+Papers presented by the contributors of W. Mejbaum-Katzenellenbogen's Melecular Biology Seminars.
Abstract This review describes the structure of the adenomatous polyposis coli (APC) gene with expressed proteins and their possible tissue interactions. The functions of APC in animals and humans are discussed. The mutational spectrum with clinical impact on colon pathology and gynaecological pathology similarities are presented, along with a discussion of a highly sensitive molecular biology test.
Abstract The design of liposomes with a hydrophilic/steric barrier at their bilayer surface allows the modification of their pharmacokinetics and reduces the uptake by the RES. Liposomes can be coated by hydrophilic molecules such as polysaccharides, which disguise the vesicle surface by creating a threedimensional matrix near them and prevent the binding of plasma proteins and their recognition by some cellular receptors. All these considerations, and previous results obtained in our laboratory showing the formation of stable GAGliposome complexes, have lead us to think about the use of the negatively charged glycosaminoglycans (GAGs), alternately to other molecules such as the monosialoganglioside GM1, more expensive, or polyethylene glycol (PEG-PE) that can disturb the structural organization of the bilayer. The present paper describes the effect of the incorporation of GAGs to phospholipid vesicles, in relation to their electrical and permeability properties. The results obtained show that there is an effective coating of the bilayer surface when glycosaminoglycans are added to liposome suspensions. The shielding of the negative surface charge by the neutral hyaluronic acid, in the absence of calcium, and the increase in the negative charge when the negative polyelectrolytes chondroitin sulfate, heparin or dextran sulfate are added to calcium-containing liposome suspensions account for the formation of stable liposome-GAG complexes. Moreover, the reduced permeability of the GAG-coated liposomes points out on their ability to hold encapsulated drugs and, so, their potential usefulness as drug-sustained release carriers. The hydrophilic coating will give to these liposomal carriers long-circulating properties.
Abstract The hydration of a variety of phospholipids has been studied, gravimetrically. The water adsorption isotherms obtained have been analyzed by the use of BET methodology. The major findings are as follows: Phospholipids containing choline, as part of their head group and having a glycerol backbone are "strong" water adsorbers; the nature and fluidity of the hydrocarbon chains do not control the "strong" vs "weak" nature of the water binding but do modulate the amount of adsorbed water. The number of methyl groups present on the N + moiety of the head groups determines both the nature of the water binding as well as the amount of water adsorbed. The electrical charge on the phospholipid head group does not, in itself, determine the nature of the water binding; it does, however affect the amount of water bound. All of the phospholipids studied exhibit a very rapid rise in electrical conductivity as water is adsorbed; this rise reaches a limiting saturation value at or close to that for the completion of the first monolayer of water adsorbed. These findings are discussed in terms of the molecular structure of the phospholipids.
Abstract O-alkyl derivatives of dioleoylphosphatidylcholine (DOPC) have been previously described as effective DNA transfection reagents. This communication reports the effects of the neutral helper lipid dioleoylphosphatidylethanolamine (DOPE) on the efficiency of transfection of BHK cells mediated by the O-ethyl-, O-hexyl-, and O-octadecyl- DOPC derivatives, compounds that by themselves are known to exhibit lyotropic phase preferences of lamellar, lamellar or cubic (depending on conditions) and inverse hexagonal, respectively. The effect of DOPE on transfection efficiency was found to be inhibition of the ethyl compound, stimulation or inhibition (depending on amount of DOPE) of the hexyl compound and stimulation in the case of the octadecyl compound, i.e., DOPE had a beneficial effect on the lipids that formed non-lamellar phases. X-ray diffraction was used to determine the lyotropic phase of the lipid-DOPE mixtures and of the lipid-DNA complex. DNA-lipid complexes tended to be lamellar unless the lipids had a very strong tendency toward the hexagonal phase, in which case the DNA complex was also hexagonal. Thus, a mixture of equal amounts of DOPE and hexyl-DOPC formed a lamellar complex with DNA, although the lipids on their own assumed the hexagonal phase. Octadecyl-DOPC formed a hexagonal phase with DOPE and the 1:1 DOPE mixture formed a hexagonal phase DNA complex; however, if smaller amounts of DOPE were included, the complex had a lamellar structure, in contrast to the hexagonal phase of the lipids by themselves. For these cationic phospholipids, there was not necessarily a benefit to transfection of generating a hexagonal phase lipid-DNA complex.
Abstract Interaction of two series of new surfactants with an incorporated antioxidant functional group with erythrocytes and planar lipid membranes was studied. Surfactants were synthesized in order to be potentially used as common bio-cides or as agents protecting biological and/or model membranes against lipid peroxidation. Both applications need the use of such bifunctional surfactants in significantly different concentrations. The aim of this work was to find the concentration range in which surfactants studied could be used as biocides. Two different models were chosen in order to do it; pig erythrocyte and asolectin planar membranes. The studied parameters of these models were hemolysis of red blood cells and stability of BLM in the presence of the compounds studied, i. e., the parameters describing mechanical properties of model membranes used. Additionally, the role of the counterions in the interaction of bifunctional surfactants with model membranes was studied. It was found that both homologous series of the surfactants influence model membranes to different degree depending on the length of their hydrophobic part and the kind of counterion. In the latter case it seems that the differences in the hydrated radii of bromide and chloride ions, and hence the differences in their ability to modify electrostatic interaction between the lipid polar heads and compounds studied, are responsible for the effects o bserved.
Abstract Malachite green (MG) consisting of green crystals with a metallic lustre, is very soluble in water and is highly cytotoxic to mammalian cells and also acts as a liver tumor promoter. In view of its industrial importance and possible exposure to human beings, MG poses a potential environmental health hazard. The malignant transformation of Syrian hamster embryo (SHE) cells by MG has been reported earlier. In this study, an attempt has been made to study the levels of vimentin, vimentin phosphorylation and the expression of PCNA and BrdU incorporation in MG transformed cells compared to control cells. Immunohistochemical and immunoprecipitation studies showed enhanced levels of vimentin in transformed cells compared to normal cells. Metabolic labelling studies showed an overall increase in phosphorylation of total cellular proteins as well as hyperphosphorylation of vimentin in transformed cells. Transformed cells also showed an increased doubling time, PCNA expression and BrdU incorporation. This study indicates a close relationship between vimentin levels, hyperphosphorylation of vimentin and increased cell proliferation associated with the malignant transformation of SHE cells.
the mutational variability in primary structure within this family is not consistent with the Markovian model of amino acid replacement.
Abstract The process of self-assembly and the electromechanical properties of bilayer lipid membranes (BLM) were investigated. The membranes were made of phosphatidylcholine with the addition of bolaamphiphilic steroid dimer. The membranes were formed using the Mueller-Rudin method. Membrane formation in the presence of the dimer was much faster and they were more stable than those formed in the absence of the dimer. The membranes formed by this method usually contain residues of a solvent used in the formation process which increases membrane thickness. Thus, the membranes formed from pure phosphatidylcholine had an average thickness of 5.9 nm. The addition of steroid dimer to the forming solution caused the thickness to decrease to 3.9 nm. An external voltage applied to the bilayer lipid membranes caused electrocompression. The presence of bolaamphiphilic steroid dimer in the membranes decreased the electrocompressibility by approximately 20 times. The dimer molecules â€žspannedâ€ť both monolayers of the membranes and caused the membrane thickness to decrease during their formation. The presence of the dimer in the membrane limited the mobility of solvent inside the membrane. The membranes formed with the dimer have such properties as thickness, stability, resistance, breakdown voltage, electrocompressibility, and time of formation more adequate for their application as a biomembrane model and support for sensors based on biomembrane molecules.
Abstract Many anti-cancer and antiviral drugs currently used are either unable or inefficient in their ability to pass through the blood brain-barrier and to enter and maintain therapeutic drug levels in brain. The low bioavailability of these drugs is a limiting factor in their use. In order to overcome these limitations, we ester-linked various anti-cancer and antiviral drugs to ceramide and phosphatidylcholine and created prodrugs possessing therapeutic attributes lacking in the parent compounds. This resulted in greater cellular uptake and prolonged retention of these prodrugs in vitro. Likewise, prodrug concentration was greater and retention time longer than the parent drug in the brain, testes and thymus of mice. Another major goal in drug development is discovering compounds that have efficacy against a specific microorganism or virus without significant side effects. For example, many potentially good drugs cannot be used because they are either toxic to uninfected cells or they cannot be restricted to a certain part of the body. If a drug could remain inert unless and until it is inside an infected cell, many of the common problems associated with drug treatments would be solved. In an attempt to address this problem we are developing a method by which a drug will be released only in cells that are infected with a particular microorganism or virus. The methodology makes use of the fact that microparticles are ingested by macrophages. Cell-specific treatment can be achieved by combining a drug with a microparticle using microorganismspecific enzyme substrates. Thus, release of active drug will occur only in the presence of enzymes specific to the target virus or microorganism. In the uninfected macrophage drug remains bound to the microparticle and is inactive. In the infected cell active drug is released by enzymatic hydrolysis. Potential applications for this technology include all diseases in which pathogens are resident in macrophages and other phagocytic cells.