliac crest biopsy specimens. One region of the FTIR spectrum of particular interest (the amide I spectral region) was resolved into its underlying components. Of these components, the relative percent area ratio of two subbands at approximately 1660 cm(-1) and approximately 1690 cm(-1) was related to collagen cross-links that are abundant in mineralized tissues (i.e., pyridinoline [Pyr] and dehydrodihydroxylysinonorleucine [deH-DHLNL]). This study shows that it is feasible to monitor Pyr and DHLNL collagen cross-links spatial distribution in mineralized tissues. The spectroscopic parameter established in this study may be used in FTIRI analyses, thus enabling the calculation of relative Pyr/DHLNL amounts in thin (approximately 5 microm) calcified tissue sections with a spatial resolution of approximately 7 microm.
Paschalis, E. P.; Verdelis, K.; Doty, S. B.; Boskey, A. L.; Mendelsohn, R.; Yamauchi, M.
21 CFR 177.2710 - Styrene-divinylbenzene resins, cross-linked.
Code of Federal Regulations, 2012 CFR
...2012-04-01 false Styrene-divinylbenzene resins, cross-linked. 177.2710 Section...177.2710 Styrene-divinylbenzene resins, cross-linked. Styrene-divinylbenzene cross-linked copolymer resins may be safely used as articles...
21 CFR 177.2710 - Styrene-divinylbenzene resins, cross-linked.
Code of Federal Regulations, 2011 CFR
...2011-04-01 false Styrene-divinylbenzene resins, cross-linked. 177.2710 Section...177.2710 Styrene-divinylbenzene resins, cross-linked. Styrene-divinylbenzene cross-linked copolymer resins may be safely used as articles...
21 CFR 177.2710 - Styrene-divinylbenzene resins, cross-linked.
Code of Federal Regulations, 2010 CFR
...2009-04-01 true Styrene-divinylbenzene resins, cross-linked. 177.2710 Section...177.2710 Styrene-divinylbenzene resins, cross-linked. Styrene-divinylbenzene cross-linked copolymer resins may be safely used as articles...
Silicification of genipin-cross-linked polypeptide hydrogels toward biohybrid materials and mesoporous oxides.
A simple and versatile approach is proposed to use cross-linked polypeptide hydrogels as templates for silica mineralization, allowing the synthesis of polypeptide-silica hybrid hydrogels and mesoporous silica (meso-SiO(2)) by subsequent calcination. The experimental data revealed that the cross-linked polypeptide hydrogels comprised of interconnected, membranous network served as templates for the high-fidelity transcription of silica replicas spanning from nanoscale to microscale, resulting in hybrid network comprised of interpenetrated polypeptide nanodomains and silica. The mechanical properties of these as-prepared polypeptide-silica hybrid hydrogels were found to vary with polypeptide chain length and composition. The synergy between cross-link, hydrophobic interaction, and silica deposition can lead to the enhancement of their mechanical properties. The polypeptide-silica hybrid hydrogel with polypeptide and silica content as low as 1.1 wt% can achieve 114 kN/m(2) of compressive strength. By removing the polypeptide nanodomains, mesoporous silicas with average pore sizes ranged between 2 nm and 6 nm can be obtained, depending on polypeptide chain length and composition. The polypeptide-silica hybrid hydrogels demonstrated good cell compatibility and can support cell attachment/proliferation. With the versatility of polymer chemistry and feasibility of amine-catalyzed sol-gel chemistry, the present method is facile for the synthesis of green nanocomposites and biomaterials. PMID:23148676
Jan, Jeng-Shiung; Chen, Pei-Shan; Hsieh, Ping-Lun; Chen, Bo-Yu
Reversible cross-linking of polyisoprene coronas in micelles, block comicelles, and hierarchical micelle architectures using Pt(0)-olefin coordination.
Previous work has established that polyisoprene (PI) coronas in cylindrical block copolymer micelles with a poly(ferrocenyldimethylsilane) (PFS) core can be irreversibly cross-linked by hydrosilylation using (HSiMe(2))(2)O in the presence of Karstedt's catalyst. We now show that treatment of cylindrical PI-b-PFS micelles with Karstedt's catalyst alone, in the absence of any silanes, leads to PI coronal cross-linking through Pt(0)-olefin coordination. The cross-linking can be reversed through the addition of 2-bis(diphenylphosphino)ethane (dppe), a strong bidentate ligand, which removes the platinum from the PI to form Pt(dppe)(2). The Pt(0) cross-linking of PI was studied with self-assembled cylindrical PI-b-PFS block copolymer micelles, where the cross-linking was found to dramatically increase the stability of the micellar structures. The Pt(0)-alkene coordination-induced cross-linking can be used to provide transmission electron microscopy contrast between PI and poly(dimethylsiloxane) (PDMS) corona domains in block comicelles as the process selectively increases the electron density of the PI regions. Moreover, following the assembly of a hierarchical scarf-shaped comicelle consisting of a PFS-b-PDMS platelet template with PI-b-PFS tassels, Pt(0)-induced cross-linking of the PI coronal regions allowed for the selective removal of the PFS-b-PDMS center, leaving behind an unprecedented hollowed-out scarf structure. The addition of Karstedt's catalyst to PI or polybutadiene homopolymer toluene/xylene solutions resulted in the formation of polymer gels which underwent de-gelation upon the addition of dppe. PMID:21967615
Rupar, Paul A; Cambridge, Graeme; Winnik, Mitchell A; Manners, Ian
System for Gel Electrophoretic Immunoassay.
National Technical Information Service (NTIS)
A micro-analytical platform for performing electrophoresis-based immunoassays was developed by integrating photopolymerized cross-linked polyacrylamide gels within a microfluidic device. The microfluidic immunoassays are performed by gel electrophoretic s...
A. E. Herr A. K. Singh D. J. Throckmorton
Cross-linking of Ordered Pluronic/Ionic Liquid Blends for Solid Polymer Electrolytes
NASA Astrophysics Data System (ADS)
Ion gels were fabricated by cross-linking PPO-PEO-PPO triblock copolymers swollen in a room temperature ionic liquid (IL). The copolymers are modified by esterification to replace the terminal hydroxyl endgroups with methacrylate endgroups. This allows the copolymer/IL blends to be cross-linked by a UV cure, forming a gel. The strong interaction of the IL with the PEO block suppresses PEO crystallization which is necessary for good ion conduction. In addition, the interaction between the IL and PEO is strongly selective for PEO, strengthening microphase separation. Despite this, the low molecular weight copolymers remain disordered in the melt even when blended with the IL. However, high molecular weight copolymers are capable of microphase separating into highly ordered block copolymer morphologies. This difference allows the effect of microphase separation on ion transport to be studied. The effect of block copolymer composition is also studied, by varying the PEO fraction of the copolymer. The resultant gels show high ionic conductivity and solid-like behavior, indicating that these materials may be effective as solid polymer electrolytes.
Miranda, Daniel; Versek, Craig; Tuominen, Mark; Watkins, James; Russell, Thomas
Functional polymer laminates from hyperthermal hydrogen induced cross-linking.
The use of a hyperthermal hydrogen induced cross-linking process to prepare laminates comprising polypropylene, poly(isobutylene-co-isoprene), and poly(vinyl acetate) is described. In this new, milder alternative to conventional plasma techniques, neutral molecular hydrogen projectiles were used to create carbon radicals on impacted surfaces by collision-induced dissociation of C-H bonds, and this process was used to cross-link polymers on a polypropylene surface. It was demonstrated that multiple layers of cross-linked materials could be added, creating polymer laminates with each layer introducing new functionalities and properties. In particular, the present work shows that the process is largely nondestructive toward ester functionalities. First, the esters were grafted to become nonleachable. Then, the esters were subsequently hydrolyzed to convert the surface from hydrophobic to hydrophilic. Afterward, the esters could be recovered by simple esterification demonstrating that further chemical transformations were possible. PMID:22060118
Thompson, David B; Trebicky, Tomas; Crewdson, Patrick; McEachran, Matthew J; Stojcevic, Goran; Arsenault, Gilles; Lau, Woon M; Gillies, Elizabeth R
Novel Photo Affinity Cross-Linking Resin for the Isolation of Heparin Binding Proteins
A new photo cross-linking reagent, 2-(4-azidophenylamino)-4-(1-ammonio-4-azabicyclo[2,2,2]oct-1-yl)-6-morpho-lino-1,3,5-triazine chloride (named AA-D) was developed, which was used for the identification of several heparin-binding proteins on the surface of intact platelets. Also a functional resin for the isolation of heparin-binding proteins from a protein mixture was prepared.Heparin was first immobilized onto a polystyrene Merrifield resin or Argo-gel� using a reductive amination reaction. Then the
Yasuo Suda; Megumi Nakamura; Shuhei Koshida; Shoichi Kusumoto; Michael Sobel
Cross-linking of two antiparallel vortex tubes
NASA Technical Reports Server (NTRS)
The detailed mechanisms in vortex cross-linking are unveiled by adequately resolved, direct numerical simulation of two viscous vortex tubes. There are three characteristic phases: (1) inviscid induction followed by core flattening and stretching; (2) bridging of the two vortices by accumulation of annihilated and then cross-linked vortex lines; and (3) threading of the remnants of the initial vortex pair in between the two bridges as they pull apart. These phases and the role of threading (along with bridging) in the mixing and the enstrophy cascade are explained, and it is shown that the mechanism is insensitive to asymmetries.
Melander, M. V.; Hussain, F.
Conformational Entropy of an Ideal Cross-Linking Polymer Chain
We present a novel analytical method to calculate conformational entropy of ideal cross-linking polymers from the configuration integral by employing a Mayer series expansion. Mayer-functions describing chemical bonds within the chain and for cross-links are sharply peaked over the temperature range of interest, and, are well approximated as statistically weighted Dirac delta-functions that enforce distance constraints. All geometrical deformations consistent with a set of distance constraints are integrated over. Exact results for a contiguous series of connected loops are employed to substantiate the validity of a previous phenomenological distance constraint model that describes protein thermodynamics successfully based on network rigidity.
Vorov, Oleg K.; Livesay, Dennis R.
Cross-Linked Nanotube Materials with Variable Stiffness Tethers
NASA Technical Reports Server (NTRS)
The constitutive properties of a cross-linked single-walled carbon nanotube material are predicted with a multi-scale model. The material is modeled as a transversely isotropic solid using concepts from equivalent-continuum modeling. The elastic constants are determined using molecular dynamics simulation. Some parameters of the molecular force field are determined specifically for the cross-linker from ab initio calculations. A demonstration of how the cross-linked nanotubes may affect the properties of a nanotube/polyimide composite is included using a micromechanical analysis.
Frankland, Sarah-Jane V.; Odegard, Gregory M.; Herzog, Matthew N.; Gates, Thomas S.; Fay, Catherine C.
Swelling of cross-linked polystyrene spheres in toluene vapor
The swelling behavior of individual micron-sized polystyrene (PS) spheres in toluene vapor was studied via mass loading by means of micromechanical cantilever sensors. For 4%-8% cross-linked PS a mass increase of 180% in saturated toluene vapor was measured. The mass of the swollen PS sphere decreases with increasing exposure time to ultraviolet light. In addition, the swelling response is significantly different between the first and the second exposure to toluene vapor. This is attributed to the formation of a cross-linked shell at the surface of the PS spheres. Shape persistent parts were observed for locally irradiated PS spheres.
Zhang, R.; Graf, K.; Berger, R. [Polymer Physics Group, Max Planck Institute for Polymer Research, Ackermannweg 10, D-55128 Mainz (Germany); Polymer Physics Group, Max Planck Institute for Polymer Research, Ackermannweg 10, D-55128 Mainz, Germany and Research Center for Micro- and Nanochemistry and Engineering, University of Siegen, D-57068 Siegen (Germany); Polymer Physics Group, Max Planck Institute for Polymer Research, Ackermannweg 10, D-55128 Mainz (Germany)
Advanced Corneal Cross-Linking System with Fluorescence Dosimetry
Purpose. This paper describes an advanced system that combines corneal cross-linking with riboflavin with fluorescence dosimetry, the ability to measure riboflavin diffusion within the cornea both before and during UVA treatment. Methods and Results. A corneal cross-linking system utilizing a digital micromirror device (DMD) was assembled and used to measure diffusion coefficients of 0.1% riboflavin in 20% dextran in porcine eyes. A value of (3.3 � 0.2) � 10?7?cm2/s was obtained for the stroma. Diffusion coefficients for the transepithelial formulation of 0.1% riboflavin in 0.44% saline and 0.02% BAK were also measured to be 4.7 � 0.3 � 10?8?cm2/s for epithelium only and (4.6 � 0.4) � 10?7?cm2/s for stroma only. Riboflavin consumption during a UVA treatment was also demonstrated. Conclusion. A new advanced corneal cross-linking system with fluorescence dosimetry of riboflavin has been demonstrated. It is hoped that this method may play a significant role in determining the underlying mechanisms of corneal cross-linking and assist with the development of additional riboflavin formulations. Moreover, dosimetry may prove valuable in providing a method to account for the biological differences between individuals, potentially informing cornea-specific UVA treatment doses in real time.
Friedman, Marc D.; Pertaub, Radha; Usher, David; Sherr, Evan; Kamaev, Pavel; Muller, David
Simulation of Fracture Nucleation in Cross-Linked Polymer Networks
NASA Astrophysics Data System (ADS)
A novel atomistic simulation method is developed whereby polymer systems can undergo strain-rate-controlled deformation while bond scission is enabled. The aim is to provide insight into the nanoscale origins of fracture. Various highly cross-linked epoxy systems including various resin chain lengths and levels of nonreactive dilution were examined. Consistent with the results of physical experiments, cured resin strength increased and ductility decreased with increasing cross-link density. An analysis of dihedral angle activity shows the locations in the molecular network that are most absorptive of mechanical energy. Bond scission occurred principally at cross-link sites as well as between phenyl rings in the bisphenol moiety. Scissions typically occurred well after yield and were accompanied by steady increases in void size and dihedral angle motion between bisphenol moieties and at cross-link sites. The methods developed here could be more broadly applied to explore and compare the atomistic nature of deformation for various polymers such that mechanical and fracture properties could be tuned in a rational way. This method and its results could become part of a solution system that spans multiple length and time scales and that could more completely represent such mechanical events as fracture.
Moller, J. C.; Barr, S. A.; Schultz, E. J.; Breitzman, T. D.; Berry, R. J.
Citric-Acid-Derived Photo-cross-Linked Biodegradable Elastomers
Citric-acid-derived thermally cross-linked biodegradable elastomers (CABEs) have recently received significant attention in various biomedical applications, including tissue-engineering orthopedic devices, bioimaging and implant coatings. However, citric-acid-derived photo-cross-linked biodegradable elastomers are rarely reported. Herein, we report a novel photo-cross-linked biodegradable elastomer, referred to as poly(octamethylene maleate citrate) (POMC), which preserves pendant hydroxyl and carboxylic functionalities after cross-linking for the potential conjugation of biologically active molecules. POMC is a low-molecular-mass pre-polymer with a molecular mass average between 701 and 1291 Da. POMC networks are soft and elastic with an initial modulus of 0.07 to 1.3 MPa and an elongation at break between 38 and 382%. FT-IR�ATR results confirmed the successful surface immobilization of type-I collagen onto POMC films, which enhanced in vitro cellular attachment and proliferation. Photo-polymerized POMC films implanted subcutaneously into Sprague�Dawley rats demonstrated minimal in vivo inflammatory responses. The development of POMC enriches the family of citric-acid-derived biodegradable elastomers and expands the available biodegradable polymers for versatile needs in biomedical applications.
Gyawali, Dipendra; Tran, Richard T.; Guleserian, Kristine J.; Tang, Liping; Yang, Jian
Load transfer mechanisms in cross-linked DWNT fibers
NASA Astrophysics Data System (ADS)
The application of carbon nanotubes (CNT) to macroscopic composite fibers has been limited by weak shear interfaces between adjacent CNT shells and composite matrix elements. A fundamental understanding of load transfer at multiple length-scales is needed to identify how the exceptional mechanical properties of CNTs can be scaled to produce high-performance fibers. Through in-situ electron microscopy tensile testing we have elucidated load transfer mechanisms across multiple scales of cross-linked double-walled nanotube (DWNT) fibers. A low density of polymer cross-links is found to increase the total energy dissipated at failure and ductility of fibers by 5 and 10X, respectively, without reducing strength. This mutiscale approach has identified a need to enhance shear interactions between individual DWNTs within the hierarchical DWNT fiber structures. Through in-situ TEM electron irradiation studies we have shown that load can be effectively transferred to inner DWNTs within bundles by covalently cross-linking the interfaces of adjacent DWNTs and shells. We have observed order of magnitude increases in strength and modulus and identified their dependence on irradiation dose. In future a combined approach of irradiation induced covalent and polymer cross-linking may lead to high-performance DWNT-based fibers and composites with tunable mechanical properties.
Filleter, T.; Naraghi, M.; Moravsky, A.; Bernal, R.; Loutfy, R. O.; Espinosa, H. D.
Cross-Linking of Human Amniotic Membrane by Glutaraldehyde
Purpose: The transplantation of cryopreserved human amniotic membrane has been introduced recently for the reconstruction of the ocular surface. However, in some diseases the transplant usually dissolves rather quickly and early detachment may occur. Therefore, we tried to stabilize the amniotic transplant by applying glutaraldehyde for collagen cross-linking of the membrane. Methods: 18 human amnions were prepared. 4 � 4
Eberhard Spoerl; Gregor Wollensak; Friedemann Reber; Lutz Pillunat
Citric-acid-derived photo-cross-linked biodegradable elastomers.
Citric-acid-derived thermally cross-linked biodegradable elastomers (CABEs) have recently received significant attention in various biomedical applications, including tissue-engineering orthopedic devices, bioimaging and implant coatings. However, citric-acid-derived photo-cross-linked biodegradable elastomers are rarely reported. Herein, we report a novel photo-cross-linked biodegradable elastomer, referred to as poly(octamethylene maleate citrate) (POMC), which preserves pendant hydroxyl and carboxylic functionalities after cross-linking for the potential conjugation of biologically active molecules. Pre-POMC is a low-molecular-mass pre-polymer with an average molecular mass between 701 and 1291 Da. POMC networks are soft and elastic with an initial modulus of 0.07 to 1.3 MPa and an elongation-at-break between 38 and 382%. FT-IR-ATR results confirmed the successful surface immobilization of type-I collagen onto POMC films, which enhanced in vitro cellular attachment and proliferation. Photo-polymerized POMC films implanted subcutaneously into Sprague-Dawley rats demonstrated minimal in vivo inflammatory responses. The development of POMC enriches the family of citric-acid-derived biodegradable elastomers and expands the available biodegradable polymers for versatile needs in biomedical applications. PMID:20557687
Gyawali, Dipendra; Tran, Richard T; Guleserian, Kristine J; Tang, Liping; Yang, Jian
Stabilization of scleral collagen by glycerol aldehyde cross-linking
The paper aims at the evaluation of prospects for using glyceraldehyde as a cross-linking agent for the scleral tissue. Stability parameters (denaturation temperature, Young's modulus, ultimate tensile stress, proteolytic resistance) and analytical parameter (fluorescence intensity) were determined during the glycation process of isolated rabbit sclera. The analysis of fluorescence spectral characteristic provided information about some glycation products. The glyceraldehyde treatment
N. A. Danilov; N. Yu. Ignatieva; E. N. Iomdina; S. A. Semenova; G. N. Rudenskaya; T. E. Grokhovskaya; V. V. Lunin
Transglutaminase: new insights into gelatin nanoparticle cross-linking.
Gelatin nanoparticles (GNPs) have demonstrated to be beneficial as a biodegradable and biocompatible delivery system. So far, nanoparticles prepared by the two-step desolvation technique were subsequently cross-linked by glutaraldehyde to guarantee storage stability. Although in�vivo and in�vitro toxicological studies have not revealed any glutaraldehyde related undesired effects, an alternative to chemical cross-linking could ease future clinical use in humans. Therefore, the recombinant enzyme microbial transglutaminase was used to examine its cross-linking abilities in nanoparticle production. Various process parameters, such as incubation time, temperature, medium, pH and the particle purification were evaluated regarding their impact on particle size and its distribution. Cross-linking reactions were best at 25�C using an ion-free solvent at a neutral pH and have been terminated after 12?h. Preliminary storage stability testing indicated adequate consistency of particle size and particle distribution making transglutaminase a potential candidate for glutaraldehyde substitution in future GNP production. PMID:21034367
Fuchs, S; Kutscher, M; Hertel, T; Winter, G; Pietzsch, M; Coester, C
Immobilization of enzymes in photochemically cross-linked polyvinyl alcohol
Invertase and amyloglucosidase were entrapped in polyvinyl alcohol membranes through UV irradiation of pendent styrylpyridinium groups. The influence of cross-linking on immobilization efficiency was studied using prepolymers with varied cross-linker density, the above mentioned enzymes of different molecular weight, and various substrates. It was found that the larger enzyme invertase is effectively immobilized even in polymers with very low contents
Thomas Uhlich; Mathias Ulbricht; Georg Tomaschewski
Quantitative continuous assay for hyaluronan synthase
A rapid, continuous, and convenient three-enzyme coupled UV absorption assay was developed to quantitate the glucuronic acid and N-acetylglucosamine transferase activities of hyaluronan synthase from Pasteurella multocida (PmHAS). Activity was measured by coupling the UDP produced from the PmHAS-catalyzed transfer of UDP-GlcNAc and UDP-GlcUA to a hyaluronic acid tetrasaccharide primer with the oxidation of NADH. Using a Xuorescently labeled primer, the products were characterized by gel electrophoresis. Our results show that a truncated soluble form of recombinant PmHAS (residues 1�703) can catalyze the glycosyl transfers in a time- and concentration-dependent manner. The assay can be used to determine kinetic parameters, inhibition constants, and mechanistic aspects of this enzyme. In addition, it can be used to quantify PmHAS during purification of the enzyme from culture media.
Krupa, Joanne C.; Shaya, David; Chi, Lianli; Linhardt, Robert J.; Cygler, Miroslaw; Withers, Stephen G.; Mort, John S.
Preparation of hyaluronan-DNA matrices and films.
Natural carbohydrate is a class of underexplored polymers for gene delivery. The noninflammatory and nonimmunogenic properties of hyaluronan (hyaluronic acid, HA) are important in clinical situations. It has a role in wound repair and has great lubricating ability. Moreover, the presence of hyaluronidase in vivo enables any vehicle fabricated from HA to be degraded by enzyme-mediated erosion. When DNA is entrapped in a cross-linked HA vehicle, HA-DNA fragments are released on digestion by hyaluronidase. These fragments could serve both as microcarriers of DNA and its protective mechanism. This protocol describes preparation of water-insoluble HA-DNA matrices and films designed for clinical applications, and assays for verification of their bioactivities. Plasmid DNA (pDNA) encoding platelet-derived growth factor (PDGF) is coupled to the matrices that could be implanted into chronic wounds to accelerate their healing. pDNA encoding hyaluronan synthase 2 (HAS2) is coupled to the film that could initially serve as a physical barrier and subsequently a pDNA reservoir for sustaining HAS2 transfection. This would lead to continual HA production for preventing postsurgical adhesion. PMID:23028077
Chen, Weiliam
Oxidized Pectin Cross-Linked Carboxymethyl Chitosan: A New Class of Hydrogels.
Oxidation of pectin was performed with sodium periodate to prepare pectin dialdehyde (PD). In this study we used the cross-linking reaction of the active aldehyde of PD and the amino of carboxymethyl chitosan (CMC) to prepare the hydrogels. By controlling the proportion of pectin dialdehyde and CMC we made different kinds of hydrogels. We systematically studied the characters of the hydrogels using Fourier transform infrared spectroscopy analysis of the pectin dialdehyde, CMC and the hydrogels, and also X-ray diffractometry and scanning electron microscopy analysis of the instrument of the hydrogels. Equilibrium swelling showed that the gels retained about 88-93% water. The water vapor transmission rate (WVTR) and the evaporation of water from gels showed that such hydrogels were optimal for maintaining a moist environment conducive for wound healing. Examination of the hemolytic potential showed that the hydrogels were nonhemolytic in nature. The hydrogels were non-toxic and blood-compatible. This hydrogel prepared from oxidized pectin and CMC without employing any extraneous cross-linking agents is expected to have potential as wound-dressing material. PMID:22133270
Fan, Lihong; Sun, Yi; Xie, Weiguo; Zheng, Hua; Liu, Shuhua
Structural characterization of Y1 and Y2 receptors for neuropeptide Y and peptide YY by affinity cross-linking
Pharmacological studies indicate that peptide YY (PYY) and neuropeptide Y interact with multiple binding sites, categorized as Y1 and Y2 subtypes. In order to identify and structurally characterize the Y1 and Y2 receptors we covalently cross-linked (125I-Tyr36)PYY to its receptors. The Y2 receptor in rat hippocampus and rabbit kidney membranes was affinity labeled using different homo- and heterobifunctional cross-linking reagents. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography resulted in a major labeled protein band of Mr = 50,000 in both hippocampal and kidney membranes, which was unaffected by reducing agents. The Y1 receptor was analyzed in membranes from the MC-IXC human neuroblastoma cell line. Autoradiography revealed two labeled bands at Mr = 70,000 and 45,000. As the intensity of the Mr = 45,000 band was reduced by protease inhibitors, it is likely that this band is a degradation product of the larger band. Labeling of these proteins was obtained only when N-5-azido-2-nitrobenzoyloxysuccinimide was employed for cross-linking followed by exposure to UV light. Labeling of the two cross-linked bands was unaffected by reducing agents. The binding of radiolabeled PYY and the intensity of the cross-linked bands, for both the Y1 and Y2 receptors, were inhibited similarly in a dose-dependent manner by increasing concentrations of unlabeled PYY. When exposed to agarose-coupled lectins, the detergent-solubilized Y1 receptor-hormone complex was completely adsorbed by wheat germ agglutinin and partially by ricin communis II. The cross-linked Y2 receptor was almost totally adsorbed by wheat germ agglutinin-agarose and partially adsorbed by concanavalin A. The adsorptions were in all cases blocked by the appropriate hapten sugar.
Sheikh, S.P.; Williams, J.A. (Univ. of Michigan, Ann Arbor (USA))
Studies on the Sorption of Palladium using Cross?Linked Poly (4?Vinylpyridine?Divinylbenzene) Resins in Nitric Acid Medium
Various cross?linked (4, 8, and 12%) gel?type weak?base poly(4?vinylpyridine) (PVP) resins were studied for palladium recovery from nitric acid medium. The sorption of palladium was found to decrease with an increase in cross?linkage of the resin. 8 and 12% PVP resins exhibited maximum DPd(II) values at 2�6�M HNO3, whereas 4% PVP resin showed maximum DPd(II) values at lower acidities (0.1�M
R. Kumaresan; K. N. Sabharwal; T. G. Srinivasan; P. R. Vasudeva Rao; Gunesh Dhekane
Surface Cross-Linked Poly (Vinyl Alcohol) Hydrogel for Colon Targeted Drug Release
A series of surface cross-linked PVA hydrogels (previously bulk cross-linked with maleic anhydride) were prepared for different cross-linker (glutaraldehyde) concentration. FTIR-ATR study revealed the cross-linking reaction. Surface cross-linking results in contraction of pores and increase in hydrophobicity, pore tortuosity around the surface of the membrane. As a result swelling, drug release decreases with increasing glutaraldehyde concentration. After surface cross-linking swelling
Piyali Basak; Basudam Adhikari; Alok Kumar Sen
Invasive fungal infections can be devastating, particularly in immunocompromised patients, and difficult to treat with systemic drugs. Furthermore, systemic administration of those medications can have severe side effects. We have developed an injectable local antifungal treatment for direct administration into existing or potential sites of fungal infection. Amphotericin B (AmB), a hydrophobic, potent, and broad-spectrum antifungal agent, was rendered water-soluble by conjugation to a dextran-aldehyde polymer. The dextran-aldehyde-AmB conjugate retained antifungal efficacy against C. albicans. Mixing carboxymethylcellulose-hydrazide with dextran-aldehyde formed a gel that cross-linked in situ by formation of hydrazone bonds. The gel provided in vitro release of antifungal activity for 11 days, and contact with the gel killed Candida for three weeks. There was no apparent tissue toxicity in the murine peritoneum and the gel caused no adhesions. Gels produced by entrapment of a suspension of AmB in CMC-dextran without conjugation of drug to polymers did not release fungicidal activity, but did kill on contact. Injectable systems of these types, containing soluble or insoluble drug formulations, could be useful for treatment of local antifungal infections, with or without concurrent systemic therapy.
Hudson, Sarah; Langer, Robert; Fink, Gerald R.; Kohane, Daniel S.
Stability of native and cross-linked crystalline glucose isomerase.
Stabilities of native and cross-linked crystalline forms of Streptomyces rubiginosus glucose isomerase were compared in buffer and in 45% glucose/fructose solutions. The cross-linked crystalline form of the enzyme was more stable in the presence of substrate while in a buffer solution the native enzyme was more stable. Inactivation of native enzyme in buffer did not obey first-order kinetics but proceeded with a rapid first phase followed by a stable phase. This stabilization is interpreted to be a result of a conformational change in the protein structure. Inactivation of the native enzyme in buffer was directly related to protein precipitation. In the presence of high substrate concentration, the inactivation was related to browning reactions between the enzyme and the reactive sugar, resulting in soluble sugar-protein complexes. PMID:10397876
Visuri, K; Pastinen, O; Wu, X; M�kinen, K; Leisola, M
Hydrogels of collagen hydrolysate cross-linked with dialdehyde starch
Processing hydrogels of collagen hydrolysate (H) cross-linked with dialdehyde starch (DAS) by dipping or casting into biodegradable\\u000a materials for various applications, is complicated by their marked tendency to aging. One-hour action by temperatures at 60�90��C\\u000a reduces sorbed water content in hydrogels by approx. 12%; dependence of the extent of this reduction on temperature (within\\u000a the mentioned range) was not detected.
F. Langmaier; M. Ml�dek; P. Mokrej�
Mucoadhesive properties of cross-linked high amylose starch derivatives
Acetate (Ac-), aminoethyl (AE-) and carboxymethyl (CM-)derivatives of cross-linked high amylose starch (HASCL-6) were previously shown to control, over more than 20h, the release of drugs from highly loaded (up to 60% drug) monolithic tablets. It was now of interest to evaluate their mucoadhesive characteristics in view of further utilization in buccal or vaginal transmucosal delivery. The present study shows
J�r�me Mulhbacher; Pompilia Ispas-Szabo; Mathieu Ouellet; Serge Alex; Mircea Alexandru Mateescu
Cross-Linking of Polythene by Pile Radiation
Polythene subjected to irradiation in the Harwell B.E.P.O. pile becomes cross-linked, and a new type of plastic is produced which does not melt at about 115 degrees C, nor dissolve in hot organic compounds. The mechanical properties are also altered, especially above 115 degrees C, when the plastic shows rubber-like elasticity. The paper is mainly confined to a study of
A. Charlesby
Estimating the Degree of Cross-Linking in Rubber
NASA Technical Reports Server (NTRS)
Degree of cross-linking or network chain concentration of rubber estimated with aid of new method. Quantity is needed in studies of mechanical behavior of rubber. New method is based on finding rubber follows different stress/ strain relationships in extension and retraction. When rubber specimen is stretched to given extension ration and released. Stress-vs-strain curve follows two paths: one for extension and other for retraction.
Fedors, R. F.
Homogeneous UVA system for corneal cross-linking treatment
NASA Astrophysics Data System (ADS)
The treatment of keratoconus and corneal ulcers by collagen cross-linking using ultraviolet type A irradiation, combined with photo-sensitizer Riboflavin (vitamin B2), is a promising technique. The standard protocol suggests instilling Riboflavin in the pre-scratched cornea every 5min for 30min, during the UVA irradiation of the cornea at 3mW/cm2 for 30 min. This process leads to an increase of the biomechanical strength of the cornea, stopping the progression, or sometimes, even reversing Keratoconus. The collagen cross-linking can be achieved by many methods, but the utilization of UVA light, for this purpose, is ideal because of its possibility of a homogeneous treatment leading to an equal result along the treated area. We have developed a system, to be clinically used for treatment of unhealthy corneas using the cross-linking technique, which consists of an UVA emitting delivery device controlled by a closed loop system with high homogeneity. The system is tunable and delivers 3-5 mW/cm2, at 365nm, for three spots (6mm, 8mm and 10mm in diameter). The electronics close loop presents 1% of precision, leading to an overall error, after the calibration, of less than 10% and approximately 96% of homogeneity.
Ayres Pereira, Fernando R.; Stefani, Mario A.; Otoboni, Jos� A.; Richter, Eduardo H.; Ventura, Liliane
Fiber optic immunosensor for cross-linked fibrin concentration
NASA Astrophysics Data System (ADS)
Working with calcium ions in the blood, platelets produce thromboplastin which transforms prothrombin into thrombin. Removing peptides, thrombin changes fibrinogen into fibrin. Cross-linked insoluble fibrin polymers are solubilized by enzyme plasmin found in blood plasma. Resulting D-dimers are elevated in patients with intravascular coagulation, deep venous thrombosis, pulmonary embolism, myocardial infarction, multiple trauma, cancer, impaired renal and liver functions, and sepsis. Consisting principally of a NIR 780 nm GaAlAs laser diode and a 800 nm avalanche photodiode (APD), the fiber-optic immunosensor can determined D-dimer concentration to levels <0.1 ng/ml. A capture monoclonal antibody to the antigen soluble cross-linked fibrin is employed. Immobilized at the tip of an optical fiber by avidin-biotin, the captured antigen is detected by a second antibody which is labeled with NN 382 fluorescent dye. An evanescent wave traveling on an excitation optical fiber excites the antibody-antigen fluorophore complex. Concentration of cross-linked fibrin is directly proportional to the APD measured intensity of fluorescence. NIR fluorescence has advantages of low background interference, short fluorescence lifetime, and large difference between excitation and emission peaks. Competitive ELISA test for D-dimer concentration requires trained personnel performing a time consuming operation.
Moskowitz, Samuel E.
The cellular response to transglutaminase-cross-linked collagen.
Collagen, type I, is a highly abundant natural protein material which has been cross-linked by a variety of methods including chemical agents, physical heating and UV irradiation with the aim of enhancing its physical characteristics such as mechanical strength, thermal stability, resistance to proteolytic breakdown, thus increasing its overall biocompatibility. However, in view of the toxicity of residual cross-linking agents, or impracticability at large scales, it would be more useful if the collagen could be cross-linked by a milder, efficient and more practical means by using enzymes as biological catalysts. We demonstrate that on treating native collagen type I (from bovine skin) with both tissue transglutaminase (TG2; tTG) and microbial transglutaminase (mTG; Streptoverticillium mobaraense) leads to an enhancement in cell attachment, spreading and proliferation of human osteoblasts (HOB) and human foreskin dermal fibroblasts (HFDF) when compared to culture on native collagen. The transglutaminase-treated collagen substrates also showed a greater resistance to cell-mediated endogenous protease degradation than the native collagen. In addition, the HOB cells were shown to differentiate at a faster rate than on native collagen when assessed by measurement of alkaline phosphatase activity and osteopontin expression. PMID:15927250
Chau, David Y S; Collighan, Russell J; Verderio, Elisabetta A M; Addy, Victoria L; Griffin, Martin
Injectable thermoreversible hyaluronan-based hydrogels for nucleus pulposus cell encapsulation
Introduction��Thermoreversible hydrogels have potential in spine research as they provide easy injectability and mild gelling mechanism\\u000a (by physical cross-link). The purpose of this study was to assess the potential of thermoreversible hyaluronan-based hydrogels\\u000a (HA-pNIPAM) (pNIPAM Mn�=�10, 20, 35�נ103�g�mol?1) as nucleus pulposus cells (NPC) carrier. \\u000a \\u000a \\u000a \\u000a \\u000a Materials and methods��Cytocompatibility (WST-1 assay), viability (trypan blue), morphology (toluidine blue), sulphated glycosaminoglycan synthesis\\u000a (DMMB assay)
Marianna Peroglio; Sibylle Grad; Derek Mortisen; Christoph Martin Sprecher; Svenja Illien-J�nger; Mauro Alini; David Eglin
Tissue integrity signals communicated by high-molecular weight hyaluronan and the resolution of inflammation
The extracellular matrix polysaccharide hyaluronan (HA) exerts size-dependent effects on leukocyte behavior. Low-molecular weight HA is abundant at sites of active tissue catabolism and promotes inflammation via effects on Toll-like receptor signaling. Conversely, high-molecular weight HA is prevalent in uninjured tissues and is anti-inflammatory. We propose that the ability of high-molecular weight but not low-molecular weight HA to cross-link CD44 functions as a novel form of pattern recognition that recognizes intact tissues and communicates �tissue integrity signals� that promote resolution of local immune responses.
Ruppert, S. M.; Hawn, T. R.; Arrigoni, A.; Wight, T. N.
Pericellular Hyaluronan and Prostate Cancer.
National Technical Information Service (NTIS)
The working hypothesis of the present project is that hyaluronan (HA) promotes tumor growth by maintaining pericellular spaces that allows the diffusion of nutrients. To test this hypothesis, we have carried out the following three tasks. First, the cDNA ...
C. B. Underhill
Pericellular Hyaluronan and Prostate Cancer.
National Technical Information Service (NTIS)
The working hypothesis of the present project is that hyaluronan (HA) promotes tumor growth by maintaining pericellular spaces that allows the diffusion of nutrients. To test this hypothesis, we have carried out the following specific aims. First, the cDN...
C. B. Underhill
Lymphocyte mechanical response triggered by cross-linking surface receptors
Using a recently developed method (Petersen, N. O., W. B. McConnaughey, and E. L. Elson, 1982, Proc. Natl. Acad. Sci. USA., 79:5327-5331), we have measured changes in the deformability of lymphocytes triggered by cross-linking cell surface proteins. Our study was motivated by two previously demonstrated phenomena: the redistribution ("capping") of cross-linked surface immunoglobulin (sIg) on B lymphocytes and the inhibition of capping and lateral diffusion ("anchorage modulation") of sIg by the tetravalent lectin Concanavalin A (Con A). Both capping and anchorage modulation are initiated by cross-linking cell surface proteins and both require participation of the cytoskeleton. We have shown that the resistance of lymphocytes to deformation strongly increased when sIg or Con A acceptors were cross-linked. We have measured changes in deformability in terms of an empirical "stiffness" parameter, defined as the rate at which the force of cellular compression increases with the extent of compression. For untreated cells the stiffness was approximately 0.15 mdyn/micron; for cells treated with antibodies against sIg or with Con A the stiffness increased to approximately 0.6 or 0.4 mdyn/micron, respectively. The stiffness decreased after completion of the capping of sIg. The increases in stiffness could be reversed to various extents by cytochalasin D and by colchicine. The need for cross-linking was demonstrated by the failure both of monovalent Fab' fragments of the antibodies against sIg and of succinylated Con A (a poor cross-linker) to cause an increase in stiffness. We conclude that capping and anchorage modulation involve changes in the lymphocyte cytoskeleton and possibly other cytoplasmic properties, which increase the cellular viscoelastic resistance to deformation. Similar increases in cell stiffness could be produced by exposing cells to hypertonic medium, azide ions, and to a calcium ionophore in the presence of calcium ions. These results shed new light on the capabilities of the lymphocyte cytoskeleton and its role in capping and anchorage modulation. They also demonstrate that measurements of cellular deformability can characterize changes in cytoskeletal functions initiated by signals originating at the cell surface.
Analysis of growth hormone and lactogenic binding sites cross-linked to iodinated human growth hormone
GH (GHR) and lactogenic receptors were analyzed after use of the cross-linking reagent ethylene glycol bis-(succinimidyl succinate) to attach covalently iodinated human GH (hGH) to binding proteins 1) on intact IM-9 lymphocytes, 2) in a partially purified GHR preparation from rabbit liver, and 3) in crude microsomal fractions from rabbit liver, rabbit mammary gland, and rat liver. The latter two microsomal preparations contain primarily lactogenic receptors, whereas in IM-9 lymphocytes and the rabbit liver preparations, GHR predominate. Cross-linked (125I)hGH-receptor complexes were solubilized, reduced, and separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of proteins cross-linked to (125I)hGH in the microsomal fraction from rabbit liver showed a specifically labeled complex with an estimated molecular weight (mol wt) of 75K. A slightly lower mol wt (71K) was determined for the complex labeled in the purified GHR preparation. In contrast to the relatively low mol wt complexes in rabbit liver, a complex that migrated with an apparent mol wt of 130K was identified in IM-9 lymphocytes. Labeled complexes were identified at 66K from rat liver and 61K from rabbit mammary gland. If it is assumed that hGH contributes 21K to the mol wt of the radiolabeled complexes, then the approximate mol wts of hGH-binding sites are 50-54K from rabbit liver, 109K from IM-9 lymphocytes, 45K from rat liver, and 40K from rabbit mammary gland.
Hughes, J.P.; Simpson, J.S.; Friesen, H.G.
Analysis of growth hormone and lactogenic binding sites cross-linked to iodinated human growth hormone.
GH (GHR) and lactogenic receptors were analyzed after use of the cross-linking reagent ethylene glycol bis-(succinimidyl succinate) to attach covalently iodinated human GH (hGH) to binding proteins 1) on intact IM-9 lymphocytes, 2) in a partially purified GHR preparation from rabbit liver, and 3) in crude microsomal fractions from rabbit liver, rabbit mammary gland, and rat liver. The latter two microsomal preparations contain primarily lactogenic receptors, whereas in IM-9 lymphocytes and the rabbit liver preparations, GHR predominate. Cross-linked [125I]hGH-receptor complexes were solubilized, reduced, and separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of proteins cross-linked to [125I]hGH in the microsomal fraction from rabbit liver showed a specifically labeled complex with an estimated molecular weight (mol wt) of 75K. A slightly lower mol wt (71K) was determined for the complex labeled in the purified GHR preparation. In contrast to the relatively low mol wt complexes in rabbit liver, a complex that migrated with an apparent mol wt of 130K was identified in IM-9 lymphocytes. Labeled complexes were identified at 66K from rat liver and 61K from rabbit mammary gland. If it is assumed that hGH contributes 21K to the mol wt of the radiolabeled complexes, then the approximate mol wts of hGH-binding sites are 50-54K from rabbit liver, 109K from IM-9 lymphocytes, 45K from rat liver, and 40K from rabbit mammary gland. PMID:6303751
Hughes, J P; Simpson, J S; Friesen, H G
Hyaluronan in Tubular and Interstitial Nephrocalcinosis
NASA Astrophysics Data System (ADS)
Hyaluronan (HA) is the major glycosaminoglycan (GAG) component of the renal medullary interstitium. HA is extremely large (up to 104 kDa) and composed of thousands repeating disaccharides of glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc). HA is synthesized by hyaluronan synthases (HASs) and degraded by hyaluronidases (Hyals). The production of HA by renomedullary interstitial cells is mediated by local osmolality. When excess water needs to be excreted, increased interstitial HA seems to antagonize water reabsorption, while the opposite occurs during water conservation. Hence, papillary interstitial HA is low and Hyal high during anti-diuresis, whereas during diuresis HA is high and Hyal low. The polyanion HA plays a role in the reabsorption of hypotonic fluid by immobilizing cations (Na+) via the carboxylate (COO-) groups of GlcUA. The binding of Ca2+ to anionic HA is probably also responsible for the fact that the papilla does not become a stone despite the extremely high interstitial phosphate and oxalate. HA is also an excellent crystal binding molecule. The expression of HA at the luminal surface of renal tubular cells leads to tubular nephrocalcinosis (tubular NC). Calcium staining methods (Von Kossa, Yasue) demonstrated that crystallization inhibitors cannot avoid the occasional precipitation of calcium phosphate in the papillary interstitium (interstitial NC). These crystals are probably immediately immobilized by the gel-like HA matrix. After ulcerating through the pelvic wall the calcified matrix becomes a Randall's plaque. The attachment of calcium oxalate crystals from the primary urine to plaque may ultimately lead to the development of clinical stones in the renal calyces (nephrolithiasis).
Verkoelen, Carl F.
Effects of Hyaluronan Supplementation on Cryopreserved Equine Spermatozoa Hyaluronan and Cryopreserved Equine Spermatozoa
Addition of hyaluronan, a nonsulfated glycosaminoglycan, to fresh and frozen thawed human semen results in substantial retention of motility over time. Hyaluronan also has been reported to preserve postthaw viability and maintain membrane stability of boar spermatozoa. Therefore, experiments were designed to investigate the use of a commercially available hyaluronan (Map-5, Bioniche Animal Health, Inc., Athens, GA) in freezing extender
J. E. Bruemmer; C. H. Wilson; M. Coutino da Silva; E. L. Squires
Vasoactive intestinal peptide receptor on liver plasma membranes: Solubilization and cross-linking
The hepatic receptor for VIP was solubilized from rat liver plasma membranes with 1.4% digitonin and shown to conserve its ability to bind to the ligand. This solubilized receptor demonstrated the high affinity and specificity for VIP (KD approximately 1 nM, binding preference: VIP greater than PHI greater than secretin greater than thymosin alpha 1) which were observed with the nonsolubilized VIP receptor on intact liver plasma membranes. {sup 125}I-VIP was next cross-linked to either the solubilized or nonsolubilized receptor using disuccinimido suberate or disuccinimido dithiobis(propionate), and the resulting complexes analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. A broad autoradiographic band which demonstrated a high affinity for VIP was identified at Mr 56,000 (53,000 in the absence of the reducing agent dithiothreitol) for both the solubilized and nonsolubilized receptors. We have thus been able to solubilize from rat liver plasma membranes a receptor with high affinity and specificity for VIP, and confirmed its structural similarity with the native VIP receptor in nonsolubilized membranes using cross-linking techniques.
Nguyen, T.D.; Kaiser, L.M. (Duke Univ., Durham, NC (USA))
Physicochemical, antimicrobial, and cytotoxic characteristics of a chitosan film cross-linked by a naturally occurring cross-linking agent, aglycone geniposidic acid.
The purpose of this study was to evaluate the characteristics of a chitosan film cross-linked by a naturally occurring compound, aglycone geniposidic acid (aGSA). This newly developed aGSA-cross-linked chitosan film may be used as an edible film. The chitosan film without cross-linking (fresh) and the glutaraldehyde-cross-linked chitosan film were used as controls. The characteristics of test chitosan films evaluated were their degree of cross-linking, swelling ratio, mechanical properties, water vapor permeability, antimicrobial capability, cytotoxicity, and enzymatic degradability. It was found that cross-linking of chitosan films by aGSA (at a concentration up to 0.8 mM) significantly increased its ultimate tensile strength but reduced its strain at fracture and swelling ratio. There was no significant difference in the antimicrobial capability between the cross-linked chitosan films and their fresh counterpart. However, the aGSA-cross-linked chitosan film had a lower cytotoxicity, a slower degradation rate, and a relatively lower water vapor permeability as compared to the glutaraldehyde-cross-linked film. These results suggested that the aGSA-cross-linked chitosan film may be a promising material as an edible film. PMID:16637687
Mi, Fwu-Long; Huang, Chin-Tsung; Liang, Hsiang-Fa; Chen, Mei-Chin; Chiu, Ya-Ling; Chen, Chun-Hung; Sung, Hsing-Wen
On the Formation and Properties of Interstrand DNA-DNA Cross-links Forged by Reaction of an Abasic Site With the Opposing Guanine Residue of 5?-CAp Sequences in Duplex DNA
We recently reported that the aldehyde residue of an abasic (Ap) site in duplex DNA can generate an interstrand cross-link via reaction with a guanine residue on the opposing strand. This finding is intriguing because the highly deleterious nature of interstrand cross-links suggests that even small amounts of Ap-derived cross-links could make a significant contribution to the biological consequences stemming from the generation of Ap sites in cellular DNA. Incubation of 21-bp duplexes containing a central 5?-CAp sequence under conditions of reductive amination (NaCNBH3, pH 5.2) generated much higher yields of cross-linked DNA than reported previously. At pH 7, in the absence of reducing agents, these Ap-containing duplexes also produced cross-linked duplexes that were readily detected on denaturing polyacrylamide gels. Cross-link formation was not highly sensitive to reaction conditions and, once formed, the cross-link was stable to a variety of work-up conditions. Results of multiple experiments including MALDI-TOF mass spectrometry, gel mobility, methoxyamine capping of the Ap aldehyde, inosine-for-guanine replacement, hydroxyl radical footprinting, and LCMS/MS were consistent with a cross-linking mechanism involving reversible reaction of the Ap aldehyde residue with the N2-amino group of the opposing guanine residue in 5?-CAp sequences to generate hemiaminal, imine, or cyclic hemiaminal cross-links (7-10) that were irreversibly converted under conditions of reductive amination (NaCNBH3/pH 5.2) to a stable amine linkage. Further support for the importance of the exocyclic N2-amino group in this reaction was provided by an experiment showing that installation of a 2-aminopurine-thymine base pair at the cross-linking site produced high yields (15-30%) of a cross-linked duplex at neutral pH, in the absence of NaCNBH3.
Johnson, Kevin M.; Price, Nathan E.; Wang, Jin; Fekry, Mostafa I.; Dutta, Sanjay; Seiner, Derrick R.; Wang, Yinsheng; Gates, Kent S.
Novel polymers with thermally controlled covalent cross-linking
NASA Astrophysics Data System (ADS)
The exploration of re-mending and self-healing of polymeric materials has become increasingly more exciting in the recent past. However, no highly cross-linked polymeric material could be healed in the absence of additional ingredients such as a catalyst, additional monomer, or special surface treatment of the fractured interface until the materials that are the subject of this dissertation were discovered. Thermally reversible Diels-Alder (DA) cycloadditions of multi-dienes and multi-dienophiles were used as cross-linking to prepare thermally re-mendable highly cross-linked polymers, in which the thermal reversibility can be accomplished by the retro DA reaction. The bulk polymeric materials are tough solids at room temperature but at temperatures above 90 to 120�C, a number of "intermonomer" linkages disconnect, yet reconnect upon cooling. This process is fully reversible and can be used to restore a fractured part multiple times. DSC, TMDSC, TMA, DMA and solid state NMR were applied to study the thermal and mechanical properties of the polymers. To determine the fracture-healing efficiencies of these polymers, tests were performed using compact tension specimens. Representative recoveries were obtained between 57% and 83% of the original fracture load, after the thermal treatment of failed pieces. Scanning electron microscope (SEM) was also used to characterize healing efficiency. Efficient multiple-time healing was also observed. Novel shape memory materials with Diels-Alder groups were also developed. The shapes of materials can be changed and restored after being heated to elevated temperatures (60 to 80�C). Water-soluble cationic poly(p-phenylene vinylene) were synthesized for bio-sensing purposes.
Chen, Xiangxu
Thiomer nanoparticles: stabilization via covalent cross-linking.
The purpose of this study was the development of stable thiomer nanoparticles for mucosal drug delivery. Chitosan-thioglycolic acid (chitosan-TGA) nanoparticles (NP) were formed via ionic gelation with tripolyphosphate (TPP). In order to stabilize the NP inter- and intra-molecular disulfide bonds were formed via controlled oxidation with hydrogen peroxide (H?O?). Thereafter, stability was investigated in saline and simulated body fluids at pH 2 and pH 5.5 via optical density measurements. The mucoadhesive properties were evaluated in vitro on freshly excised porcine intestinal mucosa via the rotating cylinder method. Particles had a mean size of 158?�?8?nm and a zeta potential of ~?+?16 mV. Three different degrees of oxidation were adjusted by the addition of H?O? in final concentrations of 10.60 �mol (chitosan-TGA (ox1)), 21.21 �mol (chitosan-TGA (ox2)), and 31.81 �mol (chitosan-TGA (ox3)) leading to 60%, 75%, and 83% of oxidized thiol groups, respectively. More than 99% of chitosan-TGA (ox3) NP, 70% of chitosan-TGA (ox2) NP, and 50% of chitosan-TGA (ox1) NP were stable over a 60-min period in simulated gastric fluid. In contrast, only 10% of unmodified chitosan and chitosan-TGA NP which were just ionically cross-linked remained stable in the same experiment. The adhesion times of covalently cross-linked chitosan-TGA (ox1), chitosan-TGA (ox2), and chitosan-TGA (ox3) were ~?41-fold, 31-fold, and 25-fold longer in comparison to unmodified ionically cross-linked chitosan. The method described here might be useful for the preparation of stable nanoparticulate drug delivery systems. PMID:22111974
Barthelmes, Jan; D�nnhaupt, Sarah; Hombach, Juliane; Bernkop-Schn�rch, Andreas
Identification of tyrosine 204 as the photo-cross-linking site in the DNA-EcoRI DNA methyltransferase complex by electrospray ionization mass spectrometry.
We describe a highly sensitive strategy combining laser-induced photo-cross-linking and HPLC-based electrospray ionization mass spectrometry to identify amino acid residues involved in protein-DNA recognition. The photoactivatible cross-linking thymine isostere, 5-iodoracil, was incorporated at a single site within the sequence recognized by EcoRI DNA methyltransferase (GAATTC). UV irradiation of the DNA-protein complex at 313 nm results in a >60% cross-linking yield. SDS-polyacrylamide gel electrophoresis and mass spectrometry were used to analyze the covalent cross-linked complex. The total mass is consistent with covalent bond formation between one strand of DNA and the protein with 1:1 stoichiometry. Protease digestion of the cross-linked complex yields several peptide-DNA adducts that were purified by anion-exchange column chromatography. A combination of mass spectrometric analysis and amino acid sequencing revealed that tyrosine 204 was cross-linked to the DNA. Electrospray mass spectrometric analysis of the peptide-nucleoside adduct confirmed this assignment. Tyrosine 204 resides in a peptide motif previously thought to be involved in AdoMet binding and methyl transfer. Thus, amino acids within loop segments but outside of "DNA binding" motifs can be critical to DNA recognition. Our method provides an accurate characterization of picomole quantities of DNA-protein complexes. PMID:11112526
Wong, D L; Reich, N O
A damage-recognition protein which binds to DNA containing interstrand cross-links is absent or defective in Fanconi anemia, complementation group A, cells.
A DNA binding protein with specificity for DNA containing interstrand cross-links induced by 4,5',8-trimethylpsoralen (TMP) plus long wavelength ultraviolet (UVA) light has been identified in normal human chromatin. Protein binding to DNA was determined using a gel mobility shift assay and an oligonucleotide containing a hot spot for formation of psoralen interstrand cross-links. Specificity of the damage-recognition protein for cross-links was demonstrated both by a positive correlation between level of cross-link formation in DNA and extent of protein binding and by effective competition by treated but not undamaged DNA for the binding protein. Chromatin protein extracts from cells from individuals with the genetic disorder, Fanconi anemia, complementation group A (FA-A), which have decreased ability to repair damage produced by TMP plus UVA light, failed to show any protein binding to TMP plus UVA treated DNA. We have previously shown that these chromatin protein extracts contain a DNA endonuclease complex, pI 4.6, which specifically recognizes and incises DNA containing interstrand cross-links and which in FA-A cells is defective in its ability to incise this damaged DNA (Lambert et al. (1992) Mutation Res., 273, 57-71). Together, these findings suggest that the DNA binding protein identified is involved in recognition and repair of DNA interstrand cross-links. Images
Hang, B; Yeung, A T; Lambert, M W
Method and Apparatus for Gel Electrophoretic Immunoassay.
National Technical Information Service (NTIS)
A micro-analytical platform for performing electrophoresis-based immunoassays was developed by integrating photopolymerized cross-linked polyacrylamide gels within a microfluidic device. The microfluidic immunoassays are performed by gel electrophoretic s...
A. E. Herr A. K. Singh D. J. Throckmorton
An amino acid-based heterofunctional cross-linking reagent.
We describe the synthesis and characterization of a new lysine-based heterofunctional cross-linking reagent. It carries two readily available aminooxy functionalities and an activated and protected thiol group that is capable of generating reducible disulfides, the former enable bioorthogonal modification of ketones and aldehydes by the formation of an oxime bond. The efficacy of the linker was proven by coupling two doxorubicin molecules to the functionalized amino acid core and the subsequent bioconjugation of this drug conjugate with a thiolated antibody. PMID:24504931
Lelle, Marco; Peneva, Kalina
Development of a novel photoreactive calmodulin derivative: Cross-linking of purified adenylate cyclase from bovine brain
A novel photoreactive calmodulin (CaM) derivative was developed and used to label the purified CaM-sensitive adenylate cyclase from bovine cortex. {sup 125}I-CaM was conjugated with the heterobifunctional cross-linking agent p-nitrophenyl 3-diazopyruvate (DAPpNP). Spectral data indicated that diazopyruvoyl (DAP) groups were incorporated into the CaM molecule. Iodo-CaM-DAPs behaved like native CaM with respect to (1) Ca{sup 2+}-dependent enhanced mobility on sodium dodecyl sulfate-polyacrylamide gels and (2) Ca{sup 2+}-dependent stimulation of adenylate cyclase activity. {sup 125}I-CaM-DAP photochemically cross-linked to CaM-binding proteins in a manner that was both Ca{sup 2+} dependent and CaM specific. Photolysis of forskolin-agarose-purified adenylate cyclase from bovine cortex with {sup 125}I-CaM-DAP produced a single cross-linked product which migrates on sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular weight of approximately 140,000.
Harrison, J.K.; Lawton, R.G.; Gnegy, M.E. (Univ. of Michigan, Ann Arbor (USA))
Surface Grafted Chitosan Gels. Part II. Gel Formation and Characterization.
Responsive biomaterial hydrogels attract significant attention due to their biocompatibility and degradability. In order to make chitosan based gels, we first graft one layer of chitosan to silica, and then build a chitosan/poly(acrylic acid) multilayer using the layer-by-layer approach. After cross-linking the chitosan present in the polyelectrolyte multilayer, poly(acrylic acid) is partly removed by exposing the multilayer structure to a concentrated carbonate buffer solution at a high pH, leaving a surface-grafted cross-linked gel. Chemical cross-linking enhances the gel stability against detachment and decomposition. The chemical reaction between gluteraldehyde, the cross-linking agent, and chitosan was followed in situ using total internal reflection Raman (TIRR) spectroscopy, which provided a molecular insight into the complex reaction mechanism, as well as the means to quantify the cross-linking density. The amount of poly(acrylic acid) trapped inside the surface grafted films was found to decrease with decreasing cross-linking density, as confirmed in situ using TIRR, and ex situ by Fourier transform infrared (FTIR) measurements on dried films. The responsiveness of the chitosan-based gels with respect to pH changes was probed by quartz crystal microbalance with dissipation (QCM-D) and TIRR. Highly cross-linked gels show a small and fully reversible behavior when the solution pH is switched between pH 2.7 and 5.7. In contrast, low cross-linked gels are more responsive to pH changes, but the response is fully reversible only after the first exposure to the acidic solution, once an internal restructuring of the gel has taken place. Two distinct pKa's for both chitosan and poly(acrylic acid), were determined for the cross-linked structure using TIRR. They are associated with populations of chargeable groups displaying either a bulk like dissociation behavior or forming ionic complexes inside the hydrogel film. PMID:25006685
Liu, Chao; Thormann, Esben; Claesson, Per M; Tyrode, Eric
Blends of cross-linked high amylose starch/pectin loaded with diclofenac.
Polymers blends represent an important approach to obtain materials with modulated properties to reach different and desired properties in designing drug delivery systems in order to fulfill therapeutic needs. The aim of this work was to evaluate the influence of drug loading and polymer ratio on the physicochemical properties of microparticles of cross-linked high amylose starch-pectin blends loaded with diclofenac for further application in controlled drug delivery systems. Thermal analysis and X-ray diffractograms evidenced the occurrence of drug-polymer interactions and the former pointed also to an increase in thermal stability due to drug loading. The rheological properties demonstrated that drug loading resulted in formation of weaker gels while the increase of pectin ratio contributes to origin stronger structures. PMID:23044114
Soares, Grazielle Arantes; de Castro, Ana D�ris; Cury, Beatriz S F; Evangelista, Raul C
Optimization model for UV-Riboflavin corneal cross-linking
NASA Astrophysics Data System (ADS)
Nowadays UV-cross-linking is an established method for the treatment of keraectasia. Currently a standardized protocol is used for the cross-linking treatment. We will now present a theoretical model which predicts the number of induced crosslinks in the corneal tissue, in dependence of the Riboflavin concentration, the radiation intensity, the pre-treatment time and the treatment time. The model is developed by merging the difussion equation, the equation for the light distribution in dependence on the absorbers in the tissue and a rate equation for the polymerization process. A higher concentration of Riboflavin solution as well as a higher irradiation intensity will increase the number of induced crosslinks. However, performed stress-strain experiments which support the model showed that higher Riboflavin concentrations (> 0.125%) do not result in a further increase in stability of the corneal tissue. This is caused by the inhomogeneous distribution of induced crosslinks throughout the cornea due to the uneven absorption of the UV-light. The new model offers the possibility to optimize the treatment individually for every patient depending on their corneal thickness in terms of efficiency, saftey and treatment time.
Schumacher, S.; Wernli, J.; Scherrer, S.; Bueehler, M.; Seiler, T.; Mrochen, M.
Collagen cross-linking and ultimate tensile strength in dentin.
Several studies have indicated differences in bond strength of dental materials to crown and root dentin. To investigate the potential differences in matrix properties between these locations, we analyzed upper root and crown dentin in human third molars for ultimate tensile strength and collagen biochemistry. In both locations, tensile strength tested perpendicular to the direction of dentinal tubules (undemineralized crown = 140.4 +/- 48.6/root = 95.9 +/- 26.1; demineralized crown = 16.6 +/- 6.3/root = 29.0 +/- 12.4) was greater than that tested parallel to the tubular direction (undemineralized crown = 73.1 +/- 21.2/root = 63.2 +/- 22.6; demineralized crown = 9.0 +/- 3.9/root = 16.2 +/- 8.0). The demineralized specimens showed significantly greater tensile strength in root than in crown. Although the collagen content was comparable in both locations, two major collagen cross-links, dehydrodihydroxylysinonorleucine/its ketoamine and pyridinoline, were significantly higher in the root (by ~ 30 and ~ 55%, respectively) when compared with those in the crown. These results indicate that the profile of collagen cross-linking varies as a function of anatomical location in dentin and that the difference may partly explain the site-specific tensile strength. PMID:15381724
Miguez, P A; Pereira, P N R; Atsawasuwan, P; Yamauchi, M
Damage and fatigue in cross-linked rubbers
NASA Astrophysics Data System (ADS)
Damage and fatigue of elastomers have not been fundamentally understood because of the complex nature of these materials. All currently existing models are completely phenomenological. Therefore two problems have been investigated in this research to address those fundamental issues. The first problem was creating an innovative concept with a mathematical modeling, which would be able to describe the damage using molecular characteristics of elastomers. The second problem is developing new approaches to study fatigue, and especially impact fatigue of elastomers. The following results have been obtained in this research. A theoretical model of damage has been developed which involves the basic molecular characteristics of cross-linked elastomers and takes into account the effects of viscoelasticity and stress-induced crystallization. This model was found very reliable and successful in description of numerous quasi-static simple extension experiments for monotonous and repeating loadings. It also roughly predicts in molecular terms the failure of elastomers with various degrees of cross-linking. Quasi-impact fatigue tests with different geometry of an indenter have also been performed. Some microscopic features of rubber damage have been investigated using optical microscopy and SEM. In particular, the accumulation of a completely de-vulcanized, liquid-like substance was observed under intense, multi-cycle impacts. All the findings discovered in quasi-impact experiments are consistent with the damage model predictions.
Melnikov, Alexei
Collagen Cross-Linking: Current Status and Future Directions
Collagen cross-linking (CXL) using UVA light and riboflavin (vitamin B2) was introduced as a clinical application to stabilize the cornea by inducing cross-links within and between collagen fibers. CXL has been investigated extensively and has been shown clinically to arrest the progression of keratoconic or post-LASIK ectasia. With its minimal cost, simplicity, and proven positive clinical outcome, CXL can be regarded as a useful approach to reduce the number of penetrating keratoplasties performed. Small case series have also indicated that CXL is beneficial in corneal edema by reducing stromal swelling behavior and in keratitis by inhibiting pathogen growth. Despite these encouraging results, CXL remains a relatively new method that is potentially associated with complications. Aspects such as side effects and recurrence rates have still to be elucidated. In light of the growing interest in CXL, our paper summarizes present knowledge about this promising approach. We have intentionally endeavored to include the more relevant studies from the recent literature to provide an overview of the current status of CXL.
Hovakimyan, Marine; Guthoff, Rudolf F.; Stachs, Oliver
Sorption properties of cross-linked insoluble polyvinylpyrrolidone.
The interaction of 32 drugs of diverse chemical structure with cross-linked insoluble polyvinylpyrrolidone (crospovidone) was studied. By using a polymer to drug ratio of 10:1, the sorbed amount for 20 compounds was found to be less than 5%. After a 10-fold decrease of the polymer concentration, the sorbed amount of eight other compounds fell to or below the 5% level. Only tannic acid and hexylresorcinol exhibited a significantly stronger sorption tendency. The interaction appeared to be controlled by phenolic groups in the active ingredient. The binding can be quantified by an interaction constant Ks, whose definition is based on a bulk phase model of interaction via independent binding sites. The exceptionally strong binding of hexylresorcinol, however, apparently was caused by cooperative interacting of the hexyl groups in the bound state. Desorption studies revealed that the binding was fully reversible in all cases. Therefore, the presence of cross-linked polyvinylpyrrolidone as a disintegrant in pharmaceutical preparations is not expected to interfere with GI drug absorption. PMID:7264917
Fr�mming, K H; Ditter, W; Horn, D
Radiation synthesis of low swelling acrylamide based hydrogels and determination of average molecular weight between cross-links
NASA Astrophysics Data System (ADS)
A comparative analysis of determination of cross-link density ( ?e) of hydrogels by using swelling tests and mechanical measurements has been made. Poly(acrylamide/methacrylamide) P(AAm/MAAm) and poly(acrylamide/hydroxyethyl methacrylate) P(AAm/HEMA) hydrogels were prepared by using gamma rays and used as model hydrogel systems. The uniaxial compression test was applied to cylindrical gel samples in the swollen state at pH 7. Stress-strain curves of hydrogels were evaluated to calculate the shear modulus values. The average molecular weight between cross-links (M) and ?e obtained from mechanical measurements were significantly different than the values obtained from swelling experiments. Large differences were attributed to the uncertainty on the value of the ? parameter used in the Flory-Rehner equation. �1% change in this parameter doubled or reduced the M value of hydrogel to half value.
Mahmudi, Naim; ?en, Murat; Rendevski, Stojan; G�ven, Olgun
Effects of acetic acid and lactic acid on physicochemical characteristics of native and cross-linked wheat starches.
The effects of two common organic acids; lactic and acetic acids (150 mg/kg) on physicochemical properties of native and cross-linked wheat starches were investigated prior and after gelatinization. These acids caused formation of some cracks and spots on the granules. The intrinsic viscosity of both starches decreased in the presence of the acids particularly after gelatinization. Water solubility increased while water absorption reduced after addition of the acids. The acids caused reduction in gelatinization temperature and enthalpy of gelatinization of both starches. The starch gels became softer, less cohesive, elastic and gummy when acids were added. These changes may indicate the degradation of the starch molecules by the acids. Cross-linked wheat starch was more resistant to the acids. However, both starches became more susceptible to the acids after gelatinization. The effect of lactic acid on physicochemical properties of both starches before and after gelatinization was greater than acetic acid. PMID:24206724
Majzoobi, Mahsa; Beparva, Paniz
New Aptes Cross-linked Polymers from Poly(ethylene oxide)s and Cyanuric Chloride for Lithium Batteries
NASA Technical Reports Server (NTRS)
A new series of polymer electrolytes for use as membranes for lithium batteries are described. Electrolytes were made by polymerization between cyanuric chloride and diamino-terminated poly(ethylene oxide)s, followed by cross-linking via a sol-gel process. Thermal analysis and lithium conductivity of freestanding polymer films were studied. The effects of several variables on conductivity were investigated, such as length of backbone PEO chain, length of branching PEO chain, extent of branching, extent of cross-linking, salt content, and salt counterion. Polymer films with the highest percentage of PEO were found to be the most conductive, with a maximum lithium conductivity of 3.9 x 10(exp -5) S/cm at 25 C. Addition of plasticizer to the dry polymers increased conductivity by an order of magnitude.
Tigelaar, Dean M.; Meador, Mary Ann B.; Kinder, James D.; Bennett, William R.
Hyaluronan and versican in the control of human T lymphocyte adhesion and migration
The ability of lymphocytes to migrate freely through connective tissues is vital to efficient immune function. How the extracellular matrix (ECM) may affect T cell adhesion and migration is not well understood. We have examined the adhesion and migration of activated human T lymphocytes on extracellular matrix made by fibroblast-like synoviocytes and lung fibroblasts. These cells were minimally interactive until treated with a viral mimetic, Poly I:C. This treatment promoted myofibroblast formation and engendered a higher-order structured ECM rich in versican and hyaluronan to which T cells avidly adhered in a hyaluronidase-sensitive manner. This Poly I:C-induced matrix impeded T-cell spreading and migration on and through synoviocyte monolayers, while hyaluronidase treatment or adding versican antibody during matrix formation reversed the effect on T-cell migration. Hyaluronidase also reversed the spread myofibroblast morphology. These data suggest that the viscous hyaluronan- and versican-rich matrix binds and constrains T lymphocytes. Using purified matrix components and solid state matrices of defined composition we uncovered a role for versican in modulating HA - T-cell interactions. Versican prevented T-cell binding to soluble hyaluronan, as well as the amoeboid shape change on hyaluronan coated dishes and T-cell penetration of collagen gels. Together, these data suggest that hyaluronan and versican play a role in T-cell trafficking and function in inflamed tissues.
Evanko, Stephen P.; Potter-Perigo, Susan; Bollyky, Paul L.; Nepom, Gerald T.; Wight, Thomas N.
Cross-linked comb-shaped anion exchange membranes with high base stability.
A unique one-step cross-linking strategy that connects quaternary ammonium centers using Grubbs II-catalyzed olefin metathesis was developed. The cross-linked anion exchange membranes showed swelling ratios of less than 10% and hydroxide conductivities of 18 to 40 mS cm(-1). Cross-linking improved the membranes' stability to hydroxide degradation compared to their non-cross-linked analogues. PMID:24618920
Li, Nanwen; Wang, Lizhu; Hickner, Michael
Biodegradation of differently cross-linked collagen membranes: an experimental study in the rat
The aim of the present study was to compare the biodegradation of differently cross-linked collagen membranes in rats. Five commercially available and three experimental membranes (VN) were included: (1) BioGide (BG) (non-cross-linked porcine type I and III collagens), (2) BioMend (BM), (3) BioMendExtend (BME) (glutaraldehyde cross-linked bovine type I collagen), (4) Ossix (OS) (enzymatic-cross-linked bovine type I collagen), (5) TutoDent
Daniel Rothamel; Frank Schwarz; Martin Sager; Monika Herten; Anton Sculean; Jurgen Becker
Gelatin hydrogels cross-linked with bis(vinylsulfonyl)methane (BVSM): 1. The chemical networks.
This paper deals with chemical gelation of gelatin in the presence of a cross-linker, bis(vinylsulfonyl)methane (BVSM), which is able to create covalent C-N bonds with amine groups. The investigation is performed at 40 degrees C, where no triple helices are present. Gelatin is in random coil conformation. The influence of various parameters (gelatin concentration, cross-linker concentration, and pH (number of reacting sites along the gelatin chain)) was examined. Gel formation was followed by rheological and thermodynamic measurements (microcalorimetry) versus time (kinetic measurements). Furthermore, the storage moduli were compared to the number of links formed in the course of gelation. The experiments show that, within the experimental range investigated, a fully homogeneous network is not reached; the chemical gels, even upon completion of the reactions, are still in the critical domain, near the threshold. A power law behavior was put in evidence for the shear modulus versus the distance to the gel point, expressed as the concentration of links per gelatin chain. The exponent (f = 3.4 +/- 0.3) is close to that expected for the vulcanization of long chains. The storage moduli can be superposed on a single curve where the abscissa is the product of the number of C-N links per unit volume and the gelatin concentration at an exponent equal to -0.76 +/- 0.03. This exponent suggests the role of entanglements for interchain cross-linking. PMID:16981770
Hellio-Serughetti, Dominique; Djabourov, Madeleine
Site-specific cross-linking of collagen peptides by lysyl advanced glycation endproducts.
Cross-linking of proteins by advanced glycation endproducts (AGEs) causes a host of pathological conditions but their exact roles are unknown. Cross-linking lysyl AGEs were synthesized and incorporated into two types of collagen peptides. The utility of these cross-linked peptides for biochemical investigations was demonstrated by proteolysis studies and circular dichroism. PMID:24705519
Kamalov, M; Harris, P W R; Cooper, G J S; Brimble, M A
Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes.
The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches. PMID:24938783
Leitner, Alexander; Joachimiak, Lukasz A; Unverdorben, Pia; Walzthoeni, Thomas; Frydman, Judith; F�rster, Friedrich; Aebersold, Ruedi
Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes
The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches.
Leitner, Alexander; Joachimiak, Lukasz A.; Unverdorben, Pia; Walzthoeni, Thomas; Frydman, Judith; Forster, Friedrich; Aebersold, Ruedi
Semisynthetic resorbable materials from hyaluronan esterification
In recent years, research on new, biocompatible, degradable materials has seen the development of a series of modified natural polymers. Among these, a new class of materials consisting of different hyaluronan derivatives promises to be useful in a whole range of clinical applications thanks to their varied biological properties. These new materials are obtained by chemical modification of purified hyaluronan
Davide Campoccia; Patrick Doherty; Marco Radice; Paola Brun; Giovanni Abatangelo; David F. Williams
Comparison of Cross-linked and Non-Cross-linked Acellular Porcine Dermal Scaffolds for Long-term Full-Thickness Hernia Repair in a Small Animal Model
Background: This study compared the strength of incorporation and biocompatibility of 2 porcine-derived grafts (cross-linked and non�cross-linked) in a rat hernia model. Methods: A standardized 2 � 4 cm2 fascial defect was created in 30 Wistar rats and repaired with either a cross-linked or a non�cross-linked graft. The rats were killed 3, 6, and 12 months later. The strength of incorporation, vascularization, cellular invasion, foreign body reaction, and capsule formation were evaluated. Results: Both graft materials showed cellular ingrowth and neovascularization by 3 months postimplantation. The average level of cellularization was significantly higher in the non�cross-linked grafts than in the cross-linked grafts at 6 months (2 vs 1; P = .029). Vascularization was significantly higher in the non�cross-linked grafts than in the cross-linked grafts at 6 months postimplantation (2 vs 1; P = .029) and insignificant at 3 months (2 vs 1.75; P = .311) and 12 months (1 vs 0.67; P = 1). The maximum load and breaking strength of both biomaterials increased during the study period. Overall, the strength of incorporation of the non�cross-linked grafts increased from 3 months (0.75 MPa) to 12 months (3.06 MPa) postimplantation. The strength of incorporation of the cross-linked grafts also increased from 3 months (0.59 MPa) to 12 months (1.58 MPa) postimplantation. Conclusions: The results of our study suggest that non�cross-linked grafts may be slightly more biocompatible and allow a more rapid and higher degree of cellular penetration and vascularization, resulting in stronger attachment to the tissues.
Mestak, Ondrej; Spurkova, Zuzana; Benkova, Kamila; Vesely, Pavel; Hromadkova, Veronika; Miletin, Jakub; Juzek, Robert; Molitor, Martin; Sukop, Andrej
Encapsulation of cobalt nanoparticles in cross-linked-polymer cages
NASA Astrophysics Data System (ADS)
Nanoparticles embedded in polymeric cages give rise to interesting applications ranging from nanocatalysis to drug-delivery systems. In this context, we report on synthesis of cobalt (Co) nanoparticles trapped in polyvinyl alcohol (PVA) matrix to yield self-supporting magnetic films in PVA slime. A 20 nm, Co formed in FCC geometry encapsulated with a weak citrate coat when caged in PVA matrix exhibited persistence of magnetism and good radio-frequency response. Cross-linking of PVA chains to form cage-like structures to arrest Co nanoparticles therein, is believed to be the reason for oxide-free nature of Co, promising applications in biomedicine as well as in radio-frequency shielding.
Hatamie, Shadie; Dhole, S. D.; Ding, J.; Kale, S. N.
[Keratoconus treatment by corneal cross-linking (CLX)].
Keratoconus is a disease of the cornea that usually begins during puberty and progressively weakens its biomechanical structure. Keratoconic eyes show a conic shape and progressive thinning, both leading to irregular astigmatism and reduced vision that cannot be corrected by glasses. In early cases, special contact lens can partly compensate for the visual loss while they do not stop disease progression. Until recently, the only treatment option was a corneal transplant. In 1999, a technique called corneal collagen cross-linking (CXL) was used in human corneas suffering from keratoconus for the first time. CXL uses a process called photopolymerization to halt the progression of keratoconus with an efficacy of more than 95%. Today our challenge is to screen and identify patients early enough to offer a treatment on time before irreversible vision loss develops. PMID:25004773
Hammer, Arthur; Tabibian, David; Richoz, Olivier; Hafezi, Farhad
Customized pachymetric guided epithelial debridement for corneal collagen cross linking
Backround We describe a modified method for deepitheliazation prior to corneal cross linking (CXL). The technique may overcome the current corneal pachymetric limitation parameter (over 400 microns) that is necessary for the safety of the procedure without affecting the overall benefits. Methods In a series of two patients, with inferior topographic steepening and regional thinning (less than 400 microns corresponding to the area of corneal steepening), CXL after customized epithelial removal was performed. Results There were no intra- or postoperative adverse events seen by the nine month follow up examination. Stabilization of the corneal ectasia was observed up to nine months post-costumized pachymetric-guided epithelial removal. Conclusion The technique of customized pachymetric-guided epithelial removal is easy to perform and may overcome the limitations of the preoperative corneal pachymetry expanding the application of the procedure in patients with regional corneal thinning.
Kymionis, George D; Diakonis, Vasilios F; Coskunseven, Efekan; Jankov, Mirco; Yoo, Sonia H; Pallikaris, Ioannis G
Dynamic Role of Cross-Linking Proteins in Actin Rheology
We develop a computational model to compare the relative importance of unbinding and unfolding of actin cross-linking proteins (ACPs) in the dynamic properties of the actin cytoskeleton. We show that in the strain-stiffening regime with typical physiological and experimental strain rates, unbinding events are predominant with negligible unfolding. ACPs unbound by greater forces experience larger displacements, with a tendency to rebind to different filaments. At constant strain, stress relaxes to physiological levels by unbinding only�not unfolding�of ACPs, which is consistent with experiments. Also, rebinding of ACPs dampens full relaxation of stress. When the network is allowed to return to a stress-free state after shear deformation, plastic deformation is observed only with unbinding. These results suggest that despite the possibility of unfolding, unbinding of ACPs is the major determinant for the rheology of the actin network.
Kim, Taeyoon; Hwang, Wonmuk; Kamm, Roger D.
Novel covalently cross-linked poly(etheretherketone) ionomer membranes
NASA Astrophysics Data System (ADS)
Sulfinated PEEK was synthesized via reduction of sulfochlorinated PEEK. The degree of sulfination was determined by redox titration. By partial reduction of sulfochlorinated PEEK, PEEK carrying both SO 2Cl and sulfinate groups could be obtained. From these polymers, covalently cross-linked ionomer membranes were prepared by reaction with the cross-linker diiodobutane and subsequent hydrolysis of the sulfochloride groups by aqueous post-treatment. The membranes show strongly reduced swelling connected with good H +-conductivity. Moreover, due to the fact that in these membranes all macromolecules are integrated into the covalent network, no leaching out of soluble substance took place during immersion in 90 �C hot DMAc for 5 days. One of the membranes was tested in a DMFC and exhibited stable performance up to T = 130 �C.
Zhang, Wei; Gogel, Viktor; Friedrich, K. Andreas; Kerres, Jochen
Roles of fibrin ?- and ?-chain specific cross-linking by FXIIIa in fibrin structure and function.
Factor XIII is responsible for the cross-linking of fibrin ?-chains in the early stages of clot formation, whilst ?-chain cross-linking occurs at a slower rate. Although ?- and ?-chain cross-linking was previously shown to contribute to clot stiffness, the role of cross-linking of both chains in determining clot structure is currently unknown. Therefore, the aim of this study was to determine the role of individual ?- and ?-chain cross-linking during clot formation, and its effects on clot structure. We made use of a recombinant fibrinogen (?Q398N/Q399N/K406R), which does not allow for ?-chain cross-linking. In the absence of cross-linking, intact D-D interface was shown to play a potential role in fibre appearance time, clot stiffness and elasticity. Cross-linking of the fibrin ?-chain played a role in the thickening of the fibrin fibres over time, and decreased lysis rate in the absence of ?2-antiplasmin. We also showed that ?-chain cross-linking played a role in the timing of fibre appearance, straightening fibres, increasing clot stiffness and reducing clot deformation. Cross-linking of the ?-chain played a role in fibrin fibre appearance time and fibre density. Our results show that ?- and ?-chain cross-linking play independent and specific roles in fibrin clot formation and structure. PMID:24430058
Duval, C; Allan, P; Connell, S D A; Ridger, V C; Philippou, H; Ari�ns, R A S
Measuring the Formaldehyde Protein-DNA Cross-Link Reversal Rate.
Protein-DNA binding interactions play critical roles in important cellular processes such as gene expression, cell division, and chromosomal organization. Techniques to identify and characterize these interactions often utilize formaldehyde cross-linking for stabilization of the complexes. Advantages of formaldehyde as a cross-linking reagent include cell permeability, relatively fast cross-linking kinetics, and short cross-linker length. In addition, formaldehyde cross-links are reversible, which has the advantage of allowing complexes to be dissociated if desired but may also present a problem if undesired dissociation occurs in the course of an experiment. While the kinetics of formaldehyde cross-link formation have been well-established in numerous studies, there have been no reports of the rate of cross-link dissociation, even though it is clearly a critical variable when developing a biochemical protocol involving formaldehyde cross-linking. We present here a method for measurement of the rate of formaldehyde cross-link reversal based upon the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) procedure and use it to determine the rate of cross-link reversal for cross-linked protein-DNA complexes from yeast cell lysate. The half-life of the protein-DNA cross-links varies from 179 h at 4 �C to 11.3 h at 47 �C, with a rate that increases exponentially with temperature and is independent of salt concentration. PMID:24848408
Kennedy-Darling, Julia; Smith, Lloyd M
Isotope signatures allow identification of chemically cross-linked peptides by mass spectrometry: a novel method to determine interresidue distances in protein structures through cross-linking.
Knowledge of protein structures and protein-protein interactions is essential for understanding of biological processes. Recent advances in protein cross-linking and mass spectrometry (MS) have shown significant potential to contribute to this area. Here we report a novel method to rapidly and accurately identify cross-linked peptides based on their unique isotope signature when digested in the presence of H(2)(18)O. This method overcomes the need for specially synthesized cross-linkers and/or multiple MS runs required by other techniques. We validated our method by performing a "blind" analysis of 5 proteins/complexes of known structure. Side chain repacking calculations using Rosetta show that 17 of our 20 positively identified cross-links fit the published atomic structures. The remaining 3 cross-links are likely due to protein aggregation. The accuracy and rapid throughput of our workflow will advance the use of protein cross-linking in structural biology. PMID:20476776
Zelter, Alex; Hoopmann, Michael R; Vernon, Robert; Baker, David; MacCoss, Michael J; Davis, Trisha N
Influence of Chemical Cross-Linking on the Over Voltage Positive Temperature Coefficient of Linear Low Density Polyethylene/carbon Black/aluminum Hydroxide Nano Composites
NASA Astrophysics Data System (ADS)
The effect of cross-linking of blends on the stability of positive temperature coefficient (PTC) and the elimination of negative temperature coefficient (NTC) were investigated. Linear low density polyethylene (LLDPE) was chemically cross-linking with various amounts of dicumyl peroxide (DCP). The resulting of cross-linking structure of composites was analyzed by gel content, differential scanning calorimetry (DSC), and dynamic mechanical analysis (DMA). By the effect of DCP, not only a high PTC intensity appeared, but also was the NTC effect of the composite eliminated. The PTC intensity reached six orders of magnitude for the LLDPE/DCP(0.10 wt%)/CB/Al(OH)3 composite, and successfully passed the test of over-voltage resistance.
Huang, C. Y.; Tsai, C. S.; Tsao, K. Y.
Effect of cross-linking on physicochemical properties of tapioca starch and its application in soup product.
Physicochemical properties of cross-linked tapioca starch (CLTS) with different cross-linking levels and their application as a thickening agent in soups were studied. The CLTS was prepared by cross-linking native tapioca starch suspended in alkaline solution (41.67% (w/w), pH 11) using a mixture (99:1 (w/w) ratio) of sodium trimetaphosphate (STMP) and sodium tripolyphosphate (STPP) at different concentrations ranged from 0.25% to 6.0% (w/w of starch) at 45�C for 3h. Starch paste clarity decreased with increasing concentration of STMP/STPP mixture. Variations of swelling power, solubility, pasting, gelatinization, and rheological properties of the CLTS were found. Thermogravimetric analysis exhibited higher thermal stability for the CLTS granules compared to the native one. Among the samples, the CLTS prepared using 1.0% STMP/STPP (1.0%-CLTS) and soup containing the 1.0%-CLTS exhibited the strongest gel characteristic and the greatest shear resistant properties. The 1.0%-CLTS improved the textural properties and sensory quality of soups. PMID:24299823
Wongsagonsup, Rungtiwa; Pujchakarn, Thamonwan; Jitrakbumrung, Suparat; Chaiwat, Weerawut; Fuongfuchat, Asira; Varavinit, Saiyavit; Dangtip, Somsak; Suphantharika, Manop
A major intermolecular cross-linking site in bovine dentine collagen involving the alpha 2 chain and stabilizing the 4D overlap.
Approximately 20% of the radioactivity incorporated into the dentine collagen of unerupted bovine molars after reduction with tritiated sodium borohydride was recovered in a cyanogen bromide peptide fraction of Mr 61 000 following chromatography on agarose A5m. After rechromatography on agarose A1.5m, this fraction was resolved into ten components by gel isoelectric focusing. Of these components, nine (the most acidic) were tritiated and contained the reduced cross-links dihydroxylysinonorleucine and hydroxylysinonorleucine. The amino acid compositions were consistent with the identification of each of these components as alpha 2CB3,5 linked to one or two small peptides. By limited Edman degradation, with and without prior digestion with pyroglutamate aminopeptidase (EC 22.214.171.124), these small peptides were identified as alpha 1CB0,1 and alpha 2CB1, occurring in a ratio of approximately 2:1. Specific cleavage with cathepsin D revealed that all the cross-link was associated with the C-terminal one-third of the alpha 2 chain, thus fixing the displacement of the participating molecules at 4D. The content of the known reducible cross-links present in these peptides, calculated from the specific activity of the reductant, was sufficient to account for only 10--20% of the cross-linking actually found, suggesting that stabilization is mainly through nonreducible cross-links of as yet undetermined structure. By quantitative analysis of homoserine content and semiquantitative amino-terminal analyses, it was determined that virtually all of the alpha 2 chain of bovine dentine collagen is cross-linked in this manner. One cross-link per molecule in this location could made a major contribution to the mechanical stability of the insoluble collagen fibrils in this tissue. PMID:7470454
Scott, P G
Affinity labeling of the Fc receptor on human monocytes using bifunctional cross-linking agents.
To affinity label the Fc receptor on human monocytes, Fc fragments of monoclonal human IgG1 radiolabeled with iodine 125 were covalently bound to the surface of intact monocytes using a variety of bifunctional cross-linking agents including ethylene glycol bis(succinimidyl succinate), dithio-bis-(succinimidyl proprionate), maleimidobenzoyl N-hydroxysuccinimide, glutaraldehyde and dimethyl suberimidate. After cross-linking, cells were solubilized and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by radioautography. Each of these cross-linkers caused a portion of cell-bound Fc fragments to form a covalent complex with a monocyte membrane component. This complex migrated on electrophoresis with an apparent molecular weight of 120,000. Deducting the molecular weight of Fc fragments alone (53,000) the molecular weight of the second component of the complex therefore was about 67,000. A similar estimate of receptor size also was obtained after reduction with dithiothreitol. Complex formation was potently inhibited by unlabeled Fc fragments, IgG1 or IgG3, all of which would be expected to compete with Fc fragments for IgG Fc receptor on human monocytes, but was not inhibited by Fab fragments, IgG2 or IgG4, which do not bind avidly to this receptor. By quantitating the amount of complex formed in the presence of varying concentrations of labeled ligand, it could be demonstrated that complex formation was saturable, and that Fc fragments formed complexes with avidity comparable to that with which Fc fragments bound to receptors on intact monocytes. The findings establish the feasibility of using radiolabeled Fc fragments to affinity label the IgG Fc receptors on human leukocytes. Potential advantages of this approach to studying receptor structure are discussed. PMID:3989309
Kurlander, R; Niedel, J
Synthesis of poly(N-isopropylacrylamide) hydrogels by radiation polymerization and cross-linking
Poly(N-isopropylacrylamide) [poly(NIPAAm)] shows a typical thermal reversibility of phase transition in aqueous solutions. That is, it precipitates from solution above a critical temperature called the lower critical solution temperature (LCST) and dissolves below this temperature. When it is cross-linked, the obtained hydrogel collapses above LCST, while it swells and expands below LCST. This hydrogel has received much attention recently and has been used as a model system to demonstrate the validity of theories describing the coil-globule transition, swelling of networks, and folding and unfolding of biopolymers. It has also been proposed for various applications ranging from controlled drug delivery to solute separation. Poly(NIPAAm) hydrogel is usually synthesized at room temperature from an aqueous solution of the monomer by using a redox initiator composed of ammonium persulfate and N,N,N{prime},N{prime}-tetramethylethylenediamine in the presence of N,N{prime}-methylenebisacrylamide as a cross-linker. Since the LCST of poly(NIPAAm) is around 32 C, the polymerization at room temperature proceeds in a homogeneous solution. Recently, poly(NIPAAm) hydrogels were synthesized by starting the polymerization below the LCST and then elevating the temperature above it, by which method macroporous gels with fast temperature response were obtained. The idea is to apply a radiation--induced polymerization method for the synthesis of poly(NIPAAm) hydrogels. This method offers unique advantages for synthesis: it is a simple and additive-free process at all temperatures, and the degree of cross-linking can be easily controlled by irradiation conditions. Therefore, radiation methods are especially attractive for the synthesis of hydrogels with potential biomedical application where the residual chemical initiators may contaminate the product. It is possible to combine into one step the synthesis and sterilization of the product, and it is economically competitive.
Nagaoka, Noriyasu; Kubota, Hitoshi; Katakai, Ryoichi [Gunma Univ. (Japan). Dept. of Chemistry; Safranj, Agneza; Yoshida, Masaru; Omichi, Hideki [Japan Atomic Energy Research Inst., Gunma (Japan). Dept. of Material Development
An Investigation of Siloxane Cross-linked Hydroxyapatite-Gelatin/Copolymer Composites for Potential Orthopedic Applications().
Causes of bone deficiency are numerous, but biomimetic alloplastic grafts provide an alternative to repair tissue naturally. Previously, a hydroxyapatite-gelatin modified siloxane (HAp-Gemosil) composite was prepared by cross-linking (N, N'-bis[(3-trimethoxysilyl)propyl]ethylene diamine (enTMOS) around the HAp-Gel nanocomposite particles, to mimic the natural composition and properties of bone. However, the tensile strength remained too low for many orthopedic applications. It was hypothesized that incorporating a polymer chain into the composite could help improve long range interaction. Furthermore, designing this polymer to interact with the enTMOS siloxane cross-linked matrix would provide improved adhesion between the polymer and the ceramic composite, and improve mechanical properties. To this end, copolymers of L-Lactide (LLA), and a novel alkyne derivatized trimethylene carbonate, propargyl carbonate (PC), were synthesized. Incorporation of PC during copolymerization affects properties of copolymers such as molecular weight, T(g), and % PC incorporation. More importantly, PC monomers bear a synthetic handle, allowing copolymers to undergo post-polymerization functionalization with graft monomers to specifically tailor the properties of the final composite. For our investigation, P(LLA-co-PC) copolymers were functionalized by an azido-silane (AS) via copper catalyzed azide-alkyne cycloaddition (CuAAC) through terminal alkyne on PC monomers. The new functionalized polymer, P(LLA-co-PC)(AS) was blended with HAp-Gemosil, with the azido-silane linking the copolymer to the silsesquioxane matrix within the final composite.These HAp-Gemosil/P(LLA-co-PC)(AS) composites were subjected to mechanical and biological testing, and the results were compared with those from the HAp-Gemosil composites. This study revealed that incorporating a cross-linkable polymer served to increase the flexural strength of the composite by 50%, while maintaining the biocompatibility of HAp-Gemosil ceramics. PMID:23139457
Dyke, Jason Christopher; Knight, Kelly Jane; Zhou, Huaxing; Chiu, Chi-Kai; Ko, Ching-Chang; You, Wei
An Investigation of Siloxane Cross-linked Hydroxyapatite-Gelatin/Copolymer Composites for Potential Orthopedic Applications�
Causes of bone deficiency are numerous, but biomimetic alloplastic grafts provide an alternative to repair tissue naturally. Previously, a hydroxyapatite-gelatin modified siloxane (HAp-Gemosil) composite was prepared by cross-linking (N, N?-bis[(3-trimethoxysilyl)propyl]ethylene diamine (enTMOS) around the HAp-Gel nanocomposite particles, to mimic the natural composition and properties of bone. However, the tensile strength remained too low for many orthopedic applications. It was hypothesized that incorporating a polymer chain into the composite could help improve long range interaction. Furthermore, designing this polymer to interact with the enTMOS siloxane cross-linked matrix would provide improved adhesion between the polymer and the ceramic composite, and improve mechanical properties. To this end, copolymers of L-Lactide (LLA), and a novel alkyne derivatized trimethylene carbonate, propargyl carbonate (PC), were synthesized. Incorporation of PC during copolymerization affects properties of copolymers such as molecular weight, Tg, and % PC incorporation. More importantly, PC monomers bear a synthetic handle, allowing copolymers to undergo post-polymerization functionalization with graft monomers to specifically tailor the properties of the final composite. For our investigation, P(LLA-co-PC) copolymers were functionalized by an azido-silane (AS) via copper catalyzed azide-alkyne cycloaddition (CuAAC) through terminal alkyne on PC monomers. The new functionalized polymer, P(LLA-co-PC)(AS) was blended with HAp-Gemosil, with the azido-silane linking the copolymer to the silsesquioxane matrix within the final composite. These HAp-Gemosil/P(LLA-co-PC)(AS) composites were subjected to mechanical and biological testing, and the results were compared with those from the HAp-Gemosil composites. This study revealed that incorporating a cross-linkable polymer served to increase the flexural strength of the composite by 50%, while maintaining the biocompatibility of HAp-Gemosil ceramics.
Dyke, Jason Christopher; Knight, Kelly Jane; Zhou, Huaxing; Chiu, Chi-Kai; Ko, Ching-Chang; You, Wei
Mechanically strong, flexible polyimide aerogels cross-linked with aromatic triamine.
Polyimide gels are produced by cross-linking anhydride capped polyamic acid oligomers with aromatic triamine in solution and chemically imidizing. The gels are then supercritically dried to form nanoporous polyimide aerogels with densities as low as 0.14 g/cm(3) and surface areas as high as 512 m(2)/g. To understand the effect of the polyimide backbone on properties, aerogels from several combinations of diamine and dianhydride, and formulated oligomer chain length are examined. Formulations made from 2,2'-dimethylbenzidine as the diamine shrink the least but have among the highest compressive modulus. Formulations made using 4,4'-oxydianiline or 2,2'dimethylbenzidine can be fabricated into continuous thin films using a roll to roll casting process. The films are flexible enough to be rolled or folded back on themselves and recover completely without cracking or flaking, and have tensile strengths of 4-9 MPa. Finally, the highest onset of decomposition (above 600 �C) of the polyimide aerogels was obtained using p-phenylene diamine as the backbone diamine with either dianhydride studied. All of the aerogels are suitable candidates for high-temperature insulation with glass transition temperatures ranging from 270-340 �C and onsets of decomposition from 460-610 �C. PMID:22233638
Meador, Mary Ann B; Malow, Ericka J; Silva, Rebecca; Wright, Sarah; Quade, Derek; Vivod, Stephanie L; Guo, Haiquan; Guo, Jiao; Cakmak, Miko
Photoresponsive DNA-cross-linked hydrogels for controllable release and cancer therapy.
We have developed a photoresponsive DNA-cross-linked hydrogel that can be photoregulated by two wavelengths with a reversible sol-gel conversion. This photoinduced conversion can be further utilized for precisely controllable encapsulation and release of multiple loads. Specifically, photosensitive azobenzene moieties are incorporated into DNA strands as cross-linkers, such that their hybridization to complementary DNAs (cDNAs) responds differently to different wavelengths of light. On the basis of the rheology variation of hydrogels, it is possible to utilize this material for storing and releasing molecules and nanoparticles. To prove the concept, three different materials--fluorescein, horseradish peroxidase, and gold nanoparticles--were encapsulated inside the gel at 450 nm and then released by photons at 350 nm. Further experiments were carried out to deliver the chemotherapy drug doxorubicin in a similar manner in vitro. Our results show a net release rate of 65% within 10 min, and the released drug maintained its therapeutic effect. This hydrogel system provides a promising platform for drug delivery in targeted therapy and in biotechnological applications. PMID:21126095
Kang, Huaizhi; Liu, Haipeng; Zhang, Xiaoling; Yan, Jilin; Zhu, Zhi; Peng, Lu; Yang, Huanghao; Kim, Youngmi; Tan, Weihong
Tailoring Elastic Properties of Silica Aerogels Cross-Linked with Polystyrene
NASA Technical Reports Server (NTRS)
The effect of incorporating an organic linking group, 1,6-bis(trimethoxysilyl)hexane (BTMSH), into the underlying silica structure of a styrene cross-linked silica aerogel is examined. Vinyltrimethoxysilane (VTMS) is used to provide a reactive site on the silica backbone for styrene polymerization. Replacement of up to 88 mol 1 of the silicon from tetramethoxyorthosilicate with silicon derived from BTMSH and VTMS during the making of silica gels improves the elastic behavior in some formulations of the crosslinked aerogels, as evidenced by measurement of the recovered length after compression of samples to 251 strain. This is especially true for some higher density formulations, which recover nearly 100% of their length after compression to 251 strain twice. The compressive modulus of the more elastic monoliths ranged from 0.2 to 3 MPa. Although some of these monoliths had greatly reduced surface areas, changing the solvent used to produce the gels from methanol to ethanol increased the surface area in one instance from 6 to 220 sq m2/g with little affect on the modulus, elastic recovery, porosity, or density.
Nguyen, Baochau N.; Meador, Mary Ann B.; Tousley, Marissa E.; Shonkwiler, Brian; McCorkle, Linda; Scheiman, Daniel A.; Palczer, Anna
Structure-Property Relationships in Porous 3-D Nanostructures as a Function of Preparation Conditions: Isocyanate Cross-Linked Silica Aerogels
NASA Technical Reports Server (NTRS)
Sol-gel derived silica aerogels are attractive candidates for many unique thermal, optical, catalytic, and chemical applications because of their low density and high mesoporosity. However, their inherent fragility has restricted use of aerogel monoliths to applications where they are not subject to any load. We have previously reported cross-linking the mesoporous silica structure of aerogels with di-isocyanates, styrenes or epoxies reacting with amine decorated silica surfaces. These approaches have been shown to significantly increase the strength of aerogels with only a small effect on density or porosity. Though density is a prime predictor of properties such as strength and thermal conductivity for aerogels, it is becoming clear from previous studies that varying the silica backbone and size of the polymer cross-link independently can give rise to combinations of properties which cannot be predicted from density alone. Herein, we examine the effects of four processing parameters for producing this type of polymer cross-linked aerogel on properties of the resulting monoliths. We focus on the results of C-13 CP-MAS NMR which gives insight to the size and structure of polymer cross-link present in the monoliths, and relates the size of the cross-links to microstructure, mechanical properties and other characteristics of the materials obtained.
Meador, Mary Ann B.; Capadona, Lynn A.; McCorkle, Linda; Padadopoulos, Demetrios S.; Leventis, Nicholas
Structure-Property Relationships in Porous 3-D Nanostructures as a Function of Preparation Conditions: Isocyanate Cross-Linked Silica Aerogels
NASA Technical Reports Server (NTRS)
Sol-gel derived silica aerogels are attractive candidates for many unique thermal, optical, catalytic, and chemical applications because of their low density and high mesoporosity. However, their inherent fragility has restricted use of aerogel monoliths to applications where they are not subject to any load. We have previously reported cross-linking the mesoporous silica structure of aerogels with di-isocyanates, styrenes or epoxies reacting with amine decorated silica surfaces. These approaches have been shown to significantly increase the strength of aerogels with only a small effect on density or porosity. Though density is a prime predictor of properties such as strength and thermal conductivity for aerogels, it is becoming clear from previous studies that varying the silica backbone and size of the polymer cross-link independently can give rise to combinations of properties which cannot be predicted from density alone. Herein, we examine the effects of four processing parameters for producing this type of polymer cross-linked aerogel on properties of the resulting monoliths. We focus on the results of 13C CP-MAS NMR which gives insight to the size and structure of polymer cross-link present in the monoliths, and relates the size of the cross-links to microstructure, mechanical properties and other characteristics of the materials obtained.
Meador, Mary Ann B.; Capadona, Lynn A.; McCorkle, Linda; Papadopoulos, Demetrios S.; Leventis, Nicholas
Antisense inhibition of hyaluronan synthase-2 in human osteosarcoma cells inhibits hyaluronan retention and tumorigenicity
Osteosarcoma is a common malignant bone tumor associated with childhood and adolescence. The results of numerous studies have suggested that hyaluronan plays an important role in regulating the aggressive behavior of various types of cancer cells. However, no studies have addressed hyaluronan with respect to osteosarcomas. In this investigation, the mRNA expression copy number of three mammalian hyaluronan synthases (HAS) was determined using competitive RT-PCR in the osteoblastic osteosarcoma cell line, MG-63. MG-63 are highly malignant osteosarcoma cells with an abundant hyaluronan-rich matrix. The results demonstrated that HAS-2 is the predominant HAS in MG-63. Accumulation of intracellular hyaluronan increased in association with the proliferative phase of these cells. The selective inhibition of HAS-2 mRNA in MG-63 cells by antisense phosphorothioate oligonucleotides resulted in reduced hyaluronan accumulation by these cells. As expected, the reduction in hyaluronan disrupted the assembly of cell-associated matrices. However, of most interest, coincident with the reduction in hyaluronan, there was a substantial decrease in cell proliferation, a decrease in cell motility and a decrease in cell invasiveness. These data suggest that hyaluronan synthesized by HAS-2 in MG-63 plays a crucial role in osteosarcoma cell proliferation, motility, and invasion.
Nishida, Yoshihiro [Department of Orthopaedic Surgery, Nagoya University School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-8550 (Japan)]. E-mail: email@example.com; Knudson, Warren [Department of Biochemistry, Rush Medical College, Rush University Medical Center, 1653 W. Congress Parkway, Chicago, IL 60612 (United States); Knudson, Cheryl B. [Department of Biochemistry, Rush Medical College, Rush University Medical Center, 1653 W. Congress Parkway, Chicago, IL 60612 (United States); Ishiguro, Naoki [Department of Orthopaedic Surgery, Nagoya University School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-8550 (Japan)
Riboflavin-Ultraviolet A Corneal Cross-linking for Keratoconus
Purpose: To evaluate the safety, efficacy of riboflavin-ultraviolet A irradiation (UVA) corneal cross-linking and present refractive changes induced by the treatment in cases of keratoconus. Materials and Methods: The study includes 15 eyes of 9 patients with keratoconus with an average keratometric (K) reading less than 54 D and minimal corneal thickness greater than 420 microns. The corneal epithelium was removed manually within the central 8.5 mm diameter area and the cornea was soaked with riboflavin eye drops (0.1% in 20% dextran ?-500) for 30 minutes followed by exposure to UVA radiation (365 nm, 3 mW/cm2) for 30 minutes. During the follow-up period, uncorrected visual acuity (UCVA), best spectacle-corrected visual acuity (BSCVA), manifest refraction, slit lamp examination and topographic changes were recorded at the first week, first month, 3 and 6 months. Results: There was statistically significant improvement of UCVA from a preoperative mean of 0.11 � 0.07 (range 0.05�0.3) to a postoperative mean of 0.15 � 0.06 (range 0.1�0.3) (P < 0.05). None of the eyes lost lines of preoperative UCVA but 1 eye lost 1 line of preoperative BSCVA. The preoperative mean K of 49.97 � 2.81 D (range 47.20�51.75) changed to 48.34 � 2.64 D (range 45.75�50.40). This decrease in K readings was statistically significant (P < 0.05). All eyes developed minimal faint stromal haze that cleared in 14 eyes within 1 month. In only 1 eye, this resulted in a very faint corneal scar. Other sight threatening complications were not encountered in this series. Progression of the original disease was not seen in any of the treated eyes within 6 months of follow-up. Conclusion: Riboflavin-UVA corneal cross-linking is a safe and promising method for keratoconus. Larger studies with longer follow up are recommended.
El-Raggal, Tamer M.
Development of a Novel Cross-linking Strategy for Fast and Accurate Identification of Cross-linked Peptides of Protein Complexes*
Knowledge of elaborate structures of protein complexes is fundamental for understanding their functions and regulations. Although cross-linking coupled with mass spectrometry (MS) has been presented as a feasible strategy for structural elucidation of large multisubunit protein complexes, this method has proven challenging because of technical difficulties in unambiguous identification of cross-linked peptides and determination of cross-linked sites by MS analysis. In this work, we developed a novel cross-linking strategy using a newly designed MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). DSSO contains two symmetric collision-induced dissociation (CID)-cleavable sites that allow effective identification of DSSO-cross-linked peptides based on their distinct fragmentation patterns unique to cross-linking types (i.e. interlink, intralink, and dead end). The CID-induced separation of interlinked peptides in MS/MS permits MS3 analysis of single peptide chain fragment ions with defined modifications (due to DSSO remnants) for easy interpretation and unambiguous identification using existing database searching tools. Integration of data analyses from three generated data sets (MS, MS/MS, and MS3) allows high confidence identification of DSSO cross-linked peptides. The efficacy of the newly developed DSSO-based cross-linking strategy was demonstrated using model peptides and proteins. In addition, this method was successfully used for structural characterization of the yeast 20 S proteasome complex. In total, 13 non-redundant interlinked peptides of the 20 S proteasome were identified, representing the first application of an MS-cleavable cross-linker for the characterization of a multisubunit protein complex. Given its effectiveness and simplicity, this cross-linking strategy can find a broad range of applications in elucidating the structural topology of proteins and protein complexes.
Kao, Athit; Chiu, Chi-li; Vellucci, Danielle; Yang, Yingying; Patel, Vishal R.; Guan, Shenheng; Randall, Arlo; Baldi, Pierre; Rychnovsky, Scott D.; Huang, Lan
A surface-tethered model to assess size-specific effects of hyaluronan (HA) on endothelial cells.
Crosslinked gels (hylans) containing long-chain (MW>1 x 10(6)Da) hyaluronan (HA), a connective tissue GAG, show exceptional biocompatibility for vascular implantation but poorly interact with vascular endothelial cells (ECs). Previous studies showed in situ fragmentation of HA by UV light to bioactivate hylan gels and elicit enhanced EC responses. Since fragmented HA can be pro-inflammatory, it is important to define an optimal size distribution of HA fragments on the hylan surface that will recruit and support normally functional ECs and limit ulterior responses. Related studies have shown that exogenous models of HA do not necessarily replicate cell responses to HA scaffolds. Since scaffolds cannot be created based on fragmented HA alone, we sought to determine size-specific responses of ECs to HA substrates of defined fragment sizes by creation of HA-tethered culture surfaces. HA (1000, 200, 20 kDa) and an oligomer mixture were tethered onto an aminosilane (APTMS)-treated glass surfaces using a carbodiimide reaction. MALDI-TOF showed the HA digests to contain HA 4-8mers with a 75+/-0.4% w/w of 4mers. Immuno-fluorescence, SEM, AFM and XPS analysis revealed homogeneous amine and HA surfaces. An amine s-SDTB assay and HA fluorophore-assisted carbohydrate electrophoresis (FACE) indicated surface densities of 9+/-3 amine groups/nm(2) and 0.57+/-0.44 microg/cm(2), respectively. HA/HA fragments/oligomers were stable over 21 days of incubation in serum-free culture media. EC proliferation on these surfaces resulted was limited, a possible effect of smooth surface topography, high anionicity, and in case of 4mers, non-interaction with primary HA cell-surface receptors (CD44). This work is significant in that it allows testing of cell responses to substrates composed of single-sized fragments of HA that cannot by themselves be cross-linked into a gel. Future work in our lab will use this model to assess the effects of other HA oligomer sizes on EC behavior. PMID:17045332
Ibrahim, Samir; Joddar, Binata; Craps, Matthew; Ramamurthi, Anand
Formation of dityrosine cross-links during breadmaking.
To establish its significance during commercial breadmaking, dityrosine formation was quantified in flours and doughs of six commercial wheat types at various stages of the Chorleywood Bread Process. Dityrosine was formed mainly during mixing and baking, at the levels of nmol/g dry weight. Good breadmaking flours tended to exhibit a higher dityrosine content in the final bread than low quality ones, but no relationship was found for dityrosine as a proportion of flour protein content, indicating that the latter was still a dominant factor in the analysis. There was no correlation between gluten yield of the six wheat types and their typical dityrosine concentrations, suggesting that dityrosine cross-links were not a determinant factor for gluten formation. Ascorbic acid was found to inhibit dityrosine formation during mixing and proving, and it has no significant effect on dityrosine in the final bread. Hydrogen peroxide promoted dityrosine formation, which suggests that a radical mechanism involving endogenous peroxidases might be responsible for dityrosine formation during breadmaking. PMID:16569073
Rodriguez-Mateos, Ana; Millar, Sam J; Bhandari, Dhan G; Frazier, Richard A
Grafting of cross-linked hydrogel networks to titanium surfaces.
The performance of medical implants and devices is dependent on the biocompatibility of the interfacial region between tissue and the implant material. Polymeric hydrogels are attractive materials for use as biocompatible surface coatings for metal implants. In such systems, a factor that is critically important for the longevity of an implant is the formation of a robust bond between the hydrogel layer and the implant metal surface and the ability for this assembly to withstand physiological conditions. Here, we describe the grafting of cross-linked hydrogel networks to titanium surfaces using grit-blasting and subsequent chemical functionalization using a silane-based adhesion promoter. Metal surface characterization was carried out using profilometry, scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDX) analysis. Hydrogel layers composed of poly(ethylene glycol)-dimethacrylate (PEG-DMA), poly(2-hydroxyethylmethacrylate) (PHEMA), or poly(ethylene glycol)/poly(acrylic acid) (PEG/PAA) semi-interpenetrating polymer networks (semi-IPNs) have been prepared. The mechanical properties of these hydrogel-metal assemblies have been characterized using lap-shear measurements, and the surface morphology was studied by SEM and EDX. We have shown that both high surface roughness and chemical functionalization are critical for adhesion of the hydrogel layer to the titanium substrate. PMID:24364560
Muir, Beinn V O; Myung, David; Knoll, Wolfgang; Frank, Curtis W
Zinc cross-linked hydroxamated alginates for pulsed drug release
Introduction: Alginates can be tailored chemically to improve solubility, physicochemical, and biological properties and its complexation with metal ion is useful for controlling the drug release. Materials And Methods: Synthesized N,O-dimethyl, N-methyl, or N-Benzyl hydroxylamine derivatives of sodium alginate were subsequently complexed with zinc to form beads. Hydroxamation of sodium alginate was confirmed by Fourier transform infra-red spectroscopy (FTIR) and differential scanning calorimetry (DSC). Results: The synthesized polymeric material exhibited reduced aqueous, HCl and NaOH solubility. The hydroxamated derivatives demonstrated pulsed release where change in pH of the dissolution medium stimulated the atenolol release. Conclusion: Atenolol loaded Zn cross-linked polymeric beads demonstrated the sustained the plasma drug levels with increased half-life. Although the synthesized derivatives greatly altered the aqueous solubility of sodium alginate, no significant differences in in vitro and in vivo atenolol release behavior amongst the N,O-dimethyl, N-methyl, or N-Benzyl hydroxylamine derivatives of sodium alginate were observed.
Raut, Neha S; Deshmukh, Prasad R; Umekar, Milind J; Kotagale, Nandkishor R
Transepithelial corneal collagen cross-linking in ultrathin keratoconic corneas
Background The purpose of this paper was to report the results of transepithelial corneal collagen cross-linking (CXL) with modified riboflavin and ultraviolet A irradiation in patients affected by keratoconus, each with thinnest pachymetry values of less than 400 ?m (with epithelium) and not treatable using standard de-epithelialization techniques. Methods Sixteen patients affected by progressive keratoconus with thinnest pachymetry values ranging from 331 ?m to 389 ?m underwent transepithelial CXL in one eye using a riboflavin 0.1% solution in 15% Dextran T500 containing ethylenediamine tetra-acetic acid 0.01% and trometamol to enhance epithelial penetration. The patients underwent complete ophthalmological examination, including endothelial cell density measurements and computerized videokeratography, before CXL and at one day, one week, and one, 6, and 12 months thereafter. Results Epithelial healing was complete in all patients after one day of use of a soft bandage contact lens. No side effects or damage to the limbal region was observed during the follow-up period. All patients showed slightly improved uncorrected and spectacle-corrected visual acuity; keratometric astigmatism showed reductions (up to 5.3 D) and apical ectasia power decreased (Kmax values reduced up to 4.3 D). Endothelial cell density was unchanged. Conclusion Application of transepithelial CXL using riboflavin with substances added to enhance epithelial permeability was safe, seemed to be moderately effective in keratoconic eyes with ultrathin corneas, and applications of the procedure could be extended to patients with advanced keratoconus.
Spadea, Leopoldo; Mencucci, Rita
Mechanical characterization of cross-linked serum albumin microcapsules.
Controlling the deformation of microcapsules and capsules is essential in numerous biomedical applications. The mechanical properties of the membrane of microcapsules made of cross-linked human serum albumin (HSA) are revealed by two complementary experiments in the linear elastic regime. The first provides the surfacic shear elastic modulus Gs by the study of small deformations of a single capsule trapped in an elongational flow: Gs varies from 0.002 to 5 N m(-1). The second gives the volumic Young's modulus E of the membrane by shallow and local indentations of the membrane with an AFM probe: E varies from 20 kPa to 1 MPa. The surfacic and volumic elastic moduli increase with the size of the capsule up to three orders of magnitude and with the protein concentration of the membrane. The membrane thickness is evaluated from these two membrane mechanical characteristics and increases with the size and the initial HSA concentration from 2 to 20 ?m. PMID:24817568
de Loubens, Cl�ment; Deschamps, Julien; Georgelin, Marc; Charrier, Anne; Edwards-Levy, Florence; Leonetti, Marc
Cross-linking of polytetrafluoroethylene during room-temperature irradiation
Exposure of polytetrafluoroethylene (PTFE) to {alpha}-radiation was investigated to detennine the physical and chemical effects, as well as to compare and contrast the damage mechanisms with other radiation types ({beta}, {gamma}, or thermal neutron). A number of techniques were used to investigate the chemical and physical changes in PTFE after exposure to {alpha}-radiation. These techniques include: Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and fluorescence spectroscopy. Similar to other radiation types at low doses, the primary damage mechanism for the exposure of PTFE to {alpha}-radiation appears to be chain scission. Increased doses result in a change-over of the damage mechanism to cross-linking. This result is not observed for any radiation type other than {alpha} when irradiation is performed at room temperature. Finally, at high doses, PTFE undergoes mass-loss (via smallfluorocarbon species evolution) and defluorination. The amount and type of damage versus sample depth was also investigated. Other types of radiation yield damage at depths on the order of mm to cm into PTFE due to low linear energy transfer (LET) and the correspondingly large penetration depths. By contrast, the {alpha}-radiation employed in this study was shown to only induce damage to a depth of approximately 26 {mu}m, except at very high doses.
Pugmire, David L [Los Alamos National Laboratory; Wetteland, Chris J [Los Alamos National Laboratory; Duncan, Wanda S [Los Alamos National Laboratory; Lakis, Rollin E [Los Alamos National Laboratory; Schwartz, Daniel S [Los Alamos National Laboratory
Cross linking of collagen is increased in colonic diverticulosis.
Development of colonic diverticulosis is a function of age and declining colonic wall mechanical strength. The latter is partly a consequence of changes in the collagen structure. Collagen from unaffected human colons (n = 20, age range 20-80 years) and those with colonic diverticulosis (n = 5, age range 67-80 years) were obtained at necropsy. The total collagen content was measured as the hydroxyproline content and cross linkage by collagen solubility in weak acid was studied. The colonic total collagen content was constant with age (mean (SD) 15.8 (0.3) mg/100 mg wet weight of tissue). The acid solubility of the collagen, however, increased after the age of 40 years: at over 60 years, colonic diverticulosis was associated with an increased acid solubility ratio compared with values in unaffected colons (15.3 (0.2); compared with 9.2 (0.2), p < 0.001). The cross linking of colonic collagen increases with age. These changes seem to be a factor in the aetiology of colonic diverticulosis.
Wess, L; Eastwood, M A; Wess, T J; Busuttil, A; Miller, A
Mechanical Properties of Tyramine Substituted-Hyaluronan Enriched Fascia Extracellular Matrix
Naturally-occurring biomaterial scaffolds derived from extracellular matrix (ECM) have been the topic of recent investigation in the context of rotator cuff tendon repair. We previously reported a method to treat fascia ECM with high molecular weight tyramine substituted-hyaluronan (TS-HA) for use as a tendon augmentation scaffold. The presence of cross-linked TS-HA in fascia was associated with an increased macrophage and giant cell response compared to water treated controls after implantation in a rat abdominal wall model. The objective of this study was to determine the extent to which TS-HA treatment was associated with mechanical property changes of fascia after implantation in the rat model. Fascia samples in all groups demonstrated time-dependent decreases in mechanical properties. TS-HA treated fascia with cross-linking exhibited a lower toe modulus, a trend toward lower toe stiffness, and a higher transition strain than water treated controls not only after implantation, but also at time zero. TS-HA treatment, with or without cross-linking, had no significant effect on time-zero or post-implantation load relaxation ratio, load relaxation rate, linear-region stiffness, or linear-region modulus. Our findings demonstrated that the particular TS-HA treatment employed in this study decreased the low-load elastic mechanical properties of fascia ECM, in keeping with the heightened macrophage and giant cell host response seen previously. This work provides a starting point and guidance for investigating alternative HA treatment strategies.
Chin, LiKang; Calabro, Anthony; Walker, Esteban; Derwin, Kathleen A.
Characterization of the deoxyguanosine-lysine cross-link of methylglyoxal.
Methylglyoxal is a mutagenic bis-electrophile that is produced endogenously from carbohydrate precursors. Methylglyoxal has been reported to induce DNA-protein cross-links (DPCs) in vitro and in cultured cells. Previous work suggests that these cross-links are formed between guanine and either lysine or cysteine side chains. However, the chemical nature of the methylglyoxal induced DPC have not been determined. We have examined the reaction of methylglyoxal, deoxyguanosine (dGuo), and N(?)-acetyllysine (AcLys) and determined the structure of the cross-link to be the N(2)-ethyl-1-carboxamide with the lysine side chain amino group (1). The cross-link was identified by mass spectrometry and the structure confirmed by comparison to a synthetic sample. Further, the cross-link between methylglyoxal, dGuo, and a peptide (AcAVAGKAGAR) was also characterized. The mechanism of cross-link formation is likely to involve an Amadori rearrangement. PMID:24801980
Petrova, Katya V; Millsap, Amy D; Stec, Donald F; Rizzo, Carmelo J
Effect of glucose content on thermally cross-linked fibrous gelatin scaffolds for tissue engineering.
Thermally cross-linked glucose-containing electrospun gelatin meshes were studied as possible cell substrate materials. FTIR analysis was used to study the effect of glucose on cross-linking reactions. It was found that the presence of glucose increases the extent of cross-linking of fibrous gelatin scaffolds, which in return determines scaffold properties and their usability in tissue engineering applications. Easy to handle fabric-like scaffolds were obtained from blends containing up to 15% glucose. Maximum extent of cross-linking was reached at nearly 20% glucose content. Cross-linking effectively resulted in decreased solubility and increased resistance to enzymatic degradation. Preliminary short-term cell culture experiments indicate that such thermally cross-linked gelatin-glucose scaffolds are suitable for tissue engineering applications. PMID:25063151
Siimon, Kaido; Reemann, Paula; P�der, Annika; Pook, Martin; Kangur, Triin; Kingo, K�lli; Jaks, Viljar; M�eorg, Uno; J�rvek�lg, Martin
A role for hyaluronan in joint development.
Hyaluronan, a common connective tissue component, influences cell adhesion, migration and cytodifferentiation in vitro, and because of these properties has been postulated to have a role in morphogenesis. Its role in the development of cartilage-associated structures, such as joints, has yet to be defined. Using a biotinylated hyaluronan-binding region-link protein complex, free hyaluronan binding sites have been localised in the joint region concomitant with the first signs of cavitation (Stage 37), whereafter it is localised in the joint space and is maintained here as this enlarges. The application of our results is discussed in the context of a primary role for hyaluronan in joint cavity formation. Images Fig. 1 Fig. 2
Craig, F M; Bayliss, M T; Bentley, G; Archer, C W
Algorithms for identifying protein cross-links via tandem mass spectrometry
Cross-linking technology combined with tandem mass spectrometry (MS-MS) is a powerful method that provides a rapid solution to the discovery of protein-protein interactions and protein structures. We studied the problem of detecting cross-linked peptides and cross-linked amino acids from tandem mass spectral data. Our method consists of two steps: the first step finds two protein subsequences whose mass sum equals
Ting Chen; Jacob D. Jaffe; George M. Church
Molecular simulations predict novel collagen conformations during cross-link loading
Collagen cross-linking mechanically strengthens tissues during development and aging, but there is limited data describing how force transmitted across cross-links affects molecular conformation. We used Steered Molecular Dynamics (SMD) to model perpendicular force through a side chain. Results predicted collagen peptides have negligible bending resistance and that mechanical force causes helix disruption below covalent bond failure strength, suggesting alternative molecular conformations precede cross-link rupture and macroscopic damage during mechanical loading.
Bourne, Jonathan W.; Torzilli, Peter A.
Performance improvement of nano-silica\\/polypropylene composites through in-situ cross-linking approach
To prepare polypropylene (PP) based nanocomposites with improved mechanical performance, an in-situ cross-linking route was adopted by adding reactive monomers and cross-linking agents into the system containing nano-silica during melt compounding. In the meantime, the reactive monomers were polymerized and grafted onto to the nanoparticles, and the grafted polymer chains were cross-linked forming a network of the tiny reinforcement dispersed
Tong Hui Zhou; Wen Hong Ruan; Yu Liang Mai; Min Zhi Rong; Ming Qiu Zhang
The chemistry of natural enzyme-induced cross-links of proteins
The cross-linking of protein molecules to form stable supramolecular aggregates capable of acting as protective and supporting structures is a common feature of organisms coping with the stresses of life. These new polymeric forms range from thick rigid structures to thin flexible membranes. The formation of such cross-links must be carefully controlled since more or less than optimal cross-linking could
A. J. Bailey
Rheology and Convective Heat Transfer Properties of Borate Cross-Linked Nitrogen Foam Fracturing Fluid
This article considers the rheology and convective heat transfer characteristics of borate cross-linked guar and borate cross-linked foam fracturing fluid by conducting experiments on a large-scale test loop at 30 MPa. The results show that severe chemical degradation of borate cross-linked guar occurred as temperature increased. When temperature was higher than the threshold value, the cross-linker was almost disabled and
Xiao Sun; Shuzhong Wang; Yu Bai; Shuangshuang Liang
Proton conducting membranes based on cross-linked sulfonated poly(ether ether ketone) (SPEEK)
A new method for the preparation of proton exchange membranes (PEMs), based on cross-linked sulfonated poly(ether ether ketones) is reported. The cross-linking involves thermally activated bridging of the polymer chains with polyatomic alcohols, which makes the polymer mechanically stronger and reduces its swelling by water. Although the cross-linking occurs through the sulfonic acid group, a major portion of acid functions
Serguei D Mikhailenko; Keping Wang; Serge Kaliaguine; Peixiang Xing; Gilles P Robertson; Michael D Guiver
Plant phenolics as cross-linkers of gelatin gels and gelatin-based coacervates for use as food ingredients
Polyphenols are known to react under oxidizing conditions with side chain amino groups of peptides, leading to formation of cross-links in proteins. Plant-derived phenolic acids and flavonoids were used to prepare cross-linked gelatin gels in bulk and cross-linked gelatin�pectin coacervates in the form of microparticles for use as food ingredients. Gels cross-linked by these materials had greater mechanical strength, reduced
George Strauss; Suzanne M. Gibson
The effects of cross-linking agents on some properties of HEMA-based resins.
The use of 2-hydroxyethyl methacrylate (HEMA)-based polymer as a biocompatible material has been well-established. HEMA-based resins containing cross-linking agents have several potential clinical applications. It is hypothesized that the incorporation of cross-linking agent will improve the mechanical properties of HEMA-based polymers while reducing water absorption and solubility. The purpose of the work reported here was to test this hypothesis and to determine the most effective cross-linking agent. A relationship among flexural strength, modulus, water absorption and solubility, and concentration of cross-linking agent was demonstrated. Strength and modulus tend to increase as the cross-linking agent concentration is increased, up to about 50%, after which the values level out or begin to fall. Water absorption drops with increasing cross-linking agent over the whole range of concentrations. Solubility tends to show a small decrease initially (up to 40%), followed by a noticeable increase as cross-linking agent concentration is increased. The trends were similar for all cross-linking agents, although there were differences in the absolute values in all properties depending upon the type of cross-linking agent used. PMID:7560422
Arima, T; Hamada, T; McCabe, J F
Controlled sparse and percolating cross-linking in waterborne soft adhesives.
The effect of low levels of cross-linking on the adhesive and mechanical properties of waterborne pressure-sensitive adhesives was investigated. We have taken advantage of a core-shell latex particle morphology obtained by emulsion polymerization to create a heterogeneous structure of cross-links without major modification of the monomer composition. The latex particles comprise a shell containing cross-linkable diacetone acrylamide (DAAM) repeat units localized on the periphery of a slightly softer core copolymer of very similar composition. Adipic acid dihydrazide was added to the latex prior to film formation to react with DAAM repeat units and affect interfacial cross-linking between particles in the adhesive films. The honeycomb-like structure obtained after drying of the latex results in a good balance between the dissipative properties required for adhesion and the resistance to creep. The characterization of the mechanical properties of the films shows that the chosen cross-linking method creates a percolating lightly cross-linked network, swollen with a nearly un-cross-linked component. With this cross-linking method, the linear viscoelastic properties of the soft films are nearly unaffected by the cross-linking while the nonlinear tensile properties are greatly modified. As a result, the long-term shear resistance of the adhesive film improves very significantly while the peel force remains nearly the same. A simple rheological model is used to interpret qualitatively the changes in the material parameters induced by cross-linking. PMID:20355828
Deplace, F; Carelli, C; Langenfeld, A; Rabjohns, M A; Foster, A B; Lovell, P A; Creton, C
Direct imaging of fluctuations in a cross-linked biopolymer network
NASA Astrophysics Data System (ADS)
Cross-linked networks are ubiquitous in synthetic and biological polymer systems, such as rubbers and cytoskeletons. To model cross-linked networks, several theories have been developed on the basis of different assumptions as to fluctuations in the networks. Here we put these theories to direct test. This talk will describe direct single-molecule imaging of the dynamic fluctuations of junction points in a cross-linked semiflexible polymer (F-actin) network. The actin filaments are cross linked by biotin/avidin. The junction points are selectively labeled to allow nm spatial imaging resolution. The surprising results point to limitations of the prevailing network models.
Wang, Bo; Jiang, Lingxiang; Tsang, Boyce; Granick, Steve
Accelerated corneal collagen cross-linking for progressive keratoconus.
Abstract Purpose: To evaluate the efficacy of accelerated corneal cross-linking (CXL) procedure for progressive keratoconus. Materials and methods: Twenty-three eyes of 23 patients undergone accelerated CXL procedure were evaluated preoperatively and postoperatively at 1st, 3rd and 6th month for uncorrected distant visual acuity (UDVA), best corrected distant visual acuity (CDVA), spherical error, cylindrical error, spherical equivalent (SE), keratometric values and thinnest corneal thickness (TCT) values with corneal topography by Scheimpflug camera and endothelial cell density (ECD). Results: The mean UDVA was improved from 0.97?�?0.41 logarithm of minimal angle of resolution (logMAR) to 0.76?�?0.45 logMAR at the 6th month after CXL (p?=?0.332). The mean CDVA was improved from 0.49?�?0.30 logMAR to 0.34?�?0.22 logMAR at the 6th month after CXL (p?=?0.026). The mean sphere was decreased from -4.47?�?4.1 diopter (D) to -3.79?�?3.86?D and the mean cylinder was decreased from -5.60?�?2.2?D to -4.55?�?1.98?D and the mean SE was decreased from -7.22?�?4.48?D to -6.36?�?4.34?D at the 6th month after CXL (p?=?0.128, p?=?0.002 and p?=?0.045, respectively). Flat keratometry, steep keratometry, mean keratometry and maximum keratometry were significantly reduced at the 6th month after CXL (p?=?0.025, p?0.001, p?=?0.004 and p?=?0.03, respectively). TCT and ECD were not changed significantly the 6th month after CXL (p?=?0.135 and p?=?0.082, respectively). Conclusion: Accelerated CXL procedure was effective to stabilize progression of keratoconus with significant reduction in topographic keratometric values and significant increase in CDVA in 6 months. PMID:23879803
C?nar, Yasin; Cing�, Abdullah K�r?at; Turkcu, Fatih Mehmet; Y�ksel, Harun; Sahin, Alparslan; Y?ld?r?m, Adnan; Caca, Ihsan; C?nar, Tuba
Transglutaminases: Widespread Cross-linking Enzymes in Plants
Background Transglutaminases have been studied in plants since 1987 in investigations aimed at interpreting some of the molecular mechanisms by which polyamines affect growth and differentiation. Transglutaminases are a widely distributed enzyme family catalysing a myriad of biological reactions in animals. In plants, the post-translational modification of proteins by polyamines forming inter- or intra-molecular cross-links has been the main transglutaminase reaction studied. Characteristics of Plant Transglutaminases The few plant transglutaminases sequenced so far have little sequence homology with the best-known animal enzymes, except for the catalytic triad; however, they share a possible structural homology. Proofs of their catalytic activity are: (a) their ability to produce glutamyl-polyamine derivatives; (b) their recognition by animal transglutaminase antibodies; and (c) biochemical features such as calcium-dependency, etc. However, many of their fundamental biochemical and physiological properties still remain elusive. Transglutaminase Activity is Ubiquitous It has been detected in algae and in angiosperms in different organs and sub-cellular compartments, chloroplasts being the best-studied organelles. Possible Roles Possible roles concern the structural modification of specific protein substrates. In chloroplasts, transglutaminases appear to stabilize the photosynthetic complexes and Rubisco, being regulated by light and other factors, and possibly exerting a positive effect on photosynthesis and photo-protection. In the cytosol, they modify cytoskeletal proteins. Preliminary reports suggest an involvement in the cell wall construction/organization. Other roles appear to be related to fertilization, abiotic and biotic stresses, senescence and programmed cell death, including the hypersensitive reaction. Conclusions The widespread occurrence of transglutaminases activity in all organs and cell compartments studied suggests a relevance for their still incompletely defined physiological roles. At present, it is not possible to classify this enzyme family in plants owing to the scarcity of information on genes encoding them.
Serafini-Fracassini, Donatella; Del Duca, Stefano
Corneal cross-linking in 9 horses with ulcerative keratitis
Background Corneal ulcers are one of the most common eye problems in the horse and can cause varying degrees of visual impairment. Secondary infection and protease activity causing melting of the corneal stroma are always concerns in patients with corneal ulcers. Corneal collagen cross-linking (CXL), induced by illumination of the corneal stroma with ultraviolet light (UVA) after instillation of riboflavin (vitamin B2) eye drops, introduces crosslinks which stabilize melting corneas, and has been used to successfully treat infectious ulcerative keratitis in human patients. Therefore we decided to study if CXL can be performed in sedated, standing horses with ulcerative keratitis with or without stromal melting. Results Nine horses, aged 1 month to 16 years (median 5 years) were treated with a combination of CXL and medical therapy. Two horses were diagnosed with mycotic, 5 with bacterial and 2 with aseptic ulcerative keratitis. A modified Dresden-protocol for CXL could readily be performed in all 9 horses after sedation. Stromal melting, diagnosed in 4 horses, stopped within 24 h. Eight of nine eyes became fluorescein negative in 13.5 days (median time; range 4�26 days) days after CXL. One horse developed a bacterial conjunctivitis the day after CXL, which was successfully treated with topical antibiotics. One horse with fungal ulcerative keratitis and severe uveitis was enucleated 4 days after treatment due to panophthalmitis. Conclusions CXL can be performed in standing, sedated horses. We did not observe any deleterious effects attributed to riboflavin or UVA irradiation per se during the follow-up, neither in horses with infectious nor aseptic ulcerative keratitis. These data support that CXL can be performed in the standing horse, but further studies are required to compare CXL to conventional medical treatment in equine keratitis and to optimize the CXL protocol in this species.
Integrated Cryogenic Satellite Communications Cross-Link Receiver Experiment
NASA Technical Reports Server (NTRS)
An experiment has been devised which will validate, in space, a miniature, high-performance receiver. The receiver blends three complementary technologies; high temperature superconductivity (HTS), pseudomorphic high electron mobility transistor (PHEMT) monolithic microwave integrated circuits (MMIC), and a miniature pulse tube cryogenic cooler. Specifically, an HTS band pass filter, InP MMIC low noise amplifier, HTS-sapphire resonator stabilized local oscillator (LO), and a miniature pulse tube cooler will be integrated into a complete 20 GHz receiver downconverter. This cooled downconverter will be interfaced with customized signal processing electronics and integrated onto the space shuttle's 'HitchHiker' carrier. A pseudorandom data sequence will be transmitted to the receiver, which is in low Earth orbit (LEO), via the Advanced Communication Technology Satellite (ACTS) on a 20 GHz carrier. The modulation format is QPSK and the data rate is 2.048 Mbps. The bit error rate (BER) will be measured in situ. The receiver is also equipped with a radiometer mode so that experiment success is not totally contingent upon the BER measurement. In this mode, the receiver uses the Earth and deep space as a hot and cold calibration source, respectively. The experiment closely simulates an actual cross-link scenario. Since the receiver performance depends on channel conditions, its true characteristics would be masked in a terrestrial measurement by atmospheric absorption and background radiation. Furthermore, the receiver's performance depends on its physical temperature, which is a sensitive function of platform environment, thermal design, and cryocooler performance. This empirical data is important for building confidence in the technology.
Romanofsky, R. R.; Bhasin, K. B.; Downey, A. N.; Jackson, C. J.; Silver, A. H.; Javadi, H. H. S.
Chain gangs: new aspects of hyaluronan metabolism.
Hyaluronan is a matrix polymer prominent in tissues undergoing rapid growth, development, and repair, in embryology and during malignant progression. It reaches 10(7) Daltons in size but also exists in fragmented forms with size-specific actions. It has intracellular forms whose functions are less well known. Hyaluronan occurs in all vertebrate tissues with 50% present in skin. Hyaluronan provides a scaffold on which sulfated proteoglycans and matrix proteins are organized. These supramolecular structures are able to entrap water and ions to provide tissues with hydration and turgor. Hyaluronan is recognized by membrane receptors that trigger intracellular signaling pathways regulating proliferation, migration, and differentiation. Cell responses are often dependent on polymer size. Catabolic turnover occurs by hyaluronidases and by free radicals, though proportions between these have not been determined. New aspects of hyaluronan biology have recently become realized: involvement in autophagy, in the pathology of diabetes., the ability to modulate immune responses through effects on T regulatory cells and, in its fragmented forms, by being able to engage several toll-like receptors. It is also apparent that hyaluronan synthases and hyaluronidases are regulated at many more levels than previously realized, and that the several hyaluronidases have functions in addition to their enzymatic activities. PMID:22216413
Erickson, Michael; Stern, Robert
Type VI collagen microfibrils: evidence for a structural association with hyaluronan
Type VI collagen, a widespread structural component of connective tissues, has been isolated in abundance from fetal bovine skin by a procedure involving bacterial collagenase digestion under nonreducing, nondenaturing conditions and gel filtration chromatography. Rotary shadowing electron microscopic analysis revealed that the collagen VI was predominantly in the form of extensive intact microfibrillar arrays. These microfibrils were seen in association with hyaluronan, which was identified by its ability to bind the G1 fragment of cartilage proteoglycan. Treatment with highly purified hyaluronidase largely disrupted the collagen VI microfibrils into component tetramers, double tetramers, and short microfibrillar sections. Subsequent incubation of disrupted collagen VI in the presence of hyaluronan facilitated a partial repolymerization of the microfibrils. In vitro binding studies have also demonstrated that type VI collagen binds hyaluronan with a relatively high affinity. These studies demonstrate that a specific structural relationship exists between type VI collagen and hyaluronan. This association is likely to be of primary importance in the growth and remodeling processes of connective tissues.
Alkaline battery containing a separator of a cross-linked copolymer of vinyl alcohol and unsaturated carboxylic acid
NASA Astrophysics Data System (ADS)
A battery separator for an alkaline battery is described. The separator comprises a cross linked copolymer of vinyl alcohol units and unsaturated carboxylic acid units. The cross linked copolymer is insoluble in water, has excellent zincate diffusion and oxygen gas barrier properties and a low electrical resistivity. Cross linking with a polyaldehyde cross linking agent is preferred.
Hsu, L. C.; Philipp, W. H.; Sheibley, D. W.; Gonzalez-Sanabria, O. D.
Hyaluronan Synthase Gene and Uses Thereof, Part Two.
National Technical Information Service (NTIS)
The present invention relates to a recombinant host cell that includes an expression construct having a hyaluronan synthase gene that encodes an enzymatically active hyaluronan synthase (HAS), and to the use of these recombinant cells that produce hyaluro...
K. Kumari P. DeAngelis P. H. Weigel
Hyaluronan Synthase Gene and Uses Thereof, Part Three.
National Technical Information Service (NTIS)
The present invention relates to a recombinantly produced, enzymatically active hyaluronan synthase, wherein the recombinantly produced, enzymatically active hyaluronan synthase is a single protein that is a dual-action catalyst that utilizes UDP-GlcA and...
K. Kumari P. DeAngelis P. H. Weigel
Analytical characterisation of glutardialdehyde cross-linking products in gelatine-gum arabic complex coacervates.
Encapsulates having shells of cross-linked mixtures of proteins and polysaccharides are widely used in the food and pharmaceutical industry for controlled release of actives and flavour compounds. In order to be able to predict the behaviour and the release characteristics of the microcapsules, a better understanding of the nature and extent of the cross-linking reaction is needed. Several analytical techniques were applied for the characterisation of glutardialdehyde (GDA) cross-linked encapsulates made of gelatine and gum arabic. To allow the use of sensitive, high-resolution methods such as chromatography and mass spectrometry, the sample first had to be hydrolysed. In this way, a mixture of amino acids, small peptides and the cross-link moieties was obtained. High-resolution liquid chromatography coupled to high-resolution mass spectrometry (HPLC-MS) was applied to detect possible cross-link markers through a comparison of HPLC-MS mass-chromatograms obtained for cross-linked and non-cross-linked coacervates. HPLC-MS/MS was used to identify the species responsible for the differences. Cross-linking occurred between GDA molecules and lysine and hydroxylysine epsilon-amino groups, and up to eight cross-link products of different nature could be identified. They included pyridinium ions and Schiff bases, and also unreacted GDA condensation products. Next, based on the insight gained in the possible chemical structures present in the cross-link markers, methods for selective labelling of these functionalities were employed to allow easier detection of related reaction products. Both liquid chromatography (LC) and gas chromatography (GC) were used in these experiments. Unfortunately, these approaches failed to detect new cross-link markers, most likely as a result of the low levels at which these are present. PMID:17983779
Fuguet, Elisabet; van Platerink, Chris; Janssen, Hans-Gerd
Matching cross-linked peptide spectra: only as good as the worse identification.
Chemical cross-linking mass spectrometry identifies interacting surfaces within a protein assembly through labeling with bifunctional reagents and identifying the covalently modified peptides. These yield distance constraints that provide a powerful means to model the three-dimensional structure of the assembly. Bioinformatic analysis of cross-linked data resulting from large protein assemblies is challenging because each cross-linked product contains two covalently linked peptides, each of which must be correctly identified from a complex matrix of potential confounders. Protein Prospector addresses these issues through a complementary mass modification strategy in which each peptide is searched and identified separately. We demonstrate this strategy with an analysis of RNA polymerase II. False discovery rates (FDRs) are assessed via comparison of cross-linking data to crystal structure, as well as by using a decoy database strategy. Parameters that are most useful for positive identification of cross-linked spectra are explored. We find that fragmentation spectra generally contain more product ions from one of the two peptides constituting the cross-link. Hence, metrics reflecting the quality of the spectral match to the less confident peptide provide the most discriminatory power between correct and incorrect matches. A support vector machine model was built to further improve classification of cross-linked peptide hits. Furthermore, the frequency with which peptides cross-linked via common acylating reagents fragment to produce diagnostic, cross-linker-specific ions is assessed. The threshold for successful identification of the cross-linked peptide product depends upon the complexity of the sample under investigation. Protein Prospector, by focusing the reliability assessment on the least confident peptide, is better able to control the FDR for results as larger complexes and databases are analyzed. In addition, when FDR thresholds are calculated separately for intraprotein and interprotein results, a further improvement in the number of unique cross-links confidently identified is achieved. These improvements are demonstrated on two previously published cross-linking datasets. PMID:24335475
Trnka, Michael J; Baker, Peter R; Robinson, Philip J J; Burlingame, A L; Chalkley, Robert J
In vitro cross-linking of gluten into high-molecular-weight polymers with transglutaminase.
From the amino acid composition of gluten proteins and the substrate specificity of transglutaminases (TGase) we concluded that gluten proteins can be favourable substrates for TGases due to their high glutamine content. By use of sodium dodecyl sulfate polyacrylamide gel-electrophoresis it was demonstrated that from gluten-ES and gluten-TS high-molecular-weight proteins developed in the presence of Ca2+ and red blood cell lysate containing TGase. When ovalbumin or deamidated gluten were applied as substrates no high-molecular-weight products were formed. Upon spectrophotometric measurements we found that covalent cross-links (isopeptide bonds) formed under the effect of TGases presumably cause a change in the position of chromophore groups in the substrates. Absorption decrease was detected between 274-276 nm as a result in the case of gluten-TS and gluten-ES used as substrates for TGase. No such change occurred in ovalbumin and deamidated gluten, applied as controls, under the influence of TGase. On the basis of our experiments it is postulated that the first step in gluten toxicity is presumably the binding of gluten to the intestine mucosa. In this binding the high transglutaminase activity in the intestines of coeliac patients and the high glutamine content of gluten may have an important role. PMID:2900018
Szabolcs, M; Sipka, S; Csorba, S
Oxidation-induced unfolding facilitates Myosin cross-linking in myofibrillar protein by microbial transglutaminase.
Myofibrillar protein from pork Longissimus muscle was oxidatively stressed for 2 and 24 h at 4 �C with mixed 10 ?M FeCl(3)/100 ?M ascorbic acid/1, 5, or 10 mM H(2)O(2) (which produces hydroxyl radicals) and then treated with microbial transglutaminase (MTG) (E:S = 1:20) for 2 h at 4 �C. Oxidation induced significant protein structural changes (P < 0.05) as evidenced by suppressed K-ATPase activity, elevated Ca-ATPase activity, increased carbonyl and disulfide contents, and reduced conformational stability, all in a H(2)O(2) dose-dependent manner. The structural alterations, notably with mild oxidation, led to stronger MTG catalysis. More substantial amine reductions (19.8-27.6%) at 1 mM H(2)O(2) occurred as compared to 11.6% in nonoxidized samples (P < 0.05) after MTG treatment. This coincided with more pronounced losses of myosin in oxidized samples (up to 33.2%) as compared to 21.1% in nonoxidized (P < 0.05), which was attributed to glutamine-lysine cross-linking as suggested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PMID:22809283
Li, Chunqiang; Xiong, Youling L; Chen, Jie
Lactogen receptors in rat Leydig cells: analysis of their structure with bifunctional cross-linking reagents
(/sup 125/I)Iodohuman GH was found to bind to receptors with specificity for lactogenic hormones in a Triton X-100 extract from Leydig cell membranes displaying an affinity constant of 3.8 X 10(/sup 9/) M-1 and a binding capacity of 167 fmol/mg protein. Cross-linking of solubilized (/sup 125/I)iodohuman GH-receptor complexes with disuccinimidyl suberate followed by analysis by sodium dodecyl sulfate-gel electrophoresis in the presence of beta-mercaptoethanol and autoradiography resulted in the appearance of bands with apparent mol wt of 113,000, 103,000, 59,000, and 53,000. The appearance of these bands was prevented by incubation in the presence of lactogenic hormones. By using a two-dimensional electrophoresis technique (first dimension under nonreducing conditions; second dimension under reducing conditions), it was demonstrated that a fraction of the mol wt 59,000 species can be released from the mol wt 103,000 species upon cleavage of disulfide bonds. These results suggest the existence of lactogen receptor species with approximate mol wt of 91,000, 81,000, 37,000, and 31,000 in Triton X-100 extracts from Leydig cell membranes if the contribution of the free hormone (mol wt, 22,000) is subtracted. A fraction of the mol wt 37,000 subunits appears to be contained within the 81,000 species linked through disulfide bonds.
Bonifacino, J.S.; Dufau, M.L.
Drug release characteristics of physically cross-linked thermosensitive poly(N-vinylcaprolactam) hydrogel particles.
The effect of physical cross-linking was studied on the formation and properties of thermosensitive polymer particles of poly(N-vinylcaprolactam), PVCL, and PVCL grafted with poly(ethylene oxide) macromonomer, PVCL-graft-C(11)EO(42). Loading and release of model drugs into/from the hydrogel particles were evaluated. Thermosensitive particles were stabilized by cross-linkers, the most feasible of which was salicylic acid (SA). At 23 degrees C, below the lower critical solution temperature (LCST) of the thermosensitive polymers, stability of the hydrogels was poor, whereas at 37 degrees C stable hydrogel particles were formed. All the drugs and also the cross-linker (SA) were released more efficiently from the PVCL particles compared to the PVCL-graft-C(11)EO(42) particles. Drug concentration and pH affected clearly the rate and extent of drug release in physiological buffer. The higher drug release from the PVCL was based on the more open gel-like structure as opposed to PVCL-graft-C(11)EO(42) particles. Complex formation between the cross-linker and the polymers was due to the hydrogen bonding between the hydroxyl groups of SA and H-bond acceptors of the PVCL. In the case of PVCL-graft-C(11)EO(42), the ethylene oxide chain provided more opportunities for H-bonding in comparison to the pure PVCL, creating more stable complexes (more tightly packed particles) leading to sustained drug release. PMID:18306245
Vihola, Henna; Laukkanen, Antti; Tenhu, Heikki; Hirvonen, Jouni
Synthesis of a disulfide cross-linked polygalacturonic acid hydrogel for biomedical applications.
Polygalacturonic acid (PGA) hydrogel cross-linked via disulfide bonds was synthesized using a thiol oxidation reaction. PGA was grafted with cysteine to yield thiolated PGA (denoted PGAcys). Per gram, PGA-conjugated cysteine was 725���77�?mol, and the degree of modification was 16.24�%. A PGAcys hydrogel film was fabricated under physiological conditions, with gel content 91.6�% and water content 43.3�%. The PGAcys hydrogel was used as a drug carrier for rosmarinic acid (RA) (denoted PGAcys/RA) and to prevent postsurgical adhesion. The in vitro dynamic release behavior of RA from the PGAcys hydrogel was analyzed. The profiles showed that 80�% of the total RA was released from the hydrogel within 15�min, followed by zero-order kinetic release. Animal implant studies showed that PGAcys and PGAcys/RA hydrogel films reduced adhesion incidence by over 90�%, significantly higher than did Hyaluronate/Carboxymethylcellulose (analogous Seprafilm�) (42�%). The PGAcys/RA hydrogel film also reduced the early inflammatory reaction. PMID:23468164
Peng, Hsiu-Hui; Chen, Yi-Min; Lee, Chen-I; Lee, Ming-Wei
Radiation cross-linking of small electrical wire insulator fabricated from NR/LDPE blends
NASA Astrophysics Data System (ADS)
A low voltage, radiation-crosslinked wire insulator has been fabricated from blends of natural rubber block (STR-5L) and LDPE with phthalic anhydride (PA) as a compatibilizer. Physical properties of the NR/LDPE blend ratios of 50/50 and 60/40 with 0.5, 1.0, and 1.5 wt% PA were evaluated. The gel content increased as the radiation dose increased. Tensile at break exhibited a maximum value of 12 MPa at 120 kGy for 1.0 and 1.5 wt% PA of both blend ratios. A higher PA content yielded a higher modulus for the same blend ratio. Blends of 60/40 ratio with 1.0 wt% PA and 0.8 wt% antimony oxide flame retardant gave the highest limiting oxygen index (LOI) of >30% at above 150 kGy. Other electrical properties of the wire insulator were investigated. It was found that an insulator fabricated from a PA content of 1.0 wt% in the NR/LDPE blend ratio of 50/50, after gamma ray cross-linked at a dose of 180 kGy in low vacuum (1 mm Hg), met the Thai Industrial Standard 11-2531 for low voltage wire below 1.0 kV. To comply with the standard for vertical flame test, a more suitable flame retardant was needed for the insulator.
Siri-Upathum, Chyagrit; Punnachaiya, Suvit
Ionically cross-linked alginate hydrogels as tissue engineering scaffolds
NASA Astrophysics Data System (ADS)
Generation of living tissues through tissue engineering can be achieved via incorporation of cells into synthetic scaffolds designed to facilitate new tissue formation. Necessary characteristics of a scaffold include biocompatibility, high porosity with controllable pore size and interconnectivity, moldability, chemical and mechanical stability, and structural homogeneity. Hydrogels often possess many of the necessary characteristics and thus are favorable candidates for scaffolding. Alginate hydrogels are commonly made by ionically crosslinking with calcium ions from CaCl2 or CaSO4. These hydrogels are favored for their mild gel formation, however the gelation rate is rapid and uncontrollable (fast-gelation), resulting in varying crosslinking density throughout the gel. In this work, structurally homogeneous calcium alginate hydrogels were formed via a slow-gelation system that utilizes uniform mixing of CaCO3 with sodium alginate solution, and the addition of slowly hydrolyzing D-gluconic acid lactone to slowly release calcium ions for crosslinking. Homogeneity and mechanical properties of these hydrogels were shown to be superior to those of fast-gelled hydrogels. Gelation rate was controlled through the incorporation of CaSO4, and by varying total calcium content, polymer concentration and gelation temperature. Control over mechanical properties and diffusivity was demonstrated in the homogeneous hydrogels by adjusting compositional variables. Consistent control over solute diffusivity through gel discs reflected the structural homogeneity of the gels. To overcome the instability of ionically crosslinked gels in tissue culture medium, a method was developed to control the hydrogel dimensions by adjusting the ionic concentration of the medium. Stability of the hydrogels in this controlled environment was characterized through swelling experiments and mechanical testing. To provide for scaffold degradation and thereby promote tissue growth, alginate lyase was incorporated into the system induce enzymatically controlled alginate degradation. This alginate hydrogel system is novel in that cells are directly and uniformly incorporated into a hydrogel with controlled gelation rate and material properties. The injectable alginate hydrogels can be molded into complex shapes or injected into the body to avoid invasive surgery. Further, the ability to tailor their gelation rate, material properties and degradation enable these alginate hydrogels to be used for a variety of biomedical applications including drug or biofactor delivery.
Kuo, Catherine Kyleen
The pyrrolobenzodiazepine dimer SJG-136 forms sequence-dependent intrastrand DNA cross-links and monoalkylated adducts in addition to interstrand cross-links.
SJG-136 (1) is a sequence-selective DNA-interactive agent that is about to enter phase II clinical trials. Using a HPLC/MS-based methodology developed to evaluate the binding of DNA-interactive agents to oligonucleotides of varying length and sequence, we have demonstrated that, in addition to the previously known interstrand cross-link at Pu-GATC-Py sequences, 1 can form a longer interstrand cross-link at Pu-GAATC-Py sequences, an intrastrand cross-link at both shorter Pu-GATG-Py and longer Pu-GAATG-Py sequences, and, in addition, monoalkylated adducts at suitable PBD binding sites where neither intra- or interstrand cross-links are feasible because of the unavailability of two appropriately positioned guanines. Crucially, we have demonstrated a preference for the extended intrastrand cross-link with Pu-GAATG-Py, which forms more rapidly than the other cross-links (rank order: Pu-GAATG-Py > Pu-GATC-Py > Pu-GATG-Py and Pu-GAATC-Py). However, thermal denaturation studies suggest that the originally reported Pu-GATC-Py interstrand cross-link is more stable, consistent with the covalent joining of both strands of the duplex and a lower overall distortion of the helix according to modeling studies. These observations impact on the proposed mechanism of action of SJG-136 (1) both in vitro and in vivo, the repair of its adducts and mechanism of resistance in cells, and potentially on the type of pharmacodynamic assay used in clinical trials. PMID:19725510
Rahman, Khondaker M; Thompson, Andrew S; James, Colin H; Narayanaswamy, Mathangi; Thurston, David E
Preparation of size tunable giant vesicles from cross-linked dextran(ethylene glycol) hydrogels�
We present a novel chemically cross-linked dextran�poly(ethylene glycol) hydrogel substrate for the preparation of dense vesicle suspensions under physiological ionic strength conditions. These vesicles can be easily diluted for individual study. Modulating the degree of cross-linking within the hydrogel network results in tuning of the vesicle size distribution.
Mora, Nestor Lopez; Hansen, Jesper S.; Gao, Yue; Ronald, Andrew A.; Kieltyka, Roxanne
Cross-linked telopeptides of type I and III collagens in malignant ovarian tumours in vivo
Malignant tumours often induce a fibroproliferative response in the adjacent stroma, characterized by increased expression of type I and type III procollagens. In normal tissues, fibrillar collagens normally undergo extensive intermolecular cross-linking that provides tensile strength to the tissue. Here we set out to characterize collagen cross-linking in human ovarian carcinoma tissue in vivo. Biochemical and immunochemical methods were used for cross-linked telopeptides of type I and III collagens in samples of benign and malignant serous tumours. The locations and staining patterns of these proteins were visualized immunohistochemically. The contents of both total collagen and the cross-linked type I and type III collagens in the malignant samples were only about 20% of those in the benign tumours. The cross-linked telopeptide antigens derived from the collagens were smaller and more heterogeneous in size in the malignant than in the benign tumours, indicating a defective cross-linking process scarcely leading to the formation of mature cross-links in the collagen fibres in malignancy. Immunostaining revealed disorganized type I and type III collagen bundles in carcinomas. These findings suggest that the collagen cross-linking process is aberrant in malignant tumours, possibly resulting in increased susceptibility of tumour collagens for the proteolysis often associated with tumour invasion. � 1999 Cancer Research Campaign
Kauppila, S; Bode, M K; Stenback, F; Risteli, L; Risteli, J
Bypass of a psoralen DNA interstrand cross-link by DNA polymerases ?, ?, and ? in vitro.
Repair of DNA interstrand cross-links in mammalian cells involves several biochemically distinctive processes, including the release of one of the cross-linked strands and translesion DNA synthesis (TLS). In this report, we investigated the in vitro TLS activity of a psoralen DNA interstrand cross-link by three DNA repair polymerases, DNA polymerases ?, ?, and ?. DNA polymerase ? is capable of bypassing a psoralen cross-link with a low efficiency. Cell extracts prepared from DNA polymerase ? knockout mouse embryonic fibroblasts showed a reduced bypass activity of the psoralen cross-link, and purified DNA polymerase ? restored the bypass activity. In addition, DNA polymerase ? misincorporated thymine across the psoralen cross-link and DNA polymerase ? extended these mispaired primer ends, suggesting that DNA polymerase ? may serve as an inserter and DNA polymerase ? may play a role as an extender in the repair of psoralen DNA interstrand cross-links. The results demonstrated here indicate that multiple DNA polymerases could participate in TLS steps in mammalian DNA interstrand cross-link repair. PMID:23106263
Smith, Leigh A; Makarova, Alena V; Samson, Laura; Thiesen, Katherine E; Dhar, Alok; Bessho, Tadayoshi
Preparation and Structures of Ethyl-Cyanoethyl Cellulose/Cross-Linked Polystyrene Composites.
National Technical Information Service (NTIS)
Ethyl-cyanoethyl cellulose ((E-CE)C)/cross-linked polystyrene (PS) composites were prepared via polymerization of styrene in (E-CE)C/styrene solutions with glycol diacrylate as a cross-linking reagent. The polymerization of the solution, morphology, and m...
S. H. Jiang Y. Huang
Influence of cross-linking degree on shape memory effect of styrene copolymer
This paper is concerned about the synthesis of shape memory styrene copolymer and the investigation of the influence of cross-linking degree on its shape memory effect. As one of novel actuators in smart materials, shape memory polymers (SMPs) have been investigated intensively. Styrene copolymer with proper cross-linking degree can exhibit shape memory effect (SME). In this paper, the influence of
Dawei Zhang; Xin Lan; Yanju Liu; Jinsong Leng
Cross-Linked Envelope-related Markers for Squamous Differentiation in Human Lung Cancer Cell Lines
Lung carcinoma cell lines were analyzed in culture and in nude mouse xenograft for both morphological appearance and expression of specific proteins that participate in cross-linked envelope formation during normal squamous cell terminal differentiation. Cross-linked envelope formation, induced by artificial influx of millimolar ( :r* into the cultured cells, was an exclusive trait of squamous, adenosquamous, and mucoepidermoid carcinomas. Small
Mark L. Levitt; Adi F. Gazdar; Herbert K. Oie; Hildegarde Schuller; Scott M. Thacher
Determination of the cross-linking effect of adipic acid dihydrazide on glycoconjugate preparation
The cross-linking effect of adipic acid dihydrazide (ADH) on polysaccharide derivatization can be evaluated by applying combination of elemental analysis and colorimetric assay. Elemental analysis is used for estimation of total ADH bound to polysaccharide and a colorimetric trinitrobenzene sulfonic acid assay is used to determine the part of ADH not involved in cross-linking. The difference of values expressed as
Slavom�r Bystrick�; Eva Machov�; Anna Malov�kov�; Grigorij Kogan
A novel cross-linked human amniotic membrane for corneal implantations.
The aim of this study was to evaluate the efficacy of Al2(SO4)3 cross-linked human amniotic membrane for ocular surface reconstruction using tissue culture techniques. The human amniotic membrane was cross-linked with Al2(SO4)3, and the cross-linked human amniotic membrane was characterized for its mechanical properties, percentage of swelling in water, sterility, infrared spectroscopy and scanning electron microscopy. The potential of cross-linked human amniotic membrane to support the attachment and proliferation of corneal limbal epithelial cells was assessed in vitro, using static culture system. About 125% increase in the tensile strength was observed in the cross-linked human amniotic membrane compared to human amniotic membrane. Infrared spectroscopy studies have confirmed the cross-linking of human amniotic membrane with Al2(SO4)3. The cross-linked human amniotic membrane was found to be sterile up to 1 year. In culture studies, confluent sheets of epithelial cells were seen at the end of 14th day resembling the morphological features of limbal epithelia. The cross-linked human amniotic membrane has exhibited improved mechanical properties, and the tissue culture studies have shown its feasibility to be used as a limbal transplant. It was concluded that the crosslinked human amniotic membrane with its improved mechanical properties could be used on par with human amniotic membrane. PMID:23662337
Sekar, S; Sasirekha, K; Krishnakumar, S; Sastry, T P
A facile microwave irradiation approach that results in a cross-linking reaction of poly (vinyl alcohol) (PVA) with metallic and bimetallic systems is described. Nanocomposites of PVA cross-linked metallic systems such as Pt, Cu, and In and bimetallic systems such as Pt-In, Ag-P...
A facile method utilizing microwave irradiation is described that accomplishes the cross-linking reaction of PVA with metallic and bimetallic systems. Nanocomposites of PVA-cross-linked metallic systems such as Pt, Cu, and In and bimetallic systems such as Pt-In, Ag-Pt, Pt-Fe, Cu...
Measurement of cross linked fibrin derivatives in plasma: an immunoassay using monoclonal antibodies
Fibrinogen degradation, fibrin polymerisation, and the insertion of cross links into fibrin by fibrin stabilising factor lead to the appearance of new antigenic determinants. Antibodies against these antigenic sites may react specifically with the derivatives but not with the parent molecules. We have utilised a monoclonal antibody, which interacts with the cross linked fragment D dimer and related high molecular
A N Whitaker; M J Elms; P P Masci; P G Bundesen; D B Rylatt; A J Webber; I H Bunce
Method of Cross-Linking Polyvinyl Alcohol and Other Water Soluble Resins.
National Technical Information Service (NTIS)
A self-supporting sheet structure comprising a water soluble, non-cross-linked polymer, such as polyvinyl alcohol, is reported which is capable of being cross-linked by reaction with hydrogen atom radicals and hydroxyl molecule radicals in an aqueous solu...
D. W. Sheibley W. H. Philipp L. C. Hsu
Use of the photoaffinity cross-linking agent N-hydroxysuccinimidyl-4-azidosalicylic acid to characterize salivary-glycoprotein-bacterial interactions.
The present study has utilized the iodinatable cross-linking agent N-hydroxysuccinimidyl-4-azidosalicylic acid (ASA) to examine the specific interaction between the proline-rich glycoprotein (PRG) of human parotid saliva and Streptococcus sanguis G9B. The binding of 125I-ASA-PRG to Streptococcus sanguis G9B displayed saturation kinetics, reversibility and was inhibited by unlabelled PRG. Inhibition studies with other glycoproteins and saccharides indicated that binding was mediated by a bacterial adhesin with specificity towards N-acetylneuraminic acid, galactose, and N-acetylgalactosamine. After cross-linking, the 125I-ASA-PRG-adhesin complex could be extracted with SDS and separated from uncoupled 125I-ASA-PRG by gel filtration on Sepharose CL-6B. Approx. 1% of the 125I-ASA-PRG was cross-linked to the bacterial surface. Examination of the 125I-ASA-PRG-adhesin complex by SDS/polyacrylamide-gel electrophoresis/fluorography on 5% -(w/v)-polyacrylamide gels revealed that PRG was bound to two bacterial components. These findings support our previous suggestion that human salivary glycoproteins can specifically interact with oral streptococci and that these interactions occur between the glycoprotein's carbohydrate units and lectin(s) on the bacterial cell surface.
Bergey, E J; Levine, M J; Reddy, M S; Bradway, S D; Al-Hashimi, I
pH-induced metal-ligand cross-links inspired by mussel yield self-healing polymer networks with near-covalent elastic moduli.
Growing evidence supports a critical role of metal-ligand coordination in many attributes of biological materials including adhesion, self-assembly, toughness, and hardness without mineralization [Rubin DJ, Miserez A, Waite JH (2010) Advances in Insect Physiology: Insect Integument and Color, eds J�r�me C, Stephen JS (Academic Press, London), pp 75-133]. Coordination between Fe and catechol ligands has recently been correlated to the hardness and high extensibility of the cuticle of mussel byssal threads and proposed to endow self-healing properties [Harrington MJ, Masic A, Holten-Andersen N, Waite JH, Fratzl P (2010) Science 328:216-220]. Inspired by the pH jump experienced by proteins during maturation of a mussel byssus secretion, we have developed a simple method to control catechol-Fe(3+) interpolymer cross-linking via pH. The resonance Raman signature of catechol-Fe(3+) cross-linked polymer gels at high pH was similar to that from native mussel thread cuticle and the gels displayed elastic moduli (G') that approach covalently cross-linked gels as well as self-healing properties. PMID:21278337
Holten-Andersen, Niels; Harrington, Matthew J; Birkedal, Henrik; Lee, Bruce P; Messersmith, Phillip B; Lee, Ka Yee C; Waite, J Herbert
Collagen cross-linking of skin in patients with amyotrophic lateral sclerosis
NASA Technical Reports Server (NTRS)
Collagen cross-links of skin tissue (left upper arm) from 11 patients with amyotrophic lateral sclerosis (ALS) and 9 age-matched control subjects were quantified. It was found that patients with ALS had a significant reduction in the content of an age-related, stable cross-link, histidinohydroxylysinonorleucine, that was negatively correlated with the duration of illness. The contents of sodium borohydride-reducible labile cross-links, dehydro-hydroxylysinonorleucine and dehydro-histidinohydroxymerodesmosine, were significantly increased and were positively associated with the duration of illness (r = 0.703, p less than 0.05 and r = 0.684, p less than 0.05, respectively). The results clearly indicate that during the course of ALS, the cross-linking pathway of skin collagen runs counter to its normal aging, resulting in a "rejuvenation" phenomenon of skin collagen. Thus, cross-linking of skin collagen is affected in ALS.
Ono, S.; Yamauchi, M.
Corneal changes following collagen cross linking and simultaneous topography guided photoablation with collagen cross linking for keratoconus
Purpose: To compare the outcome of Collagen cross-linking (CXL) with that following topography-guided customized ablation treatment (T-CAT) with simultaneous CXL in eyes with progressive keratoconus. Materials and Methods: This was a prospective, non-randomized single centre study of 66 eyes with progressive keratoconus. Of these, 40 eyes underwent CXL and 26 eyes underwent T-CAT + CXL. The refractive, topographic, tomographic and aberrometric changes measured at baseline, 1, 3 and 6 months post-operatively were compared between both groups. Results: After a mean follow-up of 7.7 � 1.3 months, the mean retinoscopic cylinder decreased by 1.02 � 3.16 D in the CXL group (P = 0.1) and 2.87 � 3.22 D in the T-CAT + CXL group (P = 0.04). The Best corrected visual acuity increased by 2 lines or more in 10% of eyes in the CXL group and in 23.3% of eyes in the T-CAT + CXL group. The mean steepest-K reduced by 0.40 � 3.71 D (P = 0.77) in the CXL group and by 2.91 � 2.01D (P = 0.03) in the T-CAT + CXL group. The sag factor and surface asymmetry index showed no significant change in the CXL group but reduced by 3.59 � 5.94 D (P = 0.01) and 0.72 � 1.18 (P = 0.02) respectively in the T-CAT + CXL group. There was a significant increase in the highest posterior corneal elevation in both groups (9.57 � 14.93 ? in the CXL group and 7.85 � 9.25 ? in the T-CAT + CXL group, P ? 0.001 for both). There was significantly greater reduction of mean coma (P < 0.001) and mean higher-order aberrations (P = 0.01) following T-CAT + CXL compared to CXL. Conclusions: CAT + CXL is an effective approach to confer biomechanical stability and to improve the corneal contour in eyes with keratoconus and results in better refractive, topographic and aberrometric outcomes than CXL alone.
Padmanabhan, Prema; Radhakrishnan, Aishwaryah; Venkataraman, Abinaya Priya; Gupta, Nidhi; Srinivasan, Bhaskar
Preparation and characterization of a magneto-polymeric nanocomposite: Fe 3O 4 nanoparticles in a grafted, cross-linked and plasticized poly(vinyl chloride) matrix
NASA Astrophysics Data System (ADS)
In this work two kind of materials: (1) grafted, cross-linked and plasticized poly(vinyl chloride) (PVC) "plastic films" and (2) magnetic plastic films "magneto-polymeric nanocomposites" were prepared. Precursor solutions or "plastisols" used to obtain the plastic films were obtained by mixing PVC (emulsion grade) as polymeric matrix, di(2-ethylhexyl)phthalate (DOP) as plasticizer, a thermal stabilizer based in Ca/Zn salts, and a cross-linking agent, 3-mercaptopropyltrimethoxysilane (MTMS) or 3-aminopropyltriethoxysilane (ATES), at several concentrations. Flexible films were obtained from the plastisols using static casting. The stress-strain behavior and the gel content (determined by Soxhlet extraction with boiling THF) of the flexible films were measured in order to evaluate the effect of the cross-linking agent and their content on the degree of cross-linking. The magneto-polymeric nanocomposites were obtained by mixing the optimum composition of the plastisols (analyzed previously) with magnetite (Fe 3O 4)-based ferrofluid and DOP. Later, flexible films were obtained by static casting of the plastisol/ferrofluid systems. The magnetic films were characterized by the above-mentioned techniques and X-ray diffraction, vibrating sample magnetometry and thermogravimetrical analysis.
Rodr�guez-Fern�ndez, Oliverio S.; Rodr�guez-Calzad�az, C. A.; Y��ez-Flores, Isaura G.; Montemayor, Sagrario M.
The role of bridging ligands in determining DNA-binding ability and cross-linking patterns of dinuclear platinum(II) antitumour complexes.
The DNA binding ability and binding mode of platinum complexes are crucial factors that govern their cytotoxic activity. In this work, circular dichroism spectroscopy, gel electrophoresis and MALDI-TOF MS spectrometry combined with enzymatic degradation have been used to elucidate the role of bridging ligands in DNA-binding ability and cross-linking patterns of two dinuclear antitumour active platinum(II) complexes, {[cis-Pt(NH(3))(2)Cl](2)L1}(NO(3))(2) (1, L1= 4,4'-methylenedianiline) and {[cis-Pt(NH(3))(2)Cl](2)L2}(NO(3))(2) (2, L2 = alpha,alpha'-diamino-p-xylene). Although both complexes have two cis-diammine-Pt(II) moieties (1,1/c,c), complex 1 exhibits much higher DNA-binding ability than complex 2. The former readily forms both 1,3- and 1,4-intrastrand cross-links with DNA oligonucleotides, while the latter preferentially forms 1,4- rather than 1,3-intrastrand cross-links. Cytotoxicity studies against a human non-small-cell lung cancer cell line (A549) demonstrate that complex 1 has higher activity than 2. These results show that the linker properties play a critical role in controlling the DNA-binding and cross-linking abilities and in modulating the cytotoxicity of dinuclear platinum complexes. PMID:20023919
Zhu, Jianhui; Lin, Miaoxin; Fan, Damin; Wu, Ziyi; Chen, Yuncong; Zhang, Junfeng; Lu, Yi; Guo, Zijian
Hyaluronan and phospholipid association in biolubrication.
It is becoming increasingly clear that the outstanding lubrication of synovial joints is achieved by a sophisticated hierarchical structure of cartilage combined with synergistic actions of surface-active components present in the synovial fluid. In this work we focus on the association of two components of the synovial fluid, hyaluronan and dipalmitoyl phosphatidyl choline (DPPC), in bulk solution and at interfaces. We demonstrate that hyaluronan associates with DPPC vesicles and adsorbs to supported DPPC bilayers. The association structures formed at the interface are sufficiently stable to allow sequential adsorption of DPPC and hyaluronan, whereby promoting the formation of thick composite layers of these two components. The lubricating ability of such composite layers was probed by the AFM colloidal probe technique and found to be very favorable with low friction coefficients and high load bearing capacity. With DPPC as the last adsorbed component, a friction coefficient of 0.01 was found up to pressures significantly above what is encountered in healthy synovial joints. Hyaluronan as the last added component increases the friction coefficient to 0.03 and decreases the load bearing capacity somewhat (but still above what is needed in the synovial joint). Our data demonstrate that self-assembly structures formed by hyaluronan and phospholipids at interfaces are efficient aqueous lubricants, and it seems plausible that such self-assembly structures contribute to the exceptional lubrication of synovial joints. PMID:24171653
Wang, Min; Liu, Chao; Thormann, Esben; D?dinait?, Andra
Multiple phases of protien gels
A multiple phase transition was observed in gels made by covalently cross-linking proteins in either native or denatured state. The enzymatic activity of the gels prepared from native alpha-chymotrypsin was determined for each of the multiple phases. The reversibility of the swelling degrees and the enzymatic reaction rates upon phase transition suggests that the protein is at a free energy
Masahiko Annaka; Toyoichi Tanaka
The effect of cross-linking on latex film formation
NASA Astrophysics Data System (ADS)
The effect of the crosslinker chain lengths in the copolymerization reaction with n-BMA and the formation of the resulting latex films was investigated. Three crosslinkers; ethylene glycol dimethacrylate (EGDMA-198 g/mol), Aliphatic Urethane Acrylate (AUA-3728 g/mol) and a novel Macromonomer Crosslinker (Mac-3480 g/mol) were compared. Copolymerization was achieved via a miniemulsion process. The amount of the crosslinkers employed in the copolymers was 0.2 mol%. When Mac was used as the crosslinker in the latex, high toughness was retained which is comparable to the toughness of the PBMA homopolymer latex films. The P(BMA-co-Mac) films contained 60% gel (crosslinked network) prepared using persulfate (KPS, water-soluble) to >95% gel prepared using azo (AMBN, oil-soluble) initiators. For the P(BMA-co-AUA) copolymer latex films prepared using either of the initiators, no appreciable toughness was recorded at all. For the P(BMA-co-EGDMA) copolymer latex films, a dependence on the type of initiator was obtained. The persulfate initiator gave P(BMA-co-EGDMA) copolymer latex films appreciable toughness while the AMBN initiator gave brittle films. These copolymer latexes have gel contents >95%. In films derived from crosslinked latex particles, the interface or the continuous matrix (continuum) of the film is crucial for the film to be useful mechanically. The toughness in the PBMA homopolymer and P(BMA-co-Mac) copolymer latex films, was due to the long polymer linear chains that can interdiffused and entangled with chains in the adjacent neighboring particles. The network in P(BMA-co-Mac) copolymer latex films, are flexible and are able to deform upon load application such that these films have high extensibility. Lightly crosslinked PBMA polymer chains which are attached to the network chains may exist at the interface of the particles rendering the P(BMA-co-EGDMA) copolymer films the toughness as observed for the persulfate, and not in the AMBN-initiated system. Phase separations were postulated to occur during the synthesis of the copolymers. The phase separation is hypothesized to determine the particle morphology and hence the resulting film morphology and therefore the film properties.
Mohd. Ghazaly, Hafsah
Isocyanate Cross-Linked Silica: Structurally Strong Aerogels
NASA Technical Reports Server (NTRS)
Molecular-level synergism between the silica nanoparticles of pre-formed monoliths and molecular cross-linkers inverts the relative host-guest roles in glass-polymer composites, leading to new strong low-density materials. Attempts to load gels with variable amounts of polyurethane precursors such as di-ISO and diol end-capped polybutylene adipate followed by heat treatment, washing, and supercritical drying led to opaque materials, somewhat stronger than silica but still quite brittle and much inferior to the materials described above. Direct mixing of a diisocyanate and an alcohol-free sol has been attempted recently by Yim et al. Reportedly, that procedure leads to week-long gelation times and requires an at least equally long aging period. In our attempt to add various amounts of di-ISO in a base-catalyzed sol in PC, we also noticed a week-long gelation time. The resulting aerogels were translucent but no less brittle than native silica. According to more recent studies, if propylene carbonate is replaced with acetone, it leads not only to shorter processing times, but also to much stronger gels that can tolerate loads in excess of 40 kg in the arrangement presented. We attribute that behavior to the lower viscosity of acetone, that allows faster diffusion of the di-ISO solution within the pores before di-ISO has time to react with the surface of silica. Further studies are underway to vary the chemical identity of the diisocyanate, as well as the composition and density of silica.
Leventis, Nicholas; Sotiriou-Leventis, Chariklia; Zhang, Guo-Hui; Rawashdeh, Abdel-Monem M.
Formation of DNA-protein cross-links between gamma-hydroxypropanodeoxyguanosine and EcoRI.
The toxicity of acrolein, an alpha,beta-unsaturated aldehyde produced during lipid peroxidation, is attributable to its high reactivity toward DNA and cellular proteins. The major acrolein-DNA adduct, gamma-hydroxypropano-2'-deoxyguanosine (gamma-HOPdG), ring opens to form a reactive N(2)-oxopropyl moiety that cross-links to DNA and proteins. We demonstrate the ability of gamma-HOPdG in a duplex oligonucleotide to cross-link to a protein (EcoRI) that specifically interacts with DNA at a unique sequence. The formation of a cross-link to EcoRI was dependent on the intimate binding of the enzyme to its gamma-HOPdG-modified recognition site. Interestingly, the cross-link did not restrict the ability of EcoRI to cleave DNA substrates. However, stabilization of the cross-link by reduction of the Schiff base linkage resulted in loss of enzyme activity. This work indicates that the gamma-HOPdG-EcoRI cross-link is in equilibrium with free oligonucleotide and enzyme. Reversal of cross-link formation allows EcoRI to effect enzymatic cleavage of competitor oligonucleotides. PMID:18690724
VanderVeen, Laurie A; Harris, Thomas M; Jen-Jacobson, Linda; Marnett, Lawrence J
Formation of DNA-Protein Cross-Links Between ?-Hydroxypropanodeoxyguanosine and EcoRI
The toxicity of acrolein, an ?,?-unsaturated aldehyde produced during lipid peroxidation, is attributable to its high reactivity toward DNA and cellular proteins. The major acrolein?DNA adduct, ?-hydroxypropano-2?-deoxyguanosine (?-HOPdG), ring opens to form a reactive N2-oxopropyl moiety that cross-links to DNA and proteins. We demonstrate the ability of ?-HOPdG in a duplex oligonucleotide to cross-link to a protein (EcoRI) that specifically interacts with DNA at a unique sequence. The formation of a cross-link to EcoRI was dependent on the intimate binding of the enzyme to its ?-HOPdG-modified recognition site. Interestingly, the cross-link did not restrict the ability of EcoRI to cleave DNA substrates. However, stabilization of the cross-link by reduction of the Schiff base linkage resulted in loss of enzyme activity. This work indicates that the ?-HOPdG?EcoRI cross-link is in equilibrium with free oligonucleotide and enzyme. Reversal of cross-link formation allows EcoRI to effect enzymatic cleavage of competitor oligonucleotides.
Reactive, multifunctional polymer films through thermal cross-linking of orthogonal click groups.
The ability to produce robust and functional cross-linked materials from soluble and processable organic polymers is dependent upon facile chemistries for both reinforcing the structure through cross-linking and for subsequent decoration with active functional groups. Generally, covalent cross-linking of polymeric assemblies is brought about by the application of heat or light to generate highly reactive groups from stable precursors placed along the chains that undergo coupling or grafting reactions. Typically, these strategies suffer from a general lack of control of the cross-linking chemistry as well as the fleeting nature of the reactive species that precludes secondary chemistry. We have addressed both of these issues using orthogonal chemistries to effect both cross-linking and subsequent functionalization of polymer films by mild heating, which results in exacting control of the cross-link density as well as the density of the residual stable functional groups available for subsequent, stepwise functionalization. This methodology is exploited to develop a strategy for the independent and orthogonal triple-functionalization of cross-linked polymer thin-films through microcontact printing. PMID:21919513
Spruell, Jason M; Wolffs, Martin; Leibfarth, Frank A; Stahl, Brian C; Heo, Jinhwa; Connal, Luke A; Hu, Jerry; Hawker, Craig J
Surface cross-linked UHMWPE can enable the use of larger femoral heads in total joints.
Limiting cross-linking to the articular surfaces of ultrahigh molecular weight polyethylene (UHMWPE) to increase wear resistance while preventing detrimental effects of cross-linking on mechanical strength has been a desirable goal. A surface cross-linked UHMWPE can be achieved by blending UHMWPE with a free radical scavenger, such as vitamin E, consolidating the blend into an implant shape, extracting the vitamin E from the surface, and radiation cross-linking the surface extracted blend. This process results in high cross-link density in the vitamin E-depleted surface region because vitamin E hinders cross-linking during irradiation. In this study, we described the properties of successful extraction media and the manipulation of the wear and mechanical properties of extracted, irradiated blends. We showed that these formulations could have similar wear and significantly improved mechanical properties compared to currently available highly cross-linked UHMWPEs. We believe that these materials can enable thinner implant forms and more anatomical designs in joint arthroplasty and may provide a feasible alternative to metal-on-metal implants. PMID:22847503
Oral, Ebru; Neils, Andrew L; Lyons, Chelsea; Fung, Mitchell; Doshi, Brinda; Muratoglu, Orhun K
Synthesis and Characterization of Cross-linked Polymer Electrolyte Membranes for Supercapacitor
NASA Astrophysics Data System (ADS)
Cross-linked polyvinyl alcohol (PVA) electrolyte membranes have been synthesized by using a solution casting method. In this study, PVA was blended with oxidative cross-linked agent (zinc acetate) and nano-sized silica as filler to stabilize PVA matrix and enhance conductivity. The cross-linked membranes were immersed into lithium hydroxide (LiOH) aqueous solution to increase their ionic conductivity. Two techniques were used to characterize the resulted membranes including Fourier transform infra red (FTIR) and AC impedance spectroscopies. The results showed that absorption peaks of C-O-C group and Si-O-Si are presence in the FTIR spectra attributed to the cross-linking process. Impedance spectra indicated that the contribution of ionic dopant (LiOH) to enhance conductivity is insignificant. The highest conductivity of the studied cross-linked PVA membrane is 1.34�10-3 S cm-1 corresponding to 5% LiOH dopant concentration of cross-linked PVA-zinc acetate-nano silica membrane. The present study also suggested that the solution casting is appropriate for cross-linked membrane synthesis.
Rosi, Memoria; Ekaputra, Muhamad Prama; Abdullah, Mikrajuddin; Khairurrijal
DNA cross-linking by intermediates in the mitomycin activation cascade
The authors have assayed the cross-linking of oligonucleotides containing repeated mitomycin-reactive CpG sites in order to assess the factors that enhance activation of the carbamoyl function at C{sub 10}, yielding efficient mitomycin cross-linking. Drugs studied include mitomycin C (MC), N-methylmitomycin A (NMA), and the aziridinomitosene of NMA (MS). Drugs were reduced both by catalytic hydrogenation and by dithionite. They find that cross-linking by fully reduced NMA can be increased severalfold by addition of either excess dithionite reductant or the oxidant FeCl{sub 3}. Enhancement by FeCl{sub 3} is not seen with MC or MS, but excess dithionite increases cross-linking by all three compounds. They explain the action of Fe{sup 3+} by postulating production of the semiquinone of the monoadduct of mitomycin reacted at the C{sub 1}-position; according to this mechanism, departure of the carbamate from C{sub 10} is more efficient for the semiquinone than for the hydroquinone. However, the results imply that the hydroquinone can also function as a cross-linking agent. Excess dithionite beyond that required for stoichiometric reduction, activates the carbamate 2-3-fold for cross-linking. They find that the fully reduced leucoaziridinomitosene is highly unstable in solution, yet it produces efficient cross-linking. Hence, this compound is highly reactive in DNA alkylation and a good candidate for the role of primary alkylating agent.
Cera, C.; Egbertson, M.; Teng, S.P.; Crothers, D.M.; Danishefsky, S.J. (Yale Univ., New Haven, CT (USA))
Hyaluronan as an Immune Regulator in Human Diseases
Accumulation and turnover of extracellular matrix components are the hallmarks of tissue injury. Fragmented hyaluronan stimulates the expression of inflammatory genes by a variety of immune cells at the injury site. Hyaluronan binds to a number of cell surface proteins on a variety of cell types. Hyaluronan fragments signal through both Toll-like receptor (TLR) 4 and TLR2 as well as CD44 to stimulate inflammatory genes in inflammatory cells. Hyaluronan is also present on the cell surface of epithelial cells and provides protection against tissue damage by interacting with TLR2 and TLR4 on these parenchymal cells. Hyaluronan and hyaluronan-binding proteins regulate inflammation, tissue injury and repair through regulating inflammatory cell recruitment, release of inflammatory cytokines, and stem cell migration. This review focuses on the role of hyaluronan as an immune regulator in human diseases.
Differences between binding of one-chain and two-chain tissue plasminogen activators to non-cross-linked and cross-linked fibrin clots
Interaction of tissue plasminogen activator (t-PA) with fibrin plays a key role in regulation of plasminogen activation and clot dissolution. Previous investigations of t-PA-fibrin interaction, using incorporation of t-PA into polymerizing fibrin clots, have suggested that no significant differences exist in the binding of one-chain or two-chain t-PA to non-cross-linked or cross-linked fibrin. In the present study, binding of 125I-labeled and affinity-purified one-chain and two-chain forms of t-PA to preformed non-cross-linked or cross-linked, sonicated suspension of fibrin was investigated. Interaction of one-chain t-PA with cross-linked fibrin involved a single type of binding site with dissociation constant (kd) of 0.58 mumol/L and a stoichiometry (n) of 1.5. Interaction of one-chain t-PA with non-cross-linked fibrin, however, involved two classes of binding sites with dissociation constants of 0.32 and 1.5 mumol/L and corresponding number of binding sites equal to 0.57 and 2.0, respectively. In contrast to the binding of one-chain t-PA to cross-linked fibrin by a limited number of sites, two-chain t-PA appeared to involve a considerably greater number of sites (minimum six) whose dissociation constant was 3.2 mumol/L. Interaction of two-chain t-PA with non-cross-linked fibrin also showed the presence of many binding sites (minimum seven) with approximate dissociation constant of 6.4 mumol/L, as well as a few (n = 0.012) high-affinity sites with a kd of 0.011 mumol/L epsilon-Aminocaproic acid did not completely reverse the binding of either one-chain t-PA or two-chain t-PA to fibrin. The present findings suggest that the fibrin-binding properties of t-PA undergo considerable changes on proteolytic conversion from one-chain to two-chain t-PA, catalyzed under physiologic conditions by plasmin.
Husain, S.S.; Hasan, A.A.; Budzynski, A.Z. (Temple Univ. School of Medicine, Philadelphia, PA (USA))
Quantification of Protein-Protein Interactions with Chemical Cross-Linking and Mass Spectrometry
Chemical cross-linking in combination with mass spectrometry has largely been used to study protein structures and protein�protein interactions. Typically, it is used in a qualitative manner to identify cross-linked sites and provide a low-resolution topological map of the interacting regions of proteins. Here, we investigate the capability of chemical cross-linking to quantify protein�protein interactions using a model system of calmodulin and substrates melittin and mastoparan. Calmodulin is a well-characterized protein which has many substrates. Melittin and mastoparan are two such substrates which bind to calmodulin in 1:1 ratios in the presence of calcium. Both the calmodulin�melittin and calmodulin�mastoparan complexes have had chemical cross-linking strategies successfully applied in the past to investigate topological properties. We utilized an excess of immobilized calmodulin on agarose beads and formed complexes with varying quantities of mastoparan and melittin. Then, we applied disuccinimidyl suberate (DSS) chemical cross-linker, digested and detected cross-links through an LC-MS analytical method. We identified five interpeptide cross-links for calmodulin�melittin and three interpeptide cross-links for calmodulin�mastoparan. Using cross-linking sites of calmodulin�mastoparan, we demonstrated that mastoparan also binds in two orientations to calmodulin. We quantitatively demonstrated that both melittin and mastoparan preferentially bind to calmodulin in a parallel fashion, which is opposite to the preferred binding mode of the majority of known calmodulin binding peptides. Wealso demonstrated that the relative abundances of cross-linked peptide products quantitatively reflected the abundances of the calmodulin peptide complexes formed.
Chavez, Juan D.; Liu, Neal L.; Bruce, James E.
Silk cocoon of Bombyx mori: proteins and posttranslational modifications--heavy phosphorylation and evidence for lysine-mediated cross links.
Although silk is used to produce textiles and serves as a valuable biomaterial in medicine, information on silk proteins of the cocoon is limited. Scanning electron microscopy was applied to morphologically characterise the sample and the solubility of cocoon in lithium thiocyanate and 2-DE was carried out with multi-enzyme in-gel digestion followed by MS identification of silk-peptides. High-sequence coverage of the silk cocoon proteins fibroin light and heavy chain, sericins and fibrohexamerins was revealed and PTMs as heavy phosphorylation of silk fibroin heavy chain; lysine hydroxylation and Lys->allysine formation have been observed providing evidence for lysine-mediated cross linking of silk as found in collagens, which has not been reported so far. Tyrosine oxidation verified the presence of di-tyrosine cross links. A high degree of sequence conflicts probably representing single-nucleotide polymorphisms was observed. PTM and sequence conflicts may be modulating structure and physicochemical properties of silk. PMID:20029844
Chen, Wei-Qiang; Priewalder, Helga; John, Julius Paul Pradeep; Lubec, Gert
Retention of the original LLC structure in a cross-linked poly(ethylene glycol) diacrylate hydrogel with reinforcement from a silica network.
Cross-linked poly(ethylene glycol) diacrylate (PEGDA) hydrogels with uniformly controlled nanoporous structures templated from hexagonal lyotropic liquid crystals (LLC) represent separation membrane materials with potentially high permeability and selectivity due to their high pore density and narrow pore size distribution. However, retaining LLC templated nanostructures is a challenge as the polymer gels are not strong enough to sustain the surface tension during the drying process. In the current study, cross-linked PEGDA gels were reinforced with a silica network synthesized via an in situ sol-gel method, which assists in the retention of the hexagonal LLC structure. The silica precursor does not obstruct the formation of hexagonal phases. After surfactant removal and drying, these hexagonal structures in samples with a certain amount of tetraethoxysilane (TEOS) loading are well retained while the nanostructures are collapsed in samples without silica reinforcement, leading to the hypothesis that the reinforcement provided by the silica network stabilizes the LLC structure. The study examines the conditions necessary for a sufficient and well dispersed silica network in PEGDA gels that contributes to the retention of original LLC structures, which potentially enables broad applications of these gels as biomedical and membrane materials. PMID:24916196
Zhang, Juan; Xie, Zongli; Hoang, Manh; Hill, Anita J; Cong, Weiwei; She, Feng Hua; Gao, Weimin; Kong, Ling Xue
Ingested hyaluronan moisturizes dry skin
Hyaluronan (HA) is present in many tissues of the body and is essential to maintain moistness in the skin tissues, which contain approximately half the body�s HA mass. Due to its viscosity and moisturizing effect, HA is widely distributed as a medicine, cosmetic, food, and, recently marketed in Japan as a popular dietary supplement to promote skin moisture. In a randomized, double-blind, placebo-controlled clinical study it was found that ingested HA increased skin moisture and improved treatment outcomes for patients with dry skin. HA is also reported to be absorbed by the body distributed, in part, to the skin. Ingested HA contributes to the increased synthesis of HA and promotes cell proliferation in fibroblasts. These effects show that ingestion of HA moisturizes the skin and is expected to improve the quality of life for people who suffer from dry skin. This review examines the moisturizing effects of dry skin by ingested HA and summarizes the series of mechanisms from absorption to pharmacological action.
Revisiting glutaraldehyde cross-linking: the case of the Arg-Lys intermolecular doublet
In addition to the common use of glutaraldehyde to nonspecifically cross-link protein crystals through lysine residues disposed on the surface of the protein, the use of gentle vapour diffusion of glutaraldehyde offers a convenient way to limit polymerization and to allow slow diffusion throughout the crystal. In the case of trimeric barnase crystals, a specific cross-link was observed between an lysine side chain and an arginine side chain that were spatially disposed at the ideal distance on the protein surface in the three monomers. Here, the direct observation of a specific Lys�Arg cross-link site is reported and a mechanism is proposed for the reaction.
Salem, Michele; Mauguen, Yves; Prange, Thierry
A diiron protein autogenerates a valine-phenylalanine cross-link.
All known internal covalent cross-links in proteins involve functionalized groups having oxygen, nitrogen, or sulfur atoms present to facilitate their formation. Here, we report a carbon-carbon cross-link between two unfunctionalized side chains. This valine-phenyalanine cross-link, produced in an oxygen-dependent reaction, is generated by its own carboxylate-bridged diiron center and serves to stabilize the metallocenter. This finding opens the door to new types of posttranslational modifications, and it demonstrates new catalytic potential of diiron centers. PMID:21596985
Cooley, Richard B; Rhoads, Timothy W; Arp, Daniel J; Karplus, P Andrew
Structural, mechanical and osmotic properties of injectable hyaluronan-based composite hydrogels
The osmotic and scattering properties of hyaluronan-based composite hydrogels composed of stiff biopolymer chains (carboxymethylated thiolated hyaluronan (CMHA-S)) crosslinked by a flexible polymer (polyethylene glycol diacrylate (PEGDA)) are investigated and analyzed in terms of the scaling theory. The total pre-gel polymer weight concentration is varied between 0.5 wt.% and 3.2 wt.%, while the mole ratio between the reactive PEG chain ends and the thiolated HA moieties is changed between 0.15 and 1.0. The shear modulus G of the fully swollen gels exhibits a stronger dependence on pre-gel concentration than on the crosslink density. Osmotic deswelling measurements reveal that the osmotic mixing pressure depends on the weight ratio CMHA-S/PEGDA, and is practically unaffected by the pre-gel concentration. Small-angle neutron scattering observations indicate that the thermodynamic properties of these composite gels are governed by total polymer concentration, i.e., specific interactions between the two polymeric components do not play a significant role.
Horkay, Ferenc; Magda, Jules; Alcoutlabi, Mataz; Atzet, Sarah; Zarembinski, Thomas
Effector-sensitive cross-linking of phosphorylase b kinase by the novel cross-linker 4-phenyl-1,2,4-triazoline-3,5-dione.
The dienophile 4-phenyl-1,2,4-triazoline-3,5-dione (PTD) was identified as a novel protein cross-linker, and utilized as a conformational probe of phosphorylase b kinase (PhK), a hexadecameric enzyme with the subunit composition (alphabetagammadelta)4. In its reaction with this enzyme, PTD produced five major cross-linked conjugates as resolved by denaturing gel electrophoresis: alphabeta, betagammagamma, alphagamma and a doublet of differently migrating homodimers, betabeta1 and betabeta2. Cross-linking in the presence of six different activators of the kinase targeted to its various subunits caused substantial changes in the amounts of three of the conjugates. The formation of alphagamma was increased by all of the activators but the largest enhancement was caused by exogenous Ca2+/calmodulin. All except one of the activators decreased the amount of betagammagamma formed, with Mg2+ having the greatest effect, and all except two increased the amount of betabeta1, with Mg2+ again having the largest influence. From the overall similarity of the changes in cross-linking by PTD induced by the various activators, we conclude that, even though they are targeted to different sites and subunits, they induce activated conformations of PhK that have certain structural features in common. Regarding the mechanism of cross-linking by PTD, its reaction with a model nucleophile suggests that its initial reaction with a side chain nucleophile of PhK involves a 1,4-conjugate addition to form a urazole adduct, with the secondary cross-linking reaction occurring through an as yet unknown pathway.
Ayers, N A; Nadeau, O W; Read, M W; Ray, P; Carlson, G M
Effector-sensitive cross-linking of phosphorylase b kinase by the novel cross-linker 4-phenyl-1,2,4-triazoline-3,5-dione.
The dienophile 4-phenyl-1,2,4-triazoline-3,5-dione (PTD) was identified as a novel protein cross-linker, and utilized as a conformational probe of phosphorylase b kinase (PhK), a hexadecameric enzyme with the subunit composition (alphabetagammadelta)4. In its reaction with this enzyme, PTD produced five major cross-linked conjugates as resolved by denaturing gel electrophoresis: alphabeta, betagammagamma, alphagamma and a doublet of differently migrating homodimers, betabeta1 and betabeta2. Cross-linking in the presence of six different activators of the kinase targeted to its various subunits caused substantial changes in the amounts of three of the conjugates. The formation of alphagamma was increased by all of the activators but the largest enhancement was caused by exogenous Ca2+/calmodulin. All except one of the activators decreased the amount of betagammagamma formed, with Mg2+ having the greatest effect, and all except two increased the amount of betabeta1, with Mg2+ again having the largest influence. From the overall similarity of the changes in cross-linking by PTD induced by the various activators, we conclude that, even though they are targeted to different sites and subunits, they induce activated conformations of PhK that have certain structural features in common. Regarding the mechanism of cross-linking by PTD, its reaction with a model nucleophile suggests that its initial reaction with a side chain nucleophile of PhK involves a 1,4-conjugate addition to form a urazole adduct, with the secondary cross-linking reaction occurring through an as yet unknown pathway. PMID:9512471
Ayers, N A; Nadeau, O W; Read, M W; Ray, P; Carlson, G M
Entanglement Networks of 1,2-Polybutadiene Cross-Linked in States of Strain. I. Cross-Linking at 0 deg.
National Technical Information Service (NTIS)
Linear 1,2-polybudadiene is cross-linked by gamma-irradiation while strained in simple extension, with extension ratios from 1.2 to 2.0. After release, the sample retracts to a state of ease at room temperature. The molar concentrations of network strands...
O. Kramer R. L. Carpenter V. Ty J. D. Ferry
Fabrication of a novel pH-sensitive glutaraldehyde cross-linked pectin nanogel for drug delivery.
A novel pH-sensitive nanogel based on pectin cross-linked with glutaraldehyde (PT-GA) was designed and synthesized for drug delivery. Transmission electron microscope observation shows that the nano-sized gel particles exhibit a spherical morphology. The optical absorbance study of nanogel suspension reveals its pH sensitivity. Cytotoxicity study shows that the nanogel has no apparent inhibitory effect on cells. The in vitro drug-release behavior of the drug-loaded nanogel particles in three kinds of media, i.e., simulated gastric fluid, simulated intestine fluid and simulated colon fluid, was studied. PT-GA nanogel exhibits a faster release at a high pH, and the release could be further accelerated in the presence of pectinolytic enzyme, indicating that the nanogel may be used for colon-specific drug delivery. PMID:17988522
Chang, Cong; Wang, Zong-Chun; Quan, Chang-Yun; Cheng, Han; Cheng, Si-Xue; Zhang, Xian-Zheng; Zhuo, Ren-Xi
Reversible, self cross-linking nanowires from thiol-functionalized polythiophene diblock copolymers.
Poly(3-hexylthiophene)-block-poly(3-(3-thioacetylpropyl) oxymethylthiophene) (P3HT)-b-(P3TT) diblock copolymers were synthesized and manipulated by solvent-induced crystallization to afford reversibly cross-linked semiconductor nanowires. To cross-link the nanowires, we deprotected the thioacetate groups to thiols and they subsequently oxidized to disulfides. Cross-linked nanowires maintained their structural integrity in solvents that normally dissolve the polymers. These robust nanowires could be reduced to the fully solvated polymer, representing a novel, reversible cross-linking procedure for functional P3HT-based nanowire fibrils. Field-effect transistor measurements were carried out to determine the charge transport properties of these nanostructures. PMID:24735371
Hammer, Brenton A G; Reyes-Martinez, Marcos A; Bokel, Felicia A; Liu, Feng; Russell, Thomas P; Hayward, Ryan C; Briseno, Alejandro L; Emrick, Todd
Cross-linking proteins with bimetallic tetracarboxylate compounds of transition metals
Stable cross-linked complexes of transition-metal tetracarboxylates and proteins are formed. The preferred transition-metal is rhodium. The protein may be collagen or an enzyme such as a proteolytic enzyme.
Kostic, Nenad M. (Ames, IA); Chen, Jian (Ames, IA)
Cross-linking proteins with bimetallic tetracarboxylate compounds of transition metals
Stable cross-linked complexes of transition-metal tetracarboxylates and proteins are formed. The preferred transition-metal is rhodium. The protein may be collagen or an enzyme such as a proteolytic enzyme. No Drawings
Kostic, N.M.; Chen, J.
Determination of the cross-linking effect of adipic acid dihydrazide on glycoconjugate preparation.
The cross-linking effect of adipic acid dihydrazide (ADH) on polysaccharide derivatization can be evaluated by applying combination of elemental analysis and colorimetric assay. Elemental analysis is used for estimation of total ADH bound to polysaccharide and a colorimetric trinitrobenzene sulfonic acid assay is used to determine the part of ADH not involved in cross-linking. The difference of values expressed as molar ratios (per repeating unit) provides information on the amount of ADH involved in cross-linking the polysaccharides. Carboxymethylated polysaccharides were derivatized with different amounts of ADH to test the procedure. Analytical results showed that excess of ADH in the reaction only slightly decreased the cross-linking. The number of carboxyl groups remained unmodified even at high excess of ADH and high concentration of carbodiimide (EDC) coupling reagent. PMID:11003553
Bystrick�, S; Machov�, E; Malov�kov�, A; Kogan, G
Enhanced mechanical properties and blood compatibility of PDMS/liquid crystal cross-linked membrane materials.
A novel polydimethylsiloxane/liquid crystal cross-linked membrane (PDMS/LC) was prepared by using PDMS containing vinyl groups and LCs containing unsaturated linkages as matrix materials. Mechanical properties, liquid crystalline performance and blood compatibility of the PDMS/LC cross-linked membrane containing different LC contents and LC groups were investigated, respectively. The results showed that mechanical properties of the membrane increased more significantly than those of pure PDMS membranes. The PDMS/LC cross-linked membrane also possessed better membrane-forming ability, lower hemolysis rate, less platelets adhesion and more favorable anti-coagulant properties. Additionally, mechanical properties and blood compatibility of the membrane can be enhanced simultaneously and obviously due to the introduction of the cholesteric liquid crystals and the application of the preferred cross-linked reaction without byproducts. PMID:23453826
Rao, Huaxin; Zhang, Ziyong; Liu, Fanna
Inverted bulk-heterojunction solar cell with cross-linked hole-blocking layer
We have developed a hole-blocking layer for bulk-heterojunction solar cells based on cross-linked polyethylenimine (PEI). We tested five different ether-based cross-linkers and found that all of them give comparable solar cell efficiencies. The initial idea that a cross-linked layer is more solvent resistant compared to a pristine PEI layer could not be confirmed. With and without cross-linking, the PEI layer sticks very well to the surface of the indium�tin�oxide electrode and cannot be removed by solvents used to process PEI or common organic semiconductors. The cross-linked PEI hole-blocking layer functions for multiple donor�acceptor blends. We found that using cross-linkers improves the reproducibility of the device fabrication process.
Udum, Yasemin; Denk, Patrick; Adam, Getachew; Apaydin, Dogukan H.; Nevosad, Andreas; Teichert, Christian; S. White, Matthew.; S. Sariciftci, Niyazi.; Scharber, Markus C.
18. Physical Cross Links in Amorphous PET, Influence of Cooling Rate and Ageing
NASA Astrophysics Data System (ADS)
A Continuous Cooling Transformation (CCT) procedure can be used to distinguish the initial "state" of the amorphous PET samples produced upon solidification from the melt at different cooling rates. The material frozen at this stage behaves as a rubber when brought above the Tg due to the onset of physical cross links. The rubber is not a stable network, however, since physical cross links may eventually dissolve. Their size distribution, and possibly their number, depend on cooling rate and ageing. Some may be even stable above the glass transition and act as nuclei for further crystallization from the glass. Upon increasing cooling rate, size distribution becomes smaller and stability of physical cross links decreases. Ageing may give rise to the onset of further physical cross links or may affect the stability of the ones already set in depending on cooling rate, i.e. on how is constrained the initial network structure.
Piccarolo, S.; Vassileva, E.; Kiflie, Z.
Physiologically relevant oxidative degradation of oligo(proline) cross-linked polymeric scaffolds.
Chronic inflammation-mediated oxidative stress is a common mechanism of implant rejection and failure. Therefore, polymer scaffolds that can degrade slowly in response to this environment may provide a viable platform for implant site-specific, sustained release of immunomodulatory agents over a long time period. In this work, proline oligomers of varying lengths (P(n)) were synthesized and exposed to oxidative environments, and their accelerated degradation under oxidative conditions was verified via high performance liquid chromatography and gel permeation chromatography. Next, diblock copolymers of poly(ethylene glycol) (PEG) and poly(?-caprolactone) (PCL) were carboxylated to form 100 kDa terpolymers of 4%PEG-86%PCL-10%cPCL (cPCL = poly(carboxyl-?-caprolactone); i% indicates molar ratio). The polymers were then cross-linked with biaminated PEG-P(n)-PEG chains, where P(n) indicates the length of the proline oligomer flanked by PEG chains. Salt-leaching of the polymeric matrices created scaffolds of macroporous and microporous architecture, as observed by scanning electron microscopy. The degradation of scaffolds was accelerated under oxidative conditions, as evidenced by mass loss and differential scanning calorimetry measurements. Immortalized murine bone-marrow-derived macrophages were then seeded on the scaffolds and activated through the addition of ?-interferon and lipopolysaccharide throughout the 9-day study period. This treatment promoted the release of H(2)O(2) by the macrophages and the degradation of proline-containing scaffolds compared to the control scaffolds. The accelerated degradation was evidenced by increased scaffold porosity, as visualized through scanning electron microscopy and X-ray microtomography imaging. The current study provides insight into the development of scaffolds that respond to oxidative environments through gradual degradation for the controlled release of therapeutics targeted to diseases that feature chronic inflammation and oxidative stress. PMID:22017359
Yu, Shann S; Koblin, Rachel L; Zachman, Angela L; Perrien, Daniel S; Hofmeister, Lucas H; Giorgio, Todd D; Sung, Hak-Joon
Intra-molecular cross-linking of acidic residues for protein structure studies.
Intra-molecular cross-linking has been suggested as a method of obtaining distance constraints that would be useful in developing structural models of proteins. Recent work published on intra-molecular cross-linking for protein structural studies has employed commercially available primary amine selective reagents that can cross-link lysine residues to other lysine residues or the amino terminus. Previous work using these cross-linkers has shown that for several proteins of known structure, the number of cross-links that can be obtained experimentally may be small compared to what would be expected from the known structure, due to the relative reactivity, distribution, and solvent accessibility of the lysines in the protein sequence. To overcome these limitations we have investigated the use of cross-linking reagents that can react with other reactive sidechains in proteins. We used 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to activate the carboxylic acid containing residues, aspartic acid (D), glutamic acid (E), and the carboxy terminus (O), for cross-linking reactions. Once activated, the DEO sidechains can react to form 'zero-length' cross-links with nearby primary amine containing resides, lysines (K) and the amino terminus (X), via the formation of a new amide bond. We also show that the EDC-activated DEO sidechains can be cross-linked to each other using dihydrazides, two hydrazide moieties connected by an alkyl cross-linker ann of variable length. Using these reagents, we have found three new 'zero-length' cross-links in ubiquitin consistent with its known structure (M1-E16, M1-E18, and K63-E64). Using the dihydrazide cross-linkers, we have identified 2 new cross-links (D21-D32 and E24-D32) unambiguously. Using a library of dihydrazide cross-linkers with varying arm length, we have shown that there is a minimum arm length required for the DEO-DEO cross-links of 5.8 angstroms. These results show that additional structural information can be obtained by exploiting new cross-linker chemistry, increasing the probability that the protein target of choice will yield sufficient distance constraints to develop a structural model.
Kruppa, Gary Hermann; Young, Malin M.; Novak, Petr; Schoeniger, Joseph S.
Interpretation of experiments on ZDDP anti-wear films through pressure-induced cross-linking
We review a recently developed molecular-level theory for the formation and functionality of zinc dialkyldithiophosphate anti-wear films [N. J. Mosey, M. H. M�ser and T. K. Woo, Science 307 (2005) 1612]. This theory is based on the idea that pressure-induced cross-linking leads to chemically connected networks. The formation of cross-links modifies the mechanical properties of the films such that wear
N. J. Mosey; T. K. Woo; M. Kasrai; P. R. Norton; G. M. Bancroft; M. H. M�ser
Novel epoxy-based cross-linked polybenzimidazole for high temperature proton exchange membrane fuel cells
An approach has been proposed to prepare the reinforced phosphoric acid (PA) doped cross-linked polybenzimidazole membranes for high temperature proton exchange membrane fuel cells (HT-PEMFCs), using 1,3-bis(2,3-epoxypropoxy)-2,2-dimethylpropane (NGDE) as the cross-linker. FT-IR measurement and solubility test showed the successful completion of the crosslinking reaction. The resulting cross-linked membranes exhibited improved mechanical strength, making it possible to obtain higher phosphoric acid
Shuang Wang; Gang Zhang; Miaomiao Han; Hongtao Li; Yang Zhang; Jing Ni; Wenjia Ma; Mingyu Li; Jing Wang; Zhongguo Liu; Liyuan Zhang; Hui Na
Dynamic mechanical and thermal properties of cross-linked polystyrene\\/glass fiber composites
Cross-linked polystyrene\\/glass fiber composites were fabricated using cross-linked polystyrene (CLPS) as matrix and E-glass\\u000a fiber as the reinforcement. Surfaces of E-glass fibers were modified by vinyl triethoxysilane (VTES), vinyl trimethoxysilane\\u000a (VTMS) and ?-methacryloylpropyl trimethoxysilane (MPS). The treated glass fibers were analyzed by fourier transform infrared spectroscopy\\u000a (FTIR). Dynamic mechanical thermal analysis (DMTA) and thermo-gravimetric analysis (TGA) were employed to investigate
Quanyao Zhu; Fei Wu; Qing Yang; Jun Wang; Wen Chen
Constant c10 Ring Stoichiometry in the Escherichia coli ATP Synthase Analyzed by Cross-Linking?
The subunit c stoichiometry of Escherichia coli ATP synthase was studied by intermolecular cross-linking via oxidation of bi-cysteine-substituted subunit c (cA21C/cM65C). Independent of the carbon source used for growth and independent of the presence of other FoF1 subunits, an equal pattern of cross-link formation stopping at the formation of decamers was obtained.
Ballhausen, Britta; Altendorf, Karlheinz; Deckers-Hebestreit, Gabriele
Chemical and thermal cross-linking of collagen and elastin hydrolysates
Chemical and thermal cross-linking of collagen soluble in acetic acid and elastin hydrolysates soluble in water have been studied. Solutions of collagen and elastin hydrolysates were treated using variable concentrations of 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Moreover, diepoxypropylether (DEPE) has been used as cross-linking agent. Films made of collagen and elastin hydrolysates were also treated with temperature at
A. Sionkowska; J. Skopinska-Wisniewska; M. Gawron; J. Kozlowska; A. Planecka
Monodisperse and multilayer core�shell latex via surface cross-linking emulsion polymerization
We describe a method for the synthesis of phase-separated polymer latex with a monodisperse and multilayer core�shell (MMLCS) morphology by surface cross-linking emulsion polymerization. Poly(butyl acrylate)(PBA) was used for the seed and the core of the latex, the inner shell was poly(butyl acrylate-styrene) cross-linked with divinylbenzene (DVB) to avoid phase inversion, and the poly(methyl methacrylate-butyl acrylate-glycidyl methacrylate) was the outer
Yuanchun Mu; Teng Qiu; Xiaoyu Li
Controlled degradation of hydrogels using multi-functional cross-linking molecules
Hydrogels, chemically cross-linked or physically entangled, have found a number of applications as novel delivery vehicles of drugs and cells. However, the narrow ranges of degradation rates and mechanical strength currently available from many hydrogels limits their applications. We have hypothesized that utilization of multi-functional cross-linking molecules to form hydrogels could provide a wider range and tighter control over the
Kuen Yong Lee; Kamal H. Bouhadir; David J. Mooney
Bio-inspired high-performance and recyclable cross-linked polymers.
Bio-inspired molecular design and synthesis of high-performance and recyclable cross-linked polymers is reported. Reversible cross-links between hard segments are incorporated into linear segmented polyurethane via Diels-Alder reaction between maleimide pendant group and furan cross-linker. The materials form hierarchical structure and exhibit excellent properties with high stiffness, strength and toughness, and can be easily thermally reshaped and re-mended. PMID:23861235
Yu, Shen; Zhang, Rongchun; Wu, Qiang; Chen, Tiehong; Sun, Pingchuan
Polymer Networks with Slip-links: 2. Constitutive Equations for a Cross-linked Network
Stress�strain relations are derived for the mechanical response of elastomers at arbitrary three-dimensional deformations with finite strains. An elastomer is treated as an incompressible network of chains bridged by permanent (chemical cross-links and physical cross-links whose lifetime exceeds the characteristic time of deformation) and temporary (entanglements modeled as slip-links) junctions. Two types of chains are introduced in the network to
A. D. Drozdov
An antimicrobial zinc based molecule for cross linking poly-acrylic acid
A system for incorporating antimicrobial zinc into polymeric materials, in particular hydrogel type polymers has been developed. Zn(Bipy-(MMOES)2) a zinc carboxylate monomer was designed with the purpose of mimicking commercial cross linking agents such as ethylene glycol diacrylate, as well as containing antimicrobial zinc ions (Zn2+), with the intention that it can be used to cross link into any polymeric
Charlotte James; Thomas Pugh; Andrew L. Johnson; A. Tobias A. Jenkins
Hyaluronan receptor-directed assembly of chondrocyte pericellular matrix
Initial assembly of extracellular matrix occurs within a zone immediately adjacent to the chondrocyte cell surface termed the cell- associated or pericellular matrix. Assembly within the pericellular matrix compartment requires specific cell-matrix interactions to occur, that are mediated via membrane receptors. The focus of this study is to elucidate the mechanisms of assembly and retention of the cartilage pericellular matrix proteoglycan aggregates important for matrix organization. Assembly of newly synthesized chondrocyte pericellular matrices was inhibited by the addition to hyaluronan hexasaccharides, competitive inhibitors of the binding of hyaluronan to its cell surface receptor. Fully assembled chondrocyte pericellular matrices were displaced using hyaluronan hexasaccharides as well. When exogenous hyaluronan was added to matrix-free chondrocytes in combination with aggrecan, a pericellular matrix equivalent in size to an endogenous matrix formed within 30 min of incubation. Addition of hyaluronan and aggrecan to glutaraldehyde-fixed chondrocytes resulted in matrix assembly comparable to live chondrocytes. These matrices could be inhibited from assembling by the addition of excess hyaluronan hexasaccharides or displaced once assembled by subsequent incubation with hyaluronan hexasaccharides. The results indicate that the aggrecanrich chondrocyte pericellular matrix is not only on a scaffolding of hyaluronan, but actually anchored to the cell surface via the interaction between hyaluronan and hyaluronan receptors.
Dynamic OCT measurements of corneal biomechanical properties after UV cross-linking in the rabbit
NASA Astrophysics Data System (ADS)
Structural properties of the cornea determine the shape and optical quality of the eye. Keratoconus, a structural degeneration of the cornea, is often treated with UV-induced collagen cross-linking to increase tissue resistance to further deformation and degeneration. Optimal treatments would be customized to the individual and consider preexisting structural properties as well as the effects induced by treatment and this requires the capability to noninvasively measure tissue properties. The purpose of this study is to use novel methods of optical elastography to study the effects of UV-induced corneal collagen cross-linking in the rabbit eye. Low-amplitude (<1?m) elastic flexural waves were generated using focused air-pulse stimulation. Elastic wave propagation was measured over a 10x10mm area using Phase Stabilized Swept Source Optical Coherence Elastography (PhS-SSOCE) with a sensitivity of ~ 10 nm. Wave amplitude and velocity were computed and compared in tissues before and after UV cross-linking. Wave amplitude was decreased by the cross-linking treatment, while wave velocity was greater in cross-linked tissue than it was in the untreated cornea. Decreased wave amplitude and increased wave velocity after cross-linking is consistent with increased tissue stiffness. This was confirmed by conventional mechanical tension testing. These results demonstrate that the combination of the PhS-SSOCE and focused air pulse stimulation is capable of measuring low amplitude tissue motion and quantifying corneal stiffness.
Twa, Michael D.; Li, Jiasong; Manapuram, Ravi K.; Menodiado, Floredes M.; Singh, Manmohan; Aglyamov, Salavat; Emelianov, Stanislav; Larin, Kirill V.
Facile fabrication of core cross-linked micelles by RAFT polymerization and enzyme-mediated reaction.
Polymeric micelles formed in aqueous solution by assembly of amphiphilic block copolymers have been extensively investigated due to their great potential as drug carriers. However, the stability of polymeric assembly is still one of the major challenges in delivering drugs to tissues and cells. Here, we report a facile route to fabricate core cross-linked (CCL) micelles using an enzymatic polymerization as the cross-linking method. We present synthesis of poly(ethylene glycol)-block-poly(N-isopropyl acrylamide-co-N-(4-hydroxyphenethyl) acrylamide) diblock copolymer PEG-b-P(NIPAAm-co-NHPAAm) via reversible addition-fragmentation chain transfer (RAFT) polymerization. The diblock copolymer was then self-assembled into non-cross-linked (NCL) micelles upon heating above the lower critical solution temperature (LCST), and subsequently cross-linked using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) as enzyme and oxidant. The characterization of the diblock copolymer and micelles were studied by NMR, DLS, UV-vis, and fluorescence spectroscopy. The fluorescence study reveals that the cross-linking process endows the micelles with much lower critical micelle concentration (CMC). In addition, the drug release study shows that the CCL micelles have lower release amount of doxorubicin (DOX) than the NCL micelles due to the enhanced stability of the CCL micelles by core cross-linking process. PMID:24768266
Wu, Yukun; Lai, Quanyong; Lai, Shuqi; Wu, Jing; Wang, Wei; Yuan, Zhi
Dimethyl suberimidate cross-linked pericardium tissue: Raman spectroscopic and atomic force microscopy investigations
NASA Astrophysics Data System (ADS)
Chemically stabilized pericardium tissue is widely used as a tissue-derived biomaterial for the preparation of bioprostheses such as heart valves or vascular grafts. The bifunctional imidoester dimethyl suberimidate (DMS) belongs to the wide class of the cross-linking reagents and is often used to cross-link a variety of proteins, including collagen matrices and collagen-based tissues. Raman spectroscopy in the wide frequency range 200-4000 cm -1 and contact mode atomic force microscopy (AFM) have been employed to investigate the structural changes and chemical bonds in DMS cross-linked porcine pericardium tissue. It has been found, that in addition to the commonly accepted reaction with the ?-amine groups of lysine or hydroxylysine residues, DMS may interact also with the carbonyl CO and amide NH groups of the peptide bond in collagen. Our paper presents for the first time spectral evidence for the peptide contribution to the formation of DMS-collagen cross-links. The results confirm also possible competition between the hydrolysis of the free imidoester group and cross-linking reaction. Products of the partial alkaline hydrolysis of DMS have been found in the spectra. The observed changes in the surface topography of the fibrils as well as in their spatial organization in the tissue support the formation of both intra- and interfibrillar cross-links in DMS-stabilized tissue.
Jastrzebska, Maria; Zalewska-Rejdak, Justyna; Wrzalik, Roman; Kocot, Antoni; Barwi?ski, Bogdan; Mr�z, Iwona; Cwalina, Beata
Nano and micro mechanical properties of uncross-linked and cross-linked chitosan films.
The aim of this study is to determine the nano and micro mechanical properties for uncross-linked and cross-linked chitosan films. Specifically, we looked at nanoindentation hardness, microhardness, and elastic modulus. It is important to study the nano and microscale mechanical properties of chitosan since chitosan has been widely used for biomedical applications. Using the solvent-cast method, the chitosan films were prepared at room temperature on the cleaned glass plates. The chitosan solution was prepared by dissolving chitosan in acetic acid 1% (v/v). Tripolyphosphate (TPP) was used to create the cross-links between amine groups in chitosan and phosphate groups in TPP. In this study, atomic force microscopy was used to measure the nanoindentation hardness and surface topography of the uncross-linked and cross-linked chitosan films. Elastic modulus was then calculated from the nanoindentation results. The effective elastic modulus was determined by microhardness with some modifications to previous theories. The microhardness of the chitosan films were measured using Vicker's hardness meter under three different loads. Our results show that the microhardness and elastic modulus for cross-linked chitosan films are higher than the uncross-linked films. However, the cross-linked chitosan films show increased brittleness when compared to uncross-linked films. By increasing the load magnitude, the microhardness increases for both uncross-linked and cross-linked chitosan films. PMID:22100082
Aryaei, Ashkan; Jayatissa, Ahalapitiya H; Jayasuriya, A Champa
The conformational stability and flexibility of insulin with an additional intramolecular cross-link
The conformational stability and flexibility of insulin containing a cross-link between the alpha-amino group of the A-chain to the epsilon-amino group of Lys29 of the B-chain was examined. The cross-link varied in length from 2 to 12 carbon atoms. The conformational stability was determined by guanidine hydrochloride-induced equilibrium denaturation and flexibility was assessed by H2O/D2O amide exchange. The cross-link has substantial effects on both conformational stability and flexibility which depend on its length. In general, the addition of a cross-link enhances conformational stability and decreases flexibility. The optimal length for enhanced stability and decreased flexibility was the 6-carbon link. For the 6-carbon link the Gibbs free energy of unfolding was 8.0 kcal/mol compared to 4.5 kcal/mol for insulin, and the amide exchange rate decreased by at least 3-fold. A very short cross-link (i.e. the 2-carbon link) caused conformational strain that was detectable by a lack of stabilization in the Gibbs free energy of unfolding and enhancement in the amide exchange rate compared to insulin. The effect of the cross-link length on insulin hydrodynamic properties is discussed relative to previously obtained receptor binding results.
Brems, D.N.; Brown, P.L.; Nakagawa, S.H.; Tager, H.S. (Eli Lilly Co., Indianapolis, IN (USA))
Current status of corneal collagen cross-linking for keratoconus: a review.
Over the past decade, corneal collagen cross-linking has become commonplace as a treatment option for individuals with progressive keratoconus. This is based on laboratory data suggesting that cross-linking using riboflavin and ultraviolet-A irradiation increases collagen diameter and the biomechanical strength of the treated cornea. Case series and limited randomised controlled trials support these findings with data demonstrating that cross-linking slows and possibly halts the progression of keratoconus. In some patients cross-linking results in an improvement in maximum corneal curvature, visual acuity, spherical equivalent and higher-order aberrations. The number of reported complications is small. More recently, variations in the treatment protocol have been described, although they have not yet been subject to comparative studies. While the published data indicate cross-linking is effective in modifying the natural history of keratoconus, the long-term impact of this treatment is still unknown. This paper reviews the theoretical basis, pre-clinical research and clinical results of corneal collagen cross-linking in keratoconus. PMID:23414201
Chan, Elsie; Snibson, Grant R
Corneal melting after collagen cross-linking for keratoconus: a case report
Introduction Corneal collagen cross-linking is a rather new technique that uses riboflavin and ultraviolet A light for collagen fiber stabilization in keratoconus corneas. Other than reversible side effects, the preliminary results of corneal collagen cross-linking studies suggest that it is a rather safe technique. In this report, we demonstrate a case of corneal melting after corneal collagen cross-linking for keratoconus corneas associated with an acute inflammatory response. Case presentation A 23-year-old Caucasian man with keratoconus cornea stage 1 to 2 underwent uneventful corneal collagen cross-linking treatment according to the Dresden protocol. The next day the patient had intense photophobia, watering and redness of the eye, and his visual acuity was limited to counting fingers. Slit lamp biomicroscopy revealed severe corneal haze accompanied by non-specific endothelial precipitates following an acute inflammatory response. Mild inflammation could be detected in the anterior chamber. Moreover, the re-epithelialization process could barely be detected. His corneal state gradually deteriorated, resulting in descemetocele and finally perforation. Conclusion In this report, we present a case of a patient with corneal melting after standard corneal collagen cross-linking treatment for keratoconus corneas following an acute inflammatory response. Despite modifying postoperative treatment, elaboration of all apparent associated causes by the treating physicians and undergoing extensive laboratory testing, the patient developed descemetocele, which led to perforation. Our report suggests that further research is necessary regarding the safety of corneal collagen cross-linking in keratoconus corneas.
xComb: a cross-linked peptide database approach to protein-protein interaction analysis
We developed an informatic method to identify tandem mass spectra composed of chemically cross-linked peptides from those of linear peptides and to assign sequence to each of the two unique peptide sequences. For a given set of proteins the key software tool, xComb, combs through all theoretically feasible cross-linked peptides to create a database consisting of a subset of all combinations represented as peptide FASTA files. The xComb library of select theoretical cross-linked peptides may then be used as a database that is examined by a standard proteomic search engine to match tandem mass spectral datasets to identify cross-linked peptides. The database search may be conducted against as many as 50 proteins with a number of common proteomic search engines, e.g. Phenyx, Sequest, OMSSA, Mascot and X!Tandem. By searching against a peptide library of linearized, cross-linked peptides, rather than a linearized protein library, search times are decreased and the process is decoupled from any specific search engine. A further benefit of decoupling from the search engine is that protein cross-linking studies may be conducted with readily available informatics tools for which scoring routines already exist within the proteomic community.
Panchaud, Alexandre; Singh, Pragya; Shaffer, Scott A.; Goodlett, David R.
Neurofilaments are the major neuronal target of hydroxynonenal-mediated protein cross-links
Lipid peroxidation generates reactive aldehydes, most notably hydroxynonenal (HNE), which covalently binds amino acid residue side chains leading to protein inactivation and insolubility. Specific adducts of lipid peroxidation have been demonstrated to be intimately associated with pathological lesions of Alzheimer�s disease (AD), suggesting that oxidative stress is a major component in the disease. Here, we examined the HNE-cross-linking modifications by using an antibody specific for a lysine�lysine cross-link. Since in a prior study we noted no immunolabeling of neuritic plaques or neurofibrillary tangles but instead found strong labeling of axons, we focused this study on axons. Axonal labeling was examined in mouse sciatic nerve, and immunoblotting showed the cross-link was restricted to neurofilament heavy and medium subunits, which while altering migration, did not indicate larger NF aggregates, indicative of intermolecular cross-links. Examination of mice at various ages showed the extent of modification remaining relatively constant through the life span. These findings demonstrate lipid-cross-linking peroxidation primarily involves lysine-rich neurofilaments and is restricted to intramolecular cross-links.
Perry, E. A.; Castellani, R. J.; Moreira, P. I.; Nunomura, A.; Lui, Q.; Harris, P. L. R.; Sayre, L. M.; Szweda, P. A.; Szweda, L. I.; Zhu, X.; Smith, M. A.; Perry, G.
Ruthenium-catalyzed photo cross-linking of fibrin-based engineered tissue
Most cross-linking methods utilize chemistry or physical processes that are detrimental to cells and tissue development. Those that are not as harmful often do not provide a level of strength that ultimately meets the required application. The purpose of this work was to investigate the use of a ruthenium-sodium persulfate cross-linking system to form dityrosine in fibrin-based engineered tissue. By utilizing the tyrosine residues inherent to fibrin and cell-deposited proteins, at least 3-fold mechanical strength increases and 10-fold stiffness increases were achieved after cross-linking. This strengthening and stiffening effect was found to increase with culture duration prior to cross-linking such that physiologically relevant properties were obtained. Fibrin was not required for this effect as demonstrated by testing with collagen-based engineered tissue. Cross-linked tissues were implanted subcutaneously and shown to have minimal inflammation after 30 days, similar to non-cross-linked controls. Overall, the method employed is rapid, non-toxic, minimally inflammatory, and is capable of increasing strength and stiffness of engineered tissues to physiological levels.
Bjork, Jason W.; Johnson, Sandra L.; Tranquillo, Robert T.
Transglutaminase-mediated cross-linking is involved in the stabilization of extracellular matrix in human liver fibrosis
Background\\/Aims: Lysyl oxidase-mediated cross-linking contributes to the stabilization of collagen in liver fibrosis. We have investigated transglutaminase-mediated cross-linking, to determine if it participates in the stabilization of extracellular matrix in human liver fibrosis.Methods: Transglutaminase activity was assessed in vitro by incorporation of biotinylated amine into liver proteins. The product of the transglutaminase-catalyzed cross-linking reaction, N?(?-glutamyl)lysine, and the extracellular proteins cross-linked
Pascale Grenard; Solange Bresson-Hadni; Mich�le Chevallier; Dominique A. Vuitton; Sylvie Ricard-Blum
Mutational Analysis of a Conserved Glutamic Acid Required for Self-Catalyzed Cross-Linking of Bacteriophage HK97 Capsids
The capsid of bacteriophage HK97 is stabilized by 400 covalent cross-links between subunits which form without any action by external enzymes or cofactors. Cross-linking only occurs in fully assembled particles after large-scale structural changes bring together side chains from three subunits at each cross-linking site. Isopeptide cross-links form between asparagine and lysine side chains on two subunits. The carboxylate of
Lindsay E. Dierkes; Craig L. Peebles; Brian A. Firek; Roger W. Hendrix; Robert L. Duda
A comparative study of the characteristics of cross-linked, oxidized and dual-modified rice starches.
Rice starch was cross-linked with epichlorohydrin (0.3%, w/w, on a dry starch basis) and oxidized with sodium hypochlorite (2.5% w/w), respectively. Two dual-modified rice starch samples (oxidized cross-linked rice starch and cross-linked oxidized rice starch) were obtained by the oxidation of cross-linked rice starch and the cross-linking of oxidized rice starch at the same level of reagents. The physicochemical properties of native rice starch, cross-linked rice starch and oxidized rice starch were also studied parallel with those of the two dual-modified rice starch samples using rapid visco analysis (RVA), differential scanning calorimetry (DSC), dynamic rheometry and scanning electron microscopy (SEM). It was found that the levels of cross-linking and oxidation used in this study did not cause any significant changes in the morphology of rice starch granules. Cross-linked oxidized starch showed lower swelling power (SP) and solubility, and higher paste clarity in comparison with native starch. Cross-linked oxidized rice starch also had the lowest tendency of retrogradation and highest ability to resistant to shear compared with native, cross-linked, oxidized and oxidized cross-linked rice starches. These results suggest that the undesirable properties in native, cross-linked and oxidized rice starch samples could be overcome through dual-modification. PMID:22971580
Xiao, Hua-Xi; Lin, Qin-Lu; Liu, Gao-Qiang; Yu, Feng-Xiang
Immunofluoresence of rabbit corneas following collagen cross-linking treatment with Riboflavin and Ultraviolet A
Purpose To assess ultrastructural modifications in keratocytes and inflammatory cell response in rabbit corneas after riboflavin and ultraviolet A (UVA) exposure using immunoflurescence microscopy. Methods Twenty adult New Zealand albino rabbits weighing 2.0 to 3.0 kg were used in this study. Two rabbits served as controls. The animals had their epithelia removed and were cross-linked with riboflavin 0.1% solution (10mgs riboflavin-5-phosphate in 10ml 20% dextran-T-500) applied every 3 minutes for 30 minutes, and exposed to UVA (360 nm, 3 mW/cm2) for 30 minutes. Four rabbits were humanely euthanized at each time point of 1, 3 and 11 days and at 3 and 5 weeks after the procedure. Immunohistochemestry studies of thin sections of each cornea were performed using TUNEL staining, Alpha smooth muscle actin (?-SMA), CD-3, myeloperoxidase (MPO) antibodies and DAPI counterstaining. In another experiment six additional rabbits were treated as above, and after 10 days of cross-linking, 5 ?l of lipopolysaccharide (LPS) endotoxin (1?g/ml) was injected in the mid stroma. Results Cross-linked corneas showed early stromal edema. By 5 weeks, complete resolution of the edema and a pronounced highly organized anterior 200 ?m fluorescent zone was observed. TUNEL staining showed keratocyte death by both necrosis and apoptosis between day 1 and 3 after cross-linking. At day 1 the limbal area close to the cross linking zone showed some inflammatory cells as well as ?-SMA positive cells, indicative of the presence of myofibroblasts. By day 3 some myofibroblasts had migrated to the area beneath the cross linked stroma. Between day 3 and 5 weeks there was an increase in ?-SMA staining in the area surrounding the cross linked stroma. The area of cross linking remained acellular up to 5 weeks. Conclusions Collagen cross-linking results in early edema, keratocyte apoptosis and necrosis, appearance of inflammatory cells in the surrounding area of treatment and transformation of surrounding keratocytes into myofibroblasts. Compaction of anterior stroma fibers, keratocyte loss and displacement of cell nuclei including inflammatory cells may have clinical implications in the long term risk of further corneal thinning in keratoconus and in the cross linked corneal immune response.
Esquenazi, Salomon; He, Jiucheng; Li, Na; Bazan, Haydee E.P.
Intra-molecular cross-linking of acidic residues for protein structure studies.
Intra-molecular cross-linking has been suggested as a method of obtaining distance constraints that would help to develop structural models of proteins. Recent work published on intra-molecular cross-linking for protein structural studies has employed commercially available primary amine (lysine, the amino terminus) selective reagents. Previous work using these cross-linkers has shown that for several proteins of known structure, the number of cross-links that can be obtained experimentally may be small compared to what would be expected from the known structure, due to the relative reactivity, distribution and solvent accessibility of the lysines in the protein sequence. To overcome these limitations, we have investigated the use of cross-linking reagents that can react with other reactive side chains in proteins. We used 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to activate the carboxylic acid containing residues, aspartic acid (D), glutamic acid (E) and the carboxy terminus (O), for cross-linking reactions. Once activated, the DEO side chains can react to form "zero-length" cross-links with nearby primary amine containing residues, lysines (K) and the amino terminus (X), via the formation of a new amide bond. We also show that the EDC-activated DEO side chains can be cross-linked to each other using dihydrazides, two hydrazide moieties connected by an alkyl cross-linker arm of variable length. Using these reagents, we have found three new "zero-length" cross-links in ubiquitin consistent with its known structure (M1-E16, M1-E18 and K63-E64). Using the dihydrazide cross-linkers, we have identified two new cross-links (D21-D32 and E24-D32) unambiguously. Using a library of dihydrazide cross-linkers with varying arm length, we have shown that there is a minimum arm length required for the DEO-DEO cross-links of 5.8 A. These results show that additional structural information can be obtained by exploiting new cross-linker chemistry, increasing the probability that the protein target will yield sufficient distance constraints to develop a structural model. PMID:19136724
Novak, Petr; Kruppa, Gary H
Recognition of DNA interstrand cross-link of antitumor cisplatin by HMGB1 protein.
Several proteins that specifically bind to DNA modified by cisplatin, including those containing HMG-domains, mediate antitumor activity of this drug. Oligodeoxyribonucleotide duplexes containing a single, site-specific interstrand cross-link of cisplatin were probed for recognition by the rat chromosomal protein HMGB1 and its domains A and B using the electrophoretic mobility-shift assay. It has been found that the full-length HMGB1 protein and its domain B to which the lysine-rich region (seven amino acid residues) of the A/B linker is attached at the N-terminus (the domain HMGB1b7) specifically recognize DNA interstrand cross-linked by cisplatin. The affinity of these proteins to the interstrand cross-link of cisplatin is not very different from that to the major 1,2-GG intrastrand cross-link of this drug. In contrast, no recognition of the interstrand cross-link by the domain B lacking this region or by the domain A with or without this lysine-rich region attached to its C-terminus is noticed under conditions when these proteins readily bind to 1,2-GG intrastrand adduct. A structural model for the complex formed between the interstrand cross-linked DNA and the domain HMGB1b7 was constructed and refined using molecular mechanics and molecular dynamics techniques. The calculated accessible areas around the deoxyribose protons correlate well with the experimental hydroxyl radical footprint. The model suggests that the only major adaptation necessary for obtaining excellent surface complementarity is extra DNA unwinding (approximately 40 degrees ) at the site of the cross-link. The model structure is consistent with the hypothesis that the enhancement of binding affinity afforded by the basic lysine-rich A/B linker is a consequence of its tight binding to the sugar-phosphate backbone of both DNA strands. PMID:12564926
Kasparkova, Jana; Delalande, Olivier; Stros, Michal; Elizondo-Riojas, Miguel-Angel; Vojtiskova, Marie; Kozelka, Jiri; Brabec, Viktor
Immune Focusing and Enhanced Neutralization Induced by HIV-1 gp140 Chemical Cross-Linking
Experimental vaccine antigens based upon the HIV-1 envelope glycoproteins (Env) have failed to induce neutralizing antibodies (NAbs) against the majority of circulating viral strains as a result of antibody evasion mechanisms, including amino acid variability and conformational instability. A potential vaccine design strategy is to stabilize Env, thereby focusing antibody responses on constitutively exposed, conserved surfaces, such as the CD4 binding site (CD4bs). Here, we show that a largely trimeric form of soluble Env can be stably cross-linked with glutaraldehyde (GLA) without global modification of antigenicity. Cross-linking largely conserved binding of all potent broadly neutralizing antibodies (bNAbs) tested, including CD4bs-specific VRC01 and HJ16, but reduced binding of several non- or weakly neutralizing antibodies and soluble CD4 (sCD4). Adjuvanted administration of cross-linked or unmodified gp140 to rabbits generated indistinguishable total gp140-specific serum IgG binding titers. However, sera from animals receiving cross-linked gp140 showed significantly increased CD4bs-specific antibody binding compared to animals receiving unmodified gp140. Moreover, peptide mapping of sera from animals receiving cross-linked gp140 revealed increased binding to gp120 C1 and V1V2 regions. Finally, neutralization titers were significantly elevated in sera from animals receiving cross-linked gp140 rather than unmodified gp140. We conclude that cross-linking favors antigen stability, imparts antigenic modifications that selectively refocus antibody specificity and improves induction of NAbs, and might be a useful strategy for future vaccine design.
Schiffner, T.; Kong, L.; Duncan, C. J. A.; Back, J. W.; Benschop, J. J.; Shen, X.; Huang, P. S.; Stewart-Jones, G. B.; DeStefano, J.; Seaman, M. S.; Tomaras, G. D.; Montefiori, D. C.; Schief, W. R.
Mass spectrometry evidence for cisplatin as a protein cross-linking reagent
Cisplatin is a potent anti-cancer drug, which functions by cross-linking adjacent DNA guanine residues. However within one day of injection, 65~98% of the platinum in the blood plasma is protein-bound. It is generally accepted that cisplatin binds to methionine and histidine residues, but what is often underappreciated is that platinum from cisplatin has a 2+ charge and can form up to four bonds. Thus, it has the potential to function as a cross-linker. In this report, the cross-linking ability of cisplatin is demonstrated by Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) with the use of standard peptides, the 16.8 kDa protein calmodulin (CaM), but was unsuccessful for the 64 kDa protein hemoglobin. The high resolution and mass accuracy of FTICR MS along with the high degree of fragmentation of large peptides afforded by collisionally activated dissociation (CAD) and electron capture dissociation (ECD) are shown to be a valuable means of characterizing cross-linking sites. Cisplatin is different from current cross-linking reagents by targeting new functional groups, thioethers, and imidazoles groups, which provides complementarity with existing cross-linkers. In addition, platinum(II) inherently has two positive charges which enhance the detection of cross-linked products. Higher charge states not only promote the detection of cross-linking products with less purification, but result in more comprehensive MS/MS fragmentation and can assist the assignment of modification sites. Moreover, the unique isotopic pattern of platinum flags cross-linking products and modification sites by mass spectrometry.
Li, Huilin; Zhao, Yao; Phillips, Hazel I. A.; Qi, Yulin; Lin, Tzu-Yung; Sadler, Peter J.; O'Connor, Peter B.
Quantitative cross-linking/mass spectrometry using isotope-labelled cross-linkers?
Dynamic proteins and multi-protein complexes govern most biological processes. Cross-linking/mass spectrometry (CLMS) is increasingly successful in providing residue-resolution data on static proteinaceous structures. Here we investigate the technical feasibility of recording dynamic processes using isotope-labelling for quantitation. We cross-linked human serum albumin (HSA) with the readily available cross-linker BS3-d0/4 in different heavy/light ratios. We found two limitations. First, isotope labelling reduced the number of identified cross-links. This is in line with similar findings when identifying proteins. Second, standard quantitative proteomics software was not suitable for work with cross-linking. To ameliorate this we wrote a basic open source application, XiQ. Using XiQ we could establish that quantitative CLMS was technically feasible. Biological significance Cross-linking/mass spectrometry (CLMS) has become a powerful tool for providing residue-resolution data on static proteinaceous structures. Adding quantitation to CLMS will extend its ability of recording dynamic processes. Here we introduce a cross-linking specific quantitation strategy by using isotope labelled cross-linkers. Using a model system, we demonstrate the principle and feasibility of quantifying cross-linking data and discuss challenges one may encounter while doing so. We then provide a basic open source application, XiQ, to carry out automated quantitation of CLMS data. Our work lays the foundations of studying the molecular details of biological processes at greater ease than this could be done so far. This article is part of a Special Issue entitled: New Horizons and Applications for Proteomics [EuPA 2012].
Fischer, Lutz; Chen, Zhuo Angel; Rappsilber, Juri
Cross-linking methionine and amine residues with reactive halogen species.
Irreversible cross-links are increasingly being recognized as important posttranslational oxidative protein modifications that contribute to tissue injury during oxidative stress and inflammation. They also have a structural function in extracellular matrix proteins such as collagen IV. Likely contenders for forming such cross-links are the reactive halogen species that are generated by neutrophils and eosinophils, including hypochlorous acid, hypobromous acid, and their related haloamines. Methionine residues are kinetically preferred targets for these oxidants and oxidation can potentially result in sulfilimine (>SN-) bonds with amines. Therefore, we investigated whether oxidation of methionine in the model peptide formyl-Met-Leu-Phe-Lys (fMLFK) produces cross-links with lysine residues, using mass spectrometry to characterize the products. As expected, the sulfoxide was the major product with each reactive halogen species. However, intra- and intermolecular cross-linked products were also formed. Isomers of an intramolecular sulfilimine were readily produced by hypobromous acid and bromamines, with hypochlorous acid forming lesser amounts. The predominant cross-link with chloramines was an intermolecular bond between the sulfur of fMLFK and the amine derived from the chloramine. Reactive halogen species also formed these sulfilimine cross-links in other peptides that contain methionine. We propose that protein cross-links involving methionine and amine residues will form via this mechanism when granulocytes are activated at sites of inflammation. Our results also support the proposal that reactive halogen species generated by the peroxidase peroxidasin could be responsible for the sulfilimine bonds that are integral to the structure of collagen IV. PMID:24486343
Ronsein, Graziella E; Winterbourn, Christine C; Di Mascio, Paolo; Kettle, Anthony J
Conformational transitions of the cross-linking domains of elastin during self-assembly.
Elastin is the intrinsically disordered polymeric protein imparting the exceptional properties of extension and elastic recoil to the extracellular matrix of most vertebrates. The monomeric precursor of elastin, tropoelastin, as well as polypeptides containing smaller subsets of the tropoelastin sequence, can self-assemble through a colloidal phase separation process called coacervation. Present understanding suggests that self-assembly is promoted by association of hydrophobic domains contained within the tropoelastin sequence, whereas polymerization is achieved by covalent joining of lysine side chains within distinct alanine-rich, ?-helical cross-linking domains. In this study, model elastin polypeptides were used to determine the structure of cross-linking domains during the assembly process and the effect of sequence alterations in these domains on assembly and structure. CD temperature melts indicated that partial ?-helical structure in cross-linking domains at lower temperatures was absent at physiological temperature. Solid-state NMR demonstrated that ?-strand structure of the cross-linking domains dominated in the coacervate state, although ?-helix was predominant after subsequent cross-linking of lysine side chains with genipin. Mutation of lysine residues to hydrophobic amino acids, tyrosine or alanine, leads to increased propensity for ?-structure and the formation of amyloid-like fibrils, characterized by thioflavin-T binding and transmission electron microscopy. These findings indicate that cross-linking domains are structurally labile during assembly, adapting to changes in their environment and aggregated state. Furthermore, the sequence of cross-linking domains has a dramatic effect on self-assembly properties of elastin-like polypeptides, and the presence of lysine residues in these domains may serve to prevent inappropriate ordered aggregation. PMID:24550393
Reichheld, Sean E; Muiznieks, Lisa D; Stahl, Richard; Simonetti, Karen; Sharpe, Simon; Keeley, Fred W
Raloxifene ameliorates detrimental enzymatic and nonenzymatic collagen cross-links and bone strength in rabbits with hyperhomocysteinemia
Summary��We demonstrate a reduction in enzymatic divalent immature and trivalent pyridinium cross-links and an increase in the nonenzymatic\\u000a cross-link, pentosidine (Pen), in rabbits with methionine (Met)-induced hyperhomocysteinemia. Such detrimental cross-link\\u000a formation in bone was ameliorated by raloxifene (RLX) treatment.\\u000a \\u000a \\u000a \\u000a \\u000a Introduction��Collagen cross-links are determinants of bone quality. Homocysteine (Hcys) interferes with collagen cross-linking. Because\\u000a RLX is thought to ameliorate bone quality,
M. Saito; K. Marumo; S. Soshi; Y. Kida; C. Ushiku; A. Shinohara
[Cross-linking and neurodermitis: prolonged re-epithelisation with severe corneal vascularisation after cross-linking in a patient with neurodermitis and keratoconus].
This case report describes a patient with keratoconus and neurodermitis suffering from a significantly prolonged postoperative time interval to re-epithelisation after corneal cross-linking. The development of corneal calcifications and vascularisations additionally inhibited proper re-epithelisation. Therefore the patient received four subsequent subconjunctival injections of Bevacizumab and an additional keratectomy to remove the calcifications. This therapeutic scheme led to a significant reduction of corneal vascularisation and finally a full rehabilitation of the epithelium. PMID:24788604
G�rsch, I C; Steinberg, J; Richard, G; Katz, T; Linke, S
Characterization of the enzymatic activity of the actin cross-linking domain from the Vibrio cholerae MARTX Vc toxin.
Vibrio cholerae is a Gram-negative bacterial pathogen that exports enterotoxins, which alter host cells through a number of mechanisms resulting in diarrheal disease. Among the secreted toxins is the multifunctional, autoprocessing RTX toxin (MARTX(Vc)), which disrupts actin cytoskeleton by covalently cross-linking actin monomers into oligomers. The region of the toxin responsible for cross-linking activity is the actin cross-linking domain (ACD). In this study, we demonstrate unambiguously that ACD utilizes G- and not F-actin as a substrate for the cross-linking reaction and hydrolyzes one molecule of ATP per cross-linking event. Furthermore, major actin-binding proteins that regulate actin cytoskeleton in vivo do not block the cross-linking reaction in vitro. Cofilin inhibits the cross-linking of G- and F-actin, at a high mole ratio to actin but accelerates F-actin cross-linking at low mole ratios. DNase I completely blocks the cross-linking of actin, likely due to steric hindrance with one of the cross-linking sites on actin. In the context of the holotoxin, the inhibition of Rho by the Rho-inactivating domain of MARTX(Vc) (Sheahan, K. L., and Satchell, K. J. F. (2007) Cell. Microbiol. 9, 1324-1335) would accelerate F-actin depolymerization and provide G-actin, alone or in complex with actin-binding proteins, for cross-linking by ACD, ultimately leading to the observed rapid cell rounding. PMID:17951576
Kudryashov, Dmitri S; Cordero, Christina L; Reisler, Emil; Satchell, Karla J Fullner
Availability of fluorescence spectroscopic in the accompaniment of formation of corneal cross-linking
NASA Astrophysics Data System (ADS)
The corneal cross-linking is a method that associates riboflavin and ultraviolet light to induce a larger mechanical resistance at cornea. This method has been used for the treatment of Keratoconus. Since cross-linking is recent as treatment, there is a need to verify the effectiveness of the method. Therefore, the viability of the fluorescence spectroscopy technique to follow the cross-linking formation at cornea was studied. Corneas were divided in two measuring procedures: M1 (cornea + riboflavin), and M2 (cornea + riboflavina + light irradiation, 365nm). For fluorescence measurements, a spectrofluorimeter was used, where several wavelengths were selected (between 320nm and 370nm) for cornea excitation. Several fluorescence spectra were collected, at 10 min-interval, during 60 min. Spectra allowed one to observe two very well defined bands of fluorescence: the first one at 400nm (collagen), and the second one at 520nm (riboflavin). After spectra analyses, a decrease of collagen fluorescence was observed for both groups. For riboflavin, on the other hand, there was a fluorescence increase for M1, and a decrease for M2. Thus, it is possible to conclude that it this technique is sensitive for the detection of tissue structural changes during cross-linking treatment, encouraging subsequent studies on quantification of cross-linking promotion in tissue.
Costa, M. M.; Kurachi, C.; Bagnato, V. S.; Faria E Sousa, S. J.; Ventura, L.
Biodegradable Chitosan-Based Ambroxol Hydrochloride Microspheres: Effect of Cross-Linking Agents
The objective of this study was to investigate the influence of type of cross-linking method used on the properties of ambroxol hydrochloride microspheres such as encapsulation efficiency, particle size, and drug release. Microspheres were prepared by solvent evaporation technique using chitosan as a matrix-forming agent and cross-linked using formaldehyde and heat treatment. Morphological and physicochemical properties of microspheres were then investigated by scanning electron microscopy (SEM), X-ray diffractometry (XRD), differential scanning calorimetry (DSC), and Fourier-transform infrared spectroscopy (FTIR) spectroscopy. The cross-linking of chitosan takes place at the free amino group because of formation of imine bond as evidenced by FTIR. The DSC, XRD, and FTIR analysis showed that chitosan microspheres cross linked by heating were superior in properties and performance as compared to the microspheres cross-linked using formaldehyde. SEM results revealed that heat-treated microspheres were spherical, discrete having smooth, and porous structure. The particle size and encapsulation efficiencies of the prepared chitosan microspheres ranged between 10.83�24.11 ?m and 39.73?80.56%, respectively. The drug release was extended up to 12 h, and the kinetics of the drug release was obeying Higuchi kinetic proving diffusion-controlled drug release.
Gangurde, HH; Chavan, NV; Mundada, AS; Derle, DV; Tamizharasi, S
[The application of corneal collagen cross-linking in diseases other than keratoconus].
Corneal collagen cross-linking is a method of treatment using ultraviolet radiation UVA and photosensitizing substance riboflavin to strengthen the chemical connections between the collagen fibers of corneal stroma. This treatment is focused on halting the progression of the diasases, called ectasias, characterized by irregular curvature and diminished thickness of the cornea. The most common indication for corneal collagen cross-linking is keratoconus. However, this technique may be also applied to pathologies other than keratoconus. The aim of this paper is to review the applicilcability of corneal collagen cross-linking in other conditions than keratoconus. Specifically, the conditions such as pellucid marginal degeneration, post refractive surgery ectasia as well as combined corneal collagen cross-linking and topography-based photorefractive keratectomy for topographies indicating forme fruste keratoconus are discussed. In addition, the effects of-corneal collagen cross-linking-as an adjunctive therapy in keratitis, corneal ulcers and corneal edema in bullous keratopathy are considered. The authors highlight the importance of treatment in clinical practice and the potential application of the treatment and modification of the protocols in the treatment of corneal diseases other than keratoconus. PMID:23882746
Mrukwa-Kominek, Ewa; Drzyzga, ?ukasz; Rogowska-Godela, Anna; Porwik, Ewa
Influence of resin cross-linking on solid-phase chemistry.
A range of PS-DVB resins were prepared by suspension polymerization with styrene, p-chloromethyl styrene, and DVB. Yields of polymerization increased (from 40% to almost 80%) with increasing cross-linking. The beads exhibited the expected swelling characteristics, with the 0.3% resin swelling to almost 9 times its dry volume in CHCl3. Kinetics of cleavage of the dye Methyl Red from the range of Rink linked resins showed rate enhancements of up to 500% between the 6.0 and the 0.3% cross-linked resins. Total synthesis of Kawaguchipeptin B was carried out on the resins, and their performance during the syntheses was investigated. Contrary to expectations, the purities of the cyclic peptide product increased with increasing resin cross-linking doubling from the 0.3-6.0% resin. A Suzuki reaction showed the half-lives of reaction increased with increasing resin cross-linking with an 11-fold increase in half-life between the 0.3-2.7% resin. Surprisingly, we observed very little reaction in the case of the 3.0 and 6.0% cross-linked resins. PMID:11148058
Rana, S; White, P; Bradley, M
Cross-linking connectivity in bone collagen fibrils: the COOH-terminal locus of free aldehyde
NASA Technical Reports Server (NTRS)
Quantitative analyses of the chemical state of the 16c residue of the alpha 1 chain of bone collagen were performed on samples from fetal (4-6-month embryo) and mature (2-3 year old) bovine animals. All of this residue could be accounted for in terms of three chemical states, in relative amounts which depended upon the age of the animal. Most of the residue was incorporated into either bifunctional or trifunctional cross-links. Some of it, however, was present as free aldehyde, and the content increased with maturation. This was established by isolating and characterizing the aldehyde-containing peptides generated by tryptic digestion of NaB3H4-reduced mature bone collagen. We have concluded that the connectivity of COOH-terminal cross-linking in bone collagen fibrils changes with maturation in the following way: at first, each 16c residue in each of the two alpha 1 chains of the collagen molecule is incorporated into a sheet-like pattern of intermolecular iminium cross-links, which stabilizes the young, nonmineralized fibril as a whole. In time, some of these labile cross-links maturate into pyridinoline while others dissociate back to their precursor form. The latter is likely due to changes in the molecular packing brought about by the mineralization of the collagen fibrils. The resultant reduction in cross-linking connectivity may provide a mechanism for enhancing certain mechanical characteristics of the skeleton of a mature animal.
Otsubo, K.; Katz, E. P.; Mechanic, G. L.; Yamauchi, M.
Reversible Inter- and Intra-Microgel Cross-Linking using Disulfides
Thermoresponsive hydrogel nanoparticles composed of poly(N-isopropylmethacrylamide) (pNIPMAm) and the disulfide-based cross-linker N,N�-bis(acryloyl)cystamine (BAC) have been prepared using a redox-initiated, aqueous precipitation polymerization approach, leading to improved stability of the disulfide bond compared to traditional thermally-initiated methods. The resultant particles demonstrate complete erosion in response to reducing conditions or thiol competition. This stands in contrast to the behavior of thermally-initiated particles, which retain a cross-linked network following disulfide cleavage due to uncontrolled chain-branching and self-cross-linking side reactions. The synthetic strategy has also been combined with the non-degradable cross-linker N,N-methylenebisacrylamide (BIS) to generate �co-cross-linked� pNIPMAm-BAC-BIS microgels. These particles are redox-responsive, swell upon BAC cross-link scission and present reactive thiols. This pendant thiol functionality was demonstrated to be useful for conjugation of thiol-reactive probes and in reversible network formation by assembling particles cross-linked by disulfide linkages.
Gaulding, Jeffrey C.; Smith, Michael H.; Hyatt, John S.; Fernandez-Nieves, Alberto; Lyon, L. Andrew
Synthesis, characterization, and antibacterial activity of cross-linked chitosan-glutaraldehyde.
This present study deals with synthesis, characterization and antibacterial activity of cross-linked chitosan-glutaraldehyde. Results from this study indicated that cross-linked chitosan-glutaraldehyde markedly inhibited the growth of antibiotic-resistant Burkholderia cepacia complex regardless of bacterial species and incubation time while bacterial growth was unaffected by solid chitosan. Furthermore, high temperature treated cross-linked chitosan-glutaraldehyde showed strong antibacterial activity against the selected strain 0901 although the inhibitory effects varied with different temperatures. In addition, physical-chemical and structural characterization revealed that the cross-linking of chitosan with glutaraldehyde resulted in a rougher surface morphology, a characteristic Fourier transform infrared (FTIR) band at 1559 cm?�, a specific X-ray diffraction peak centered at 2? = 15�, a lower contents of carbon, hydrogen and nitrogen, and a higher stability of glucose units compared to chitosan based on scanning electron microscopic observation, FTIR spectra, X-ray diffraction pattern, as well as elemental and thermo gravimetric analysis. Overall, this study indicated that cross-linked chitosan-glutaraldehyde is promising to be developed as a new antibacterial drug. PMID:23670533
Li, Bin; Shan, Chang-Lin; Zhou, Qing; Fang, Yuan; Wang, Yang-Li; Xu, Fei; Han, Li-Rong; Ibrahim, Muhammad; Guo, Long-Biao; Xie, Guan-Lin; Sun, Guo-Chang
Dependence of nanomechanical modification of polymers on plasma-induced cross-linking
The nanomechanical properties of low-density polyethylene (LDPE) modified by inductively coupled, radio-frequency Ar plasma were investigated by surface force microscopy. The polymer surface was modified under plasma conditions of different ion energy fluences and radiation intensities obtained by varying the sample distance from the plasma power source. Nanoindentation results of the surface stiffness versus maximum penetration depth did not reveal discernible differences between untreated and plasma-treated LDPE, presumably due to the small thickness of the modified surface layer that resulted in a substrate effect. On the contrary, nanoscratching experiments demonstrated a significant increase in the surface shear resistance of plasma-modified LDPE due to chain cross-linking. These experiments revealed an enhancement of cross-linking with increasing ion energy fluence and radiation intensity, and a tip size effect on the friction force and dominant friction mechanisms (adhesion, plowing, and microcutting). In addition, LDPE samples with a LiF crystal shield were exposed to identical plasma conditions to determine the role of vacuum ultraviolet (VUV) and ultraviolet (UV) radiation in the cross-linking process. The cross-linked layer of plasma-treated LDPE exhibited much higher shear strength than that of VUV/UV-treated LDPE. Plasma-induced surface modification of the nanomechanical properties of LDPE is interpreted in the context of molecular models of the untreated and cross-linked polymer surfaces derived from experimental findings.
Tajima, S.; Komvopoulos, K. [Department of Mechanical Engineering, University of California, Berkeley, California 94720 (United States)
Cornifin, a cross-linked envelope precursor in keratinocytes that is down-regulated by retinoids.
In this study, we have characterized the cDNA clone SQ37 that was isolated previously from a rabbit squamous cell library. The gene encodes a 14-kDa protein that appears to function as a component of the cross-linked envelope in squamous differentiating cells. The protein, which has been named cornifin, has a high content of proline (31%), glutamine (20%), and cysteine (11%) and contains 13 repeats of an octapeptide (consensus sequence, EPCQPKVP) at its C terminus. SQ37 mRNA and protein are induced during squamous differentiation of rabbit tracheal (RbTE) cells and human epidermal keratinocytes. This induction is repressed by retinoids. Immunohistochemical studies reveal SQ37 immunoreactivity in fragmented cross-linked envelopes from squamous-differentiated RbTE cells and in the suprabasal layers of the epidermis. In situ hybridization analysis showed that the presence of SQ37 mRNA is restricted to the suprabasal layers. Treatment of RbTE cells with a Ca2+ ionophore induces cross-linking of the SQ37 protein into higher molecular weight complexes. This cross-linking reaction appears to be mediated by transglutaminase type I. Our observations suggest that the protein encoded by SQ37 participates in the assembly of the cross-linked envelope. PMID:1438308
Marvin, K W; George, M D; Fujimoto, W; Saunders, N A; Bernacki, S H; Jetten, A M
Cornifin, a cross-linked envelope precursor in keratinocytes that is down-regulated by retinoids.
In this study, we have characterized the cDNA clone SQ37 that was isolated previously from a rabbit squamous cell library. The gene encodes a 14-kDa protein that appears to function as a component of the cross-linked envelope in squamous differentiating cells. The protein, which has been named cornifin, has a high content of proline (31%), glutamine (20%), and cysteine (11%) and contains 13 repeats of an octapeptide (consensus sequence, EPCQPKVP) at its C terminus. SQ37 mRNA and protein are induced during squamous differentiation of rabbit tracheal (RbTE) cells and human epidermal keratinocytes. This induction is repressed by retinoids. Immunohistochemical studies reveal SQ37 immunoreactivity in fragmented cross-linked envelopes from squamous-differentiated RbTE cells and in the suprabasal layers of the epidermis. In situ hybridization analysis showed that the presence of SQ37 mRNA is restricted to the suprabasal layers. Treatment of RbTE cells with a Ca2+ ionophore induces cross-linking of the SQ37 protein into higher molecular weight complexes. This cross-linking reaction appears to be mediated by transglutaminase type I. Our observations suggest that the protein encoded by SQ37 participates in the assembly of the cross-linked envelope. Images
Marvin, K W; George, M D; Fujimoto, W; Saunders, N A; Bernacki, S H; Jetten, A M
Site specificity of psoralen-DNA interstrand cross-linking determined by nuclease Bal31 digestion
A novel method for determination of psoralen photo-cross-linking sites in double-stranded DNA is described, which is based on a pronounced inhibition of Bal31 exonuclease activity by psoralen-DNA interstrand cross-links. The results using a 51 base pair fragment of plasmid pUC19 and a 346 base pair fragment of pBR322 show that 5'-TA sequences are preferred cross-linking sites compared to 3'-TA sequences. They also indicate that sequences flanking the 5'-TA site influence the cross-linking efficiency at the site. The DNA photo-cross-linking by 4,5',8-trimethylpsoralen and 8-methoxypsoralen was analyzed, and these two psoralens showed identical site specificity. The 5'-TA preference is rationalized on the basis of the local DNA structure in terms of ..pi..-..pi.. electronic interaction between the thymines and the intercalated psoralens, as well as on the base tilt angles of the DNA.
Zhen, W.; Buchardt, O.; Nielsen, H.; Nielsen, P.E.
Alkali reversal of psoralen cross-link for the targeted delivery of psoralen monoadduct lesion
Psoralen intercalates into double-stranded DNA and photoreacts mainly with thymines to form monoadducts and interstrand cross-links. The authors used an oligonucleotide model to demonstrate a novel mechanism: the reversal of psoralen cross-links by base-catalyzed rearrangement at 90/sup 0/C (BCR). The BCR reaction is more efficient than the photoreversal reaction. They show that the BCR occurs predominantly on the furan side of a psoralen cross-link. The cleavage does not result in the breaking of the DNA backbone, and the thymine based freed from the cross-link by the cleavage reaction appears to be unmodified. Similarly, BCR of the furan-side monoadduct of psoralen removed the psoralen molecule and regenerated the unaltered native oligonucleotide. The pyrone-side psoralen monoadduct is relatively resistant to BCR. One can use BCR to perform efficient oligonucleotide-directed, site-specific delivery of a psoralen monoadduct. As a demonstration of this approach, they have hybridized a 19 base long oligonucleotide vehicle containing a furan-side psoralen monoadduct to a 56 base long complementary oligonucleotide target strand and formed a specific cross-link at the target site with 365-nm UV. Subsequent BCR released the oligonucleotide vehicle and deposited the psoralen at the target site.
Yeung, A.T.; Dinehart, W.J.; Jones, B.K.
4-thio-U cross-linking identifies the active site of the VS ribozyme
To identify nucleotides in or near the active site, we have used a circularly permuted version of the VS ribozyme capable of cleavage and ligation to incorporate a single photoactive nucleotide analog, 4-thio- uridine, immediately downstream of the scissile bond. Exposure to UV light produced two cross-linked RNAs, in which the 4-thio-uridine was cross-linked to A756 in the 730 loop of helix�VI. The cross-links formed only under conditions that support catalytic activity, suggesting that they reflect functionally relevant conformations of the RNA. One of the cross-linked RNAs contains a lariat, indicative of intramolecular cross-linking in the ligated RNA; the other is a branched molecule in which the scissile phosphodiester bond is cleaved, but occupies the same site in the ribozyme�substrate complex. These are the two forms of the RNA expected to be the ground state structures on either side of the transition state. This localization of the active site is consistent with previous mutational, biochemical and biophysical data, and provides direct evidence that the cleavage site in helix�I interacts with the 730 loop in helix�VI.
Hiley, Shawna L.; Sood, Vanita D.; Fan, Jennie; Collins, Richard A.
Emodin-mediated cross-linking enhancement for extracellular matrix homeostasis.
The extracellular matrix (ECM) is an essential element of mammalian organisms, and its cross-linking formation plays a vital role in ECM development and postnatal homeostasis. Defects in cross-link formation caused by aging, genetic, or environmental factors are known to cause numerous diseases in mammals. To augment the cross-linking formation of ECM, the present study established a ZsGreen reporter system controlled by the promoter of lysyl oxidase-like 1 gene (LOXL1), which serves as both a scaffold element and a cross-linking enzyme in the ECM. By using this system in a drug screen, we identified emodin as a strong enhancer of LOXL1 expression that promoted cross-linking formation of ECM in all the tested systems, including human fibroblast cells, cultured human skin tissues, and animals that received long-term emodin treatment. Collectively, the results suggest that emodin may serve as an effective drug or supplement for ECM homeostasis. PMID:24680685
Jian, Lihua; Zhang, Chen; Chen, Guangfeng; Shi, Xiujuan; Qiu, Yu; Xue, Yunyun; Yang, Shuzhang; Lu, Lixia; Yuan, Qionglan; Xu, Guotong; Ying, Ming; Liu, Xiaoqing
Synthesis, Characterization, and Antibacterial Activity of Cross-Linked Chitosan-Glutaraldehyde
This present study deals with synthesis, characterization and antibacterial activity of cross-linked chitosan-glutaraldehyde. Results from this study indicated that cross-linked chitosan-glutaraldehyde markedly inhibited the growth of antibiotic-resistant Burkholderia cepacia complex regardless of bacterial species and incubation time while bacterial growth was unaffected by solid chitosan. Furthermore, high temperature treated cross-linked chitosan-glutaraldehyde showed strong antibacterial activity against the selected strain 0901 although the inhibitory effects varied with different temperatures. In addition, physical-chemical and structural characterization revealed that the cross-linking of chitosan with glutaraldehyde resulted in a rougher surface morphology, a characteristic Fourier transform infrared (FTIR) band at 1559 cm?1, a specific X-ray diffraction peak centered at 2? = 15�, a lower contents of carbon, hydrogen and nitrogen, and a higher stability of glucose units compared to chitosan based on scanning electron microscopic observation, FTIR spectra, X-ray diffraction pattern, as well as elemental and thermo gravimetric analysis. Overall, this study indicated that cross-linked chitosan-glutaraldehyde is promising to be developed as a new antibacterial drug.
Li, Bin; Shan, Chang-Lin; Zhou, Qing; Fang, Yuan; Wang, Yang-Li; Xu, Fei; Han, Li-Rong; Ibrahim, Muhammad; Guo, Long-Biao; Xie, Guan-Lin; Sun, Guo-Chang
Cross-linked acrylic hydrogel for the controlled delivery of hydrophobic drugs in cancer therapy
Objective: To investigate cross-linked hydrogels prepared via inverse emulsion polymerization to entrap poorly aqueous soluble drugs. Polyethylene glycol cross-linked acrylic polymers were synthesized and the loading and release of curcumin, a model hydrophobic drug, was investigated. Methods: Physicochemical characteristics of hydrogels were studied with 13C nuclear magnetic resonance, Fourier transform infrared spectroscopy, transmission electron microscopy, scanning electron microscopy, differential scanning calorimetry, and swelling. Polymerization of the acrylic acid with cross-linked polyethylene glycol diacrylate was characterized with 13C nuclear magnetic resonance imaging and Fourier transform infrared spectroscopy. Results: The in vitro release rate of curcumin showed that there was a sustained release from the hydrogel with increased cross-linking; the release rate depended on the pH of the releasing medium. Intracellular and cytotoxicity studies were carried out in human cervical cancer cell lines. Conclusion: The results suggest cross-linked acrylic polymers can be used as efficient vectors for pH-sensitive, controlled delivery of hydrophobic drugs.
Deepa, G; Thulasidasan, Arun Kumar T; Anto, Ruby John; Pillai, J Jisha; Kumar, GS Vinod
Biodegradable chitosan-based ambroxol hydrochloride microspheres: effect of cross-linking agents.
The objective of this study was to investigate the influence of type of cross-linking method used on the properties of ambroxol hydrochloride microspheres such as encapsulation efficiency, particle size, and drug release. Microspheres were prepared by solvent evaporation technique using chitosan as a matrix-forming agent and cross-linked using formaldehyde and heat treatment. Morphological and physicochemical properties of microspheres were then investigated by scanning electron microscopy (SEM), X-ray diffractometry (XRD), differential scanning calorimetry (DSC), and Fourier-transform infrared spectroscopy (FTIR) spectroscopy. The cross-linking of chitosan takes place at the free amino group because of formation of imine bond as evidenced by FTIR. The DSC, XRD, and FTIR analysis showed that chitosan microspheres cross linked by heating were superior in properties and performance as compared to the microspheres cross-linked using formaldehyde. SEM results revealed that heat-treated microspheres were spherical, discrete having smooth, and porous structure. The particle size and encapsulation efficiencies of the prepared chitosan microspheres ranged between 10.83-24.11 ?m and 39.73?80.56%, respectively. The drug release was extended up to 12 h, and the kinetics of the drug release was obeying Higuchi kinetic proving diffusion-controlled drug release. PMID:21607049
Gangurde, Hh; Chavan, Nv; Mundada, As; Derle, Dv; Tamizharasi, S
Dynamic viscoelastic properties of collagen gels with high mechanical strength.
We developed a new method for the preparation of mechanically strong collagen gels by combining successively basic gel formation, followed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) cross-linking and lyophilization. Gels cross-linked three times with this method showed stronger mechanical properties (G': 3730�2060 Pa, G?: 288�35 Pa) than a conventional gel that was sequentially cross-linked with EDC once (G': 226�70 Pa, G?: 21�4.4 Pa), but not as strong as the same gel with heating for 30 min at 80�C (G': 7010�830 Pa, G?: 288�35 Pa) reported in our previous paper. The conventional collagen gel was cross-linked with EDC once, heated once, and then subjected twice to a lyophilization-gel formation-cross-linking cycle to give three-cycled gel 2. This gel had the strongest mechanical properties (G': 40,200�18,000 Pa, G?: 3090�1400 Pa, Young's modulus: 0.197�0.069 MPa) of the gels tested. These promising results suggest possible applications of the gels as scaffolds in tissue engineering research. PMID:23706205
Mori, Hideki; Shimizu, Kousuke; Hara, Masayuki
A thermodynamic model of physical gels
NASA Astrophysics Data System (ADS)
Physical gels are characterized by dynamic cross-links that are constantly created and broken, changing its state between solid and liquid under influence of environmental factors. This restructuring ability of physical gels makes them an important class of materials with many applications, such as in drug delivery. In this article, we present a thermodynamic model for physical gels that considers both the elastic properties of the network and the transient nature of the cross-links. The cross-links' reformation is captured through a connectivity tensor M at the microscopic level. The macroscopic quantities, such as the volume fraction of the monomer ?, number of monomers per cross-link s, and the number of cross-links per volume q, are defined by statistic averaging. A mean-field energy functional for the gel is constructed based on these variables. The equilibrium equations and the stress are obtained at the current state. We study the static thermodynamic properties of physical gels predicted by the model. We discuss the problems of un-constrained swelling and stress driven phase transitions of physical gels and describe the conditions under which these phenomena arise as functions of the bond activation energy Ea, polymer/solvent interaction parameter ?, and external stress p.
An, Yonghao; Solis, Francisco J.; Jiang, Hanqing