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The invention relates to a method for coupling proteins to a starch-derived modified polysaccharide. The binding interaction between the modified polysaccharide and the protein is based on a covalent bond which is the result of a coupling reaction between the terminal aldehyde group or a functional group of the modified polysaccharide molecule resulting from the chemical reaction of this aldehyde group and a functional group of the protein which reacts with the aldehyde group or with the resulting functional group of the polysaccharide molecule. The bond directly resulting from the coupling reaction can be optionally modified by a further reaction to the aforementioned covalent bond. The invention further relates to pharmaceutical compositions that comprise conjugates formed in this coupling process and to the use of conjugates and compositions for the prophylaxis or therapy of the human or animal body.
1. A hydroxyethylstarch-protein conjugate, characterized in that the binding interaction between the hydroxyethylstarch molecule and the protein is a single covalent bonding which is the result of a coupling reaction between (i) the terminal aldehyde group of the hydroxyethylstarch molecule and (ii) a primary amino group of the protein to form a Schiff's base.
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14. A method for preparing a hydroxyethvlstarch-protein conjugate in which the binding interaction between the hydroxyethylstarch molecule and the protein is a single covalent bonding which is the result of a coupling reaction between (i) the terminal aldehyde group of the hydroxyethylstarch molecule and (ii) a primary amino group of the protein to form a Schiff's base, characterized in that the coupling reaction is carried out in aqueous solution between the terminal aldehyde group of the hydroxyethylstarch molecule and a primary amino group of the protein.
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This application is a National Stage application under 35 U.S.C. § 371 and claims benefit under 35 U.S.C. § 119(a) of International Application No. PCT/EP03/02083 having an International Filing Date of Feb. 28, 2003, which claims the benefit of priority of German Patent Application Ser. No. 10209821.2 having a filing date of Mar. 6, 2002.
The rapid development in genetic engineering in recent decades has led to the new identification of a large number of genes for proteins having potential therapeutic benefits and to the possibility of producing without difficulty the corresponding gene products, pure or nearly pure in relatively large quantities, with the aid of biological expression systems.
However, it has emerged that the use of such proteins in practice, e.g. in diagnosis, therapy and for biotransformations, frequently meets with difficulties because the stability and solubility properties thereof, especially at physiological pH values, are often unsatisfactory. Two examples of such proteins are tumor necrosis factor TNF-α or interleukin-2.
Solubility problems additionally occur very frequently in the expression of glycoproteins in prokaryotic systems such as E. coli, because they are then expressed without the natural glycosylation, resulting in a considerably reduced solubility in some cases. This may make it necessary to use considerably more costly eukaryotic expression systems.
On therapeutic use in the body, many proteins are very quickly removed from the bloodstream or degraded. Systemically administered proteins having a molecular weight of more than about 70 kDa may be removed from the circulation by the reticuloendothelial system or specific interactions with cellular receptors. Smaller proteins having a molecular weight of less than about 70 kDa may in addition be removed to a large extent by the glomerular filtration in the kidney (exclusion limit about 70 kDa).
An approach followed recently to eliminate the described problems consists in coupling such problematic proteins to biocompatible polymers with good solubility in water, such as, for example, polyethylene glycol and dextran. On the one hand, it is possible by the coupling to increase the molecular weight above the threshold of 70 kDa, so that the plasma residence time of smaller proteins can be drastically increased, and on the other hand the solubility in aqueous medium can be improved by the hydrophilic polymer portion.
Further, usually beneficial effects which may be connected with coupling of proteins to such polymers are based on the masking of protease recognition sites and antigenic determinants on the protein molecule by the bound polymer. On the one hand, it is possible thereby for the therapeutic proteins substantially to escape proteolytic degradation, and on the other hand there is substantial suppression of the induction of allergenic reactions by the exogenous therapeutic protein. Beyond the increase in molecular weight, proteins are thus protected by the presence of a polymer from enzymatic degradation and, in addition, often from thermal denaturation. In many cases, the stability and in vivo half-life of the proteins is markedly increased, and the immunogenicity and antigenicity falls, thereby.
To date, most modifications have been carried out with polyethylene glycol or dextran, with PEG being generally preferred because it affords simpler products.
Dextran couplings have been described for only a few proteins such as, for example, streptokinase, plasmin, hemoglobin or aprotinin. However, dextran conjugates often show high allergenicity, presumably caused by dextran degradation products, a low metabolic stability and, in many cases, low yields in the coupling reactions. This has led to none of these dextran coupling products being approved as yet for therapeutic use in humans or animals.
Derivatizations with PEG have been carried out considerably more frequently, so that this method can now be regarded as standard for increasing the molecular weight of proteins. Some of these derivatives are in various phases of clinical trials or are already approved in the USA. PEG-hemoglobin is currently in phase III, as is a PEG adduct of superoxide dismutase (SOD), which is the protein which has been investigated most in relation to polymer couplings. PEG-coupled asparaginase is already employed in the therapy of acute lymphocytic leukemia. In 2001, PEG-interferon-α was approved for the treatment of hepatitis C patients.
On use of these PEG conjugates, however, side effects ranging from unpleasant to dangerous have also been reported, such as pruritis, hypersensitivity reactions and pancreatitis. In addition, the biological activity of the proteins after PEG coupling is often very low and the metabolism of the degradation products of PEG conjugates is still substantially unknown and possibly represents a health risk.
WO 99/49897 describes conjugates of hemoglobin which are formed by reacting the aldehyde groups of oxidatively ring-opened polysaccharides such as hydroxyethylstarch or dextran with primary amine groups of the protein. However, in this case, the employed polysaccharides act as polyfunctional reagents, resulting in a very heterogeneous product mixture with properties which are difficult to adjust.
U.S. Pat. No. 6,083,909 describes a process for coupling selectively oxidized hydroxyethylstarch to hemoglobin in DMSO. Our investigations have shown, however, that the desired product is not obtained under the stated conditions, because hemoglobin is denatured in DMSO and thus loses its biological activity.
There is thus still a need for physiologically well tolerated alternatives to dextran- or PEG-coupled proteins, with which the solubility of proteins can be improved or the plasma residence time of the proteins can be increased.
It is therefore an object of the invention to provide such alternatives and to develop simple and efficient processes for preparing such alternative protein derivatives.
This object is achieved according to the invention by hydroxyalkylstarch-protein conjugates which are characterized in that the binding interaction between the hydroxyalkylstarch molecule and the protein is based on a covalent bonding which is the result of a coupling reaction between the terminal aldehyde group, or a functional group derived from this aldehyde group by chemical reaction, of the hydroxyalkylstarch molecule and a functional group, which is able to react with this aldehyde group or functional group derived therefrom of the hydroxyalkylstarch molecule, of the protein, where the bonding resulting directly in the coupling reaction can be modified where appropriate by a further reaction to give the abovementioned covalent bonding.
The invention further includes pharmaceutical compositions which comprise these conjugates, and the use of these conjugates and compositions for the prophylactic or therapeutic treatment of the human or animal body, and methods for preparing these conjugates and compositions.
It has surprisingly been found that the reactions described above can, with a suitable choice of the conditions, be carried out in aqueous solution, thus allowing the biological activity of the proteins in many cases to be completely or partly retained.
The aqueous reaction medium for the coupling reaction is in this case preferably water or a mixture of water and an organic solvent, where the proportion of water in the mixture is at least about 70% by weight, preferably at least about 80% by weight, more preferably at least about 90% by weight.
The molar ratio of hydroxyalkylstarch (HAS) to protein in the coupling reaction is usually about 20:1 to 1:1, preferably about 5:1 to 1:1.
The remaining biological activity of the inventive hydroxyalkylstarch-protein conjugates, based on the initial activity of the protein, is usually at least 40%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90%, most preferably at least 95%.
The hydroxyalkylstarch (HAS) employed according to the invention can be prepared by a known method, e.g. hydroxyalkylation of starch at the C2 and/or C6 position of the anhydroglucose units with alkylene oxide or 2-chloroalkanol, e.g. 2-chloroethanol (see, for example, U.S. Pat. No. 5,218,108 for the hydroxyethylation of starch), with various desired molecular weight ranges and degrees of substitution. It is also possible to employ any preparations obtainable commercially. The definition of the alkyl grouping in "hydroxyalkylstarch", as used herein, includes methyl, ethyl, isopropyl and n-propyl, with particular preference for ethyl. A substantial advantage of HES is that it is already approved by the authorities as biocompatible plasma expander and is employed clinically on a large scale.
The average molecular weight of the hydroxyalkylstarch can be in the range from about 3 kDa to several million daltons, preferably about 4 kDa to about 1000 kDa, more preferably in the range from about 4 kDa to about 50 kDa or in the range from about 70 kDa to about 1000 kDa, particularly preferably about 130 kDa. For coupling to small proteins, the average molecular weight of the hydroxyalkylstarch is preferably chosen so that the abovementioned threshold of 70 kDa is exceeded with the conjugates, whereas for coupling to large proteins the molecular weight of the hydroxyalkylstarch will preferably be in the lower region of said range. Since coupling is possible at a plurality of sites in a protein, it may also be advantageous to couple a plurality of small polymer chains, instead of one of high molecular weight. The degree of substitution (ratio of the number of modified anhydroglucose units to the number of anhydroglucose units in total) may likewise vary and will frequently be in the range from about 0.2 to 0.8, preferably about 0.3 to 0.7, more preferably about 0.5. (Note: the numbers relate to the "degree of substitution", which is between 0 and 1). The ratio of C2 to C6 substitution is normally in the range from 4 to 16, preferably in the range from 8 to 12.
These parameters can be adjusted by known methods. Experience with the use of hydroxyethylstarch (HES) as blood substitute has shown that the residence time of HES in the plasma depends on the molecular weight and the degree of substitution and type of substitution (C2 substitution or C6 substitution), with a higher molecular weight, a higher degree of substitution and a higher proportion of C2 substitution increasing the residence time.
These relationships also apply to the inventive hydroxyalkylstarch-protein conjugates, so that the residence time of a particular conjugate in the plasma can be adjusted via the proportion of polysaccharide.
Hydroxyethylstarch products with an average molecular weight of 130 kDa and a degree of substitution of 0.5, and with an average molecular weight of 200 kDa and a degree of substitution of 0.25, have already been used clinically as blood substitutes and are also suitable for use in the present invention.
The protein suitable in the present invention is in principle any protein which has the necessary functional group, e.g. a free amino group, thiol group or carboxyl group, for reacting with the functional group of the HAS molecule.
A desired functional group can be introduced also by reacting the protein with a suitable, physiologically tolerated, bifunctional linker molecule. The remaining reactive functional group of the coupled-on linker molecule is then likewise regarded as "reactive functional group of the protein" for the purposes of the present invention.
Suitable linker molecules comprise at one end a grouping able to enter into a covalent bonding with a reactive functional group of the protein, e.g. an amino, thiol, or carboxyl group, and at the other end a grouping likewise able to enter into a covalent bonding with the terminal aldehyde group or a functional group derived therefrom by chemical reaction, e.g. a carboxyl group, activated carboxyl group, amino or thiol group. Between the two functional groups of the linker molecule there is a biocompatible bridging molecule of suitable length, e.g. a grouping derived from an alkane, an (oligo)alkylene glycol grouping or another suitable oligomer grouping. Preferred groupings able to react with amino groups are, for example, N-hydroxysuccinimide esters, sulfo-N-hydroxysuccinimide esters, imido esters or other activated carboxyl groups; preferred groupings able to react with thiol groups are, for example, maleimide and carboxyl groups; preferred groupings able to react with aldehyde or carboxyl groups are, for example, amino or thiol groups.
Examples of linker molecules for connecting SH and NH functions are:
Examples of linker molecules for connecting SH and SH functions are:
Examples of linker molecules for connecting NH and NH functions are:
Examples of linker molecules for connecting SH and CHO functions are:
An example of a linker molecule for connecting SH and OH functions is
Examples of linker molecules for converting an SH function into a COOH function are
Examples of linker molecules for converting an NH function into a COOH function are MSA (methyl N-succinimidyl adipate) or longer-chain homologues thereof or corresponding derivatives of ethylene glycol.
Examples of linker molecules for converting a COOH function into an NH function are DAB (1.4-diaminobutane) or longer-chain homologues thereof or corresponding derivatives of ethylene glycol.
An example of a linker molecule which reacts with an amino group of a molecule and provides a protected amino group at a larger distance from this molecule to avoid steric hindrance is TFCS(N-ε(trifluoroacetylcaproyloxy)-succinimide ester).
Further suitable linker molecules are known to skilled workers and commercially available or can be designed as required and depending on the functional groups present and desired in the HAS and the protein to be coupled on, and be prepared by known methods.
The term "protein" for the purposes of the present invention is intended to include every amino acid sequence which comprises at least 9-12 amino acids, preferably at least 15 amino acids, more preferably at least 25 amino acids, particularly preferably at least 50 amino acids, and also include natural derivatives, e.g. pre or proforms, glycoproteins, phosphoproteins, or synthetic modified derivatives, e.g. fusion proteins, neoglycoproteins, or proteins modified by genetic engineering methods, e.g. fusion proteins, proteins with amino acid exchanges to introduce preferred coupling sites.
For the prophylactic or therapeutic treatment of the human or animal body, the relevant protein will carry out a particular desired function in the body. The protein therefore preferably has, for example, a regulatory or catalytic function, a signal transmitting or transport function or a function in the immune response or induction of an immune response.
The protein may be selected for example from the group composed of enzymes, antibodies, antigens, transport proteins, bioadhesion proteins, hormones, growth factors, cytokines, receptors, suppressors, activators, inhibitors or a functional derivative or fragment thereof. "Functional derivative or fragment" means in this connection a derivative or fragment which has retained a desired biological property or activity of the parent molecule in whole or in part, e.g. to the extent of at least 10-30%, preferably more than 50%, even more preferably more than 70%, most preferably more than 90%. Particularly preferred examples of such a fragment are antibody fragments.
Specific examples are α-, β- or γ-interferon, interleukins, e.g. IL-1 to IL-18, growth factors, e.g. epidermal growth factor (EGF), platelet growth factor (PDGF), fibroblast growth factor (FGF), brain-derived growth factor (BDGF), nerve growth factor (NGF), B-cell growth factor (BCGF), brain-derived neurotrophic growth factor (BDNF), ciliary neurotrophic factor (CNTF), transforming growth factors, e.g. TGF-α or TGF-β, colony-stimulating factors (CSF), e.g. GM-CSF, G-CSF, BMP (bone morphogenic proteins), growth hormones, e.g. human growth hormone, tumor necrosis factors, e.g. TNF-α or TNF-β, somatostatin, somatotropin, somatomedins, serum proteins, e.g. clotting factors II-XIII, albumin, erythropoietin, myoglobin, hemoglobin, plasminogen activators, e.g. tissue plasminogen activator, hormones or prohormones, e.g. insulin, gonadotropin, melanocyte-stimulating hormone (α-MSH), triptorelin, hypothalamus hormones, e.g. antidiuretic hormones (ADH) and oxytocin, and liberins and statins, parathyroid hormone, thyroid hormones, e.g. thyroxine, thyrotropin, thyroliberin, prolactin, calcitonin, glucagon, glucagon-like peptides (GLP-1, GLP-2, etc.), exendins, e.g. exendin-4, leptin, vasopressin, gastrin, secretin, integrins, glycoprotein hormones (e.g. LH, FSH, etc.), pigmentary hormones, lipoproteins and apolipoproteins, e.g. Apo-B, Apo-E, Apo-La, immunoglobulins, e.g. IgG, IgE, IgM, IgA, IgD or a fragment thereof, hirudin, tissue pathway inhibitor, plant proteins, e.g. lectin or ricin, bee venom, snake venoms, immunotoxins, antigen E, butroxobina, alpha-proteinase inhibitor, ragweed allergen, melanin, oligolysine proteins, RGD proteins or, where appropriate, corresponding receptors for one of these proteins; or a functional derivative or fragment of one of these proteins or receptors.
Suitable enzymes may be selected for example from the groups of carbohydrate-specific enzymes, proteolytic enzymes, oxidases, oxidoreductases, transferases, hydrolases, lyases, isomerases, kinases and ligases. Specific, non-restrictive examples are asparaginase, arginase, arginine deaminase, adenosine deaminase, glutaminase, glutaminase-asparaginase, phenylalanine ammonia-lyase, tryptophanase, tyrosinase, superoxide dismutase, an endotoxinase, a catalase, peroxidase, kallikrein, trypsin, chymotrypsin, elastase, thermolysin, a lipase, a uricase, adenosine diphosphatase, purine-nucleoside phosphorylase, bilirubin oxidase, a glucose oxidase, glucodase, gluconate oxidase, galactosidase, glucocerebrosidase, glucuronidase, hyaluronidase, tissue factor, a tissue plasminogen activator, streptokinase, urokinase, MAP kinases, DNAses, RNAses, lactoferrin, and functional derivatives or fragments thereof.
As mentioned above, the functional group of the HAS molecule involved in the coupling reaction is the terminal aldehyde group or a group derived therefrom by chemical reaction.
One example of such a chemical reaction is the selective oxidation of this aldehyde group with a mild oxidizing agent such as, for example, iodine, bromine or some metal ions, or else by means of electrochemical oxidation to a carboxyl group or activated carboxyl group, e.g. an ester, lactone, amide, with the carboxyl group being converted where appropriate in a second reaction into the activated derivative. This carboxyl group or activated carboxyl group can then be coupled to a primary amino or thiol group of the protein to form an amide linkage or thioester linkage.
In a particularly preferred preparation method, this aldehyde group is selectively oxidized with a molar excess of iodine, preferably in a molar ratio of iodine to HAS of from 2:1 to 20:1, particularly preferably about 5:1 to 6:1, in aqueous basic solution. In the optimized method described in example 1, initially an amount of hydroxyalkylstarch is dissolved in hot distilled water, and somewhat less than 1 mole equivalent of aqueous iodine solution, preferably in a concentration of about 0.05-0.5N, particularly preferably about 0.1N, is added. After this, an aqueous NaOH solution in a molar concentration which is about 5-15 times, preferably about 10 times, that of the iodine solution is slowly added dropwise, at intervals of a plurality of minutes, to the reaction solution until the solution starts to become clear again after the addition. Somewhat less than 1 mole equivalent of the above aqueous iodine solution is again added to the reaction solution, the dropwise addition of the NaOH solution is resumed, and the addition of iodine and NaOH are repeated until approximately 5.5-6 mole equivalents of iodine solution and 11-12 mole equivalents of NaOH solution, based on the hydroxyalkylstarch, have been added. The reaction is then stopped, the reaction solution is desalted, e.g. by dialysis or ultrafiltration, subjected to a cation exchange chromatography, and the reaction product is obtained by lyophilization. In this method, virtually quantitative yields are achieved irrespective of the molecular weight of the HAS.
In a further particularly preferred embodiment, the selective oxidation takes place with alkaline stabilized solutions of metal ions, e.g. Cu++ or Ag+, likewise in approximately quantitative yields (example 2). It is preferred in this case to employ an approximately 3-10 times molar excess of the oxidizing agent.
The selectively oxidized hydroxyalkylstarch (ox-HAS) which has been formed is subsequently reacted in the presence of an activating reagent with a free amino group of the desired protein to form an amide linkage. Examples of suitable activating reagents are N-hydroxysuccinimide, N-hydroxyphthalimide, thiophenol, p-nitrophenol, o,p-dinitrophenol, trichlorophenol, trifluorophenol, pentachlorophenol, pentafluorophenol, 1-hydroxy-1H-benzotriazole (HOBt), HOOBt, HNSA, 2-hydroxypyridine, 3-hydroxypyridine, 3,4-dihydro-4-oxobenzotriazin-3-ol, 4-hydroxy-2,5-diphenyl-3(2H)-thiophenone 1,1-dioxide, 3-phenyl-1-(p-nitrophenyl)-2-pyrazolin-5-one), [1-benzotriazolyl-N-oxytris(dimethylamino)phosphonium hexafluoro-phosphate] (BOP), [1-benzotriazolyloxytripyrrolidino-phosphonium hexafluorophosphate (PyBOP), [O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), [O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU), [O-(benzotriazol-1-yl)-N,N,N′,N′-bis(pentamethylene)uronium hexafluorophosphate, [O-(benzotriazol-1-yl)-N,N,N′,N′-bis(tetramethylene)uronium hexafluorophosphate, carbonyldiimidazole (CDI), or preferably carbodiimides, e.g. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPC), particularly preferably EDC. In contrast to conventional methods described in the literature for similar coupling reactions, it has surprisingly been found in this connection that on use of a carbodiimide as a rule the use of otherwise obligatory further activators such as triazoles, e.g. HOBt, is unnecessary or even makes the yields worse. In the inventive coupling of ox-HES to various model compounds in the presence of EDC and absence of HOBt by contrast it was possible to achieve high yields substantially irrespective of the molecular weight of the HES (see examples).
Instead of the reaction of the carboxyl group or activated carboxyl group with a free primary amino group of the protein (e.g. of a lysine or arginine residue), an analogous reaction with a thiol group (of a cysteine) of the protein is also possible in principle. However, it must be taken into account in this connection that cysteines are usually involved in S—S bridges and are therefore not available for a coupling reaction. If, on the other hand, free cysteines are present, they frequently play an important part in catalysis or are involved in the contact site of subunits. A modification of these cysteines will then result in partial or complete loss of the biological activity. This problem could be eliminated by introducing free cysteines by conventional genetic engineering methods such as, for example directed mutagenesis or chemical peptide synthesis at those sites in the protein which are known to play no part in the activity. Optimal control of the coupling site is possible in this way. Targeted introduction of other reaction amino acids, e.g. Lys, His, Arg, Asp, Glu, into the protein would also be possible in the same way.
The reactive group of the hydroxyalkylstarch molecule can also be an amine or thiol group produced by chemical reaction of the terminal aldehyde group. For example, a reductive amination of the aldehyde group can be carried out by reaction with ammonia in the presence of hydrogen and a catalyst or in the presence of sodium cyanoborohydride. The resulting amino or thiol group can then react with a free carboxyl group of the protein (e.g. of an optionally activated glutamic or aspartic acid) to form an amide or thioester linkage.
A further possibility is for the terminal aldehyde group of the hydroxyalkylstarch molecule or a functional group derived therefrom by chemical reaction also to be reacted with a suitable physiologically tolerated bifunctional linker molecule. In this case, the "functional group derived from the terminal aldehyde group of the hydroxyalkylstarch molecule by chemical reaction" for the coupling reaction is the remaining reactive functional group of the bifunctional linker molecule with which the terminal aldehyde group or the functional group derived therefrom has been reacted. It is possible in this way likewise to convert the terminal aldehyde group into a desired functional group.
Suitable linker molecules comprise at one end a group able to enter into a covalent bonding with the terminal aldehyde group or a functional group derived therefrom by chemical reaction, e.g. a carboxyl group, activated carboxyl group, amino or thiol group, and at the other end a group able to enter into a covalent bonding with a reactive functional group of the protein, e.g. an amino, thiol or carboxyl group. Between the two functional groups of the linker molecule there is a biocompatible bridging molecule of suitable length, e.g. a grouping derived from an alkane, an (oligo)alkylene glycol grouping or another suitable oligomer grouping. Preferred groupings able to react with amino groups are, for example, N-hydroxysuccinimide esters, sulfo-N-hydroxysuccinimide esters, imido esters or other activated carboxyl groups; preferred groupings able to react with thiol groups are, for example, maleimide and carboxyl groups; preferred groupings able to react with aldehyde or carboxyl groups are, for example, amino or thiol groups.
A number of specific, non-restrictive examples of suitable linker molecules have already been indicated above with reference to the conjugation of linker molecules to the protein.
In an alternative inventive coupling method of the present invention, the terminal aldehyde group is reacted directly with a primary amino group (e.g. of a lysine or arginine residue or of the N-terminus) of the protein to form a Schiff's base. The formed Schiff's base is, subsequent or parallel thereto, reduced by reaction with a suitable reducing agent, resulting in a bonding which is stable in aqueous medium between protein and HAS. Preferred reducing agents are sodium borohydride, sodium cyanoborohydride, organic boron complexes, e.g. a 4-(dimethylamino)pyridine-boron complex, N-ethyldiisopropylamine-boron complex, N-ethylmorpholine-boron complex, N-methylmorpholine-boron complex, N-phenylmorpholine-boron complex, lutidine-boron complex, triethylamine-boron complex, trimethylamine-boron complex; suitable stereoselective reducing agents are, for example, sodium triacetate borohydride, sodium triethylborohydride, sodium trimethoxyborohydride, potassium tri-sec-butylborohydride (K-Selectride), sodium tri-sec-butylborohydride (N-Selectride), lithium tri-sec-butylborohydride (L-Selectride), potassium triamylborohydride (KS-Selectride) and lithium triamyl-borohydride (LS-selectride).
The yields can be improved by suitable variation of the reaction conditions. Parameters for such optimization tests are the pH of the reaction mixture (possible protein degradation by alkaline borohydride), temperature and duration of the incubation, and nature of the reducing agent for the one-pot reaction. A further alternative is the possibility of carrying out the reaction in two steps, in which case an immobilized reducing agent can be employed for the reduction step.
The products of the coupling reaction can be investigated by known methods, and the coupling efficiency can be established. Thus, for example, the free primary amino groups in the protein can be determined before and after the coupling with trinitrobenzenesulfonic acid (Habeeb, ASAF, Anal. Biochem. 14, 328-336 (1966)). The coupling yield of reactions involving primary amines could also be established by derivatization of the unreactive amines with fluorescamine and determination of the fluorescence. The molecular weight distribution can be established by SDS-PAGE and gel permeation. The protein content in the conjugate can be detected by SDS-PAGE and subsequent silver staining, while the saccharide content can be established by a glycan-specific staining of the bands separated by SDS-PAGE after blotting onto a membrane. Quantitative glycan determination is also possible. Exact identification of the coupling site on the protein is possible by peptide mapping and/or MALDI-TOF mass spectroscopy or electrospray ionization mass spectroscopy. It is possible in this way to optimize the coupling and to predetermine the molecular weight distribution and possibly (e.g. if the reactive groups on the protein differ in reactivity) even the coupling site of the products.
The conjugates of the present invention can where appropriate be employed as such or in the form of a pharmaceutical composition for the prophylactic or therapeutic treatment of the human or animal body.
Compositions of this type include a pharmaceutically effective amount of a conjugate of the invention as active ingredient, and a pharmaceutically suitable carrier and, where appropriate, other therapeutic or pharmaceutical ingredients or excipients. Excipients may include for example diluents, buffers, flavorings, binders, surface-active agents, thickeners, lubricants, preservatives (including antioxidants) and substances which serve to make the formulation isotonic with the blood of the intended recipient. A pharmaceutically effective amount is the amount sufficient to display on single or multiple administration a desired beneficial effect during a treatment to alleviate, cure or prevent a pathological condition. A pharmaceutically acceptable carrier is a carrier which is compatible both with the active pharmaceutical ingredient and with the patient's body.
The form of the composition will vary depending on the desired or suitable administration route. A preferred route is parenteral administration, e.g. subcutaneous, intramuscular, intravenous, intraarterial, intraarticular, intrathecal, extradural injection or, where appropriate, infusion. Intranasal, intratracheal or topical administration is also possible. Topical administration of growth factors conjugated according to the invention might for example speed up wound healing. The pharmaceutical compositions may beneficially be supplied in the form of a dosage unit and be produced by any method well known in the pharmacy sector.
The conjugates of the present invention can also be employed in all other sectors in which other protein-polymer conjugates, e.g. PEG-protein conjugates, have been used. Some specific, non-restrictive examples are the use of an HAS-protein conjugate as immobilized catalyst or reactant for a reaction in heterogeneous phase or as a column material for (immuno)affinity chromatography. Further possible uses will be plainly evident to the skilled worker with knowledge of the properties disclosed herein of the inventive HAS-protein conjugates.
The following examples are intended to explain the invention in more detail without, however, restricting it thereto. In particular, analogous reactions can also be carried out with hydroxymethylstarch and hydroxy-propylstarch, and similar results can be achieved.
Selective Oxidation of Hydroxyethylstarch (HES) with Iodine
10 g of HES-130 kDa were dissolved in 12 ml of deionized water by heating in a round-bottomed flask. 2 ml of an 12 solution (0.1N) were added to this solution. A pipette with 2 ml of 1.0N NaOH was connected to the flask via a 2-way connector, and the NaOH solution was added dropwise at about 1 drop every 4 minutes. The solution was decolorized after addition of approximately 0.2 ml of the NaOH solution and, at this time, a second portion of 2 ml of 0.1N iodine solution was added. The reaction was complete after addition of a total of 14 ml of iodine solution and 2.8 ml of NaOH solution. The reaction mixture was then dialyzed against deionized water.
The partially desalted solution was subjected to a chromatography on a cation exchange column (Amberlite IR-120, H+ form) in order to convert the aldonate groups into aldonic acid groups. Subsequently, the water was removed by lyophilization, and thus the lactone form was obtained.
Determination of the Degree of Oxidation:
1 ml of alkaline copper reagent (3.5 g of Na2PO4, 4.0 g of K Na tatrate in 50 ml of H2O, plus 10 ml of 1N NaOH, 8.0 ml of 10% strength (weight/volume) CuSO4 solution and 0.089 g of K iodate in 10 ml of H2O, after addition of 18 g of Na sulfate, make up to 100 ml) are pipetted in each case into 1 ml of sample solution under an N2 atmosphere. The mixture is heated at 100° C. for 45 minutes. After cooling, 0.2 ml of 2.5% strength KI solution and 0.15 ml of 1 M H2SO4 are added. After 5 min, 1 drop of phenol red indicator solution (1% weight/volume) is added, and titration is carried out with 5 mM Na2S2O3 solution until the color disappears. The concentration of unreacted aldehyde groups can be calculated from the consumption of titrant.
An approximately quantitative yield was achieved (>98%). It is possible by this procedure to oxidize hydroxyethylstarches with higher molecular weight (e.g. 130 kDa, 250 kDa, 400 kDa) just like hydroxyethylstarches with lower molecular weight (e.g. 10 kDa, 25 kDa, 40 kDa), in similarly high yields.
Selective Oxidation of HES with Cu2+ Ions
A solution of 0.24 mmol of HES-130 kD was prepared in 10 ml of deionized water with heating. This solution was heated in a 100 ml round-bottomed flask to a temperature of 70-80° C., and 1.17 mmol of stabilized Cu2+ (e.g. Rochelle salt as stabilizer or other stabilizers) and dilute aqueous NaOH solution was added (final concentration 0.1N NaOH). The temperature was then raised to 100° C., and the reaction was allowed to proceed until a reddish color had appeared. The reaction was stopped and the reaction mixture was cooled to 4° C. The reddish precipitate was removed by filtration. The filtrate was dialyzed against deionized water and then converted into the lactone as in example 1 and lyophilized. The oxidation took place quantitatively (yield >99%). It was also possible by this method to oxidize low molecular weight HES (e.g. HES-10 kDa, HES-25 kDa, HES-40 kDa) and higher molecular weight HES species.
Coupling of Selectively Oxidized High Molecular Weight HES (ox-HES-130 kDa) to Human Serum Albumin (HSA)
4.3 g of ox-HES-130 kDa and 200 mg of HSA (Sigma, Taufkirchen) were completely dissolved in water by gentle heating in a round-bottom flask with magnetic stirrer. 30 mg of ethyldimethylaminopropylcarbodiimide (EDC), dissolved in water, were added to this solution. After stirring very moderately for 2 h, a second portion of 30 mg of EDC was added. After stirring very moderately for a further two hours, a third portion of 40 mg of the carbodiimide was added. The reaction mixture was left under these conditions overnight, dialyzed against distilled water for 15 h and lyophilized. The success of the coupling was demonstrated by gel permeation chromatography, SDS-PAGE and carbohydrate-specific staining (Glyco-Dig kit from Roche-Boehringer, Basle) after blotting onto a PVDF membrane. The yield of coupling product was about 90%.
Coupling of Selectively Oxidized Low Molecular Weight HES (ox-HES-10 kDa) to Human Serum Albumin (HSA)
7.4 g of ox-HES-10 kDa and 50 mg of HSA were completely dissolved in water in a round-bottom flask with magnetic stirrer. The reaction was carried out by the method described above for high molecular weight HES, adding a total of 282 mg of EDC in three aliquots. The reaction mixture was likewise dialyzed and lyophilized as described above. Analysis (as above) showed the coupling product was obtained, but the yields were somewhat lower than in the coupling with high molecular weight ox-HES.
Coupling of ox-HES-130 kDa to Myoglobin (Mb)
4.3 g of ox-HES-130 kDa were completely dissolved in water (6-7 ml), and then 100 mg of Mb (Sigma, Taufkirchen), dissolved in 10 ml of 0.1 M phosphate buffer (pH 7.0), were added. The coupling reaction was started by adding 30 mg of EDC. Addition of EDC was repeated every 2 hours until a total of 90 mg of the carbodiimide had been consumed. The reaction mixture was then dialyzed against 50 mM phosphate buffer, pH 7.0, and lyophilized. GPC showed a definite product peak, which was detected in the hold-up volume at 450 nm. It was possible to calculate a coupling yield of 88% from this. The oxygen-binding capacity of the hesylated myoglobin was about 76% of the binding capacity of unmodified Mb.
Coupling of ox-HES-10 kDa to Superoxide Dismutase (SOD)
One part by volume of an aqueous solution of ox-HES-10 kDa (1.05 g/ml) was incubated with one part by volume of a 7 mg/ml SOD solution (Sigma, Taufkirchen) in 50 mM phosphate buffer, pH 7.6, at room temperature. The coupling reaction was initiated by adding 280 mg of EDC in 5 portions over a period of 24 h. The progress of the reaction was followed by GPC analysis in phosphate buffer and detection at 280 nm. After 24 h, 81% of the protein were found in the higher molecular weight region of the separating column, and the reaction was stopped after this time. The reaction mixture was subjected to a diafiltration with a 30 kDa membrane and then lyophilized. Mass spectrometric analysis of the product showed an average molar ratio of HES to protein of about 3:1.
Coupling of ox-HES-130 kDa to Streptokinase (SK)
3.8 kg of ox-HES-130 kDa were dissolved together with 35 mg of streptokinase (Sigma, Taufkirchen) in the minimum amount of 50 mM phosphate buffer, pH 7.2. At room temperature, 46.5 mg of EDC and 20 mg of 1-hydroxybenzotriazole hydrate (HOBt) were added, and reaction was maintained with gentle stirring for a total of 24 h. After dialysis and freeze drying, about 78% of the protein were found as HES conjugate by GPC analysis. In the SDS-PAGE with silver staining, a distinct increase in the molecular mass of the streptokinase was observable. In parallel with this, carbohydrate structures were unambiguously detectable in the high molecular waveband with the digoxigenin method.
Coupling of ox-HES-130 kDa to Human Interleukin-1 (IL-2)
45 mg of ox-HES-130 kDa were completely dissolved in 0.5 ml of 50 mM Na phosphate buffer, pH 6.5, with gentle heating. After addition of 0.25 mg of human IL-2 (Sigma, Taufkirchen), which made the solution opaque, the mixture was stirred at room temperature for 4-6 h. Then 5 mg of EDC were added in 4 portions with a time difference of 2 h for each, and stirring was continued overnight, resulting in a clear solution. GPC analysis revealed a coupling yield of about 65%.
Coupling of ox-HES-25 kDa to Human Tumor Necrosis Factor α (TNFα)
0.3 mg of hTNFα (Sigma, Taufkirchen) were added to 86 mg of ox-HES-25 kDa in about 0.4 ml of 0.1 M phosphate buffer (pH 7.0). The cloudy solution was stirred for about 2 h before 1 mg of EDC and 0.5 mg of HOBt were added. Stirring was continued for about 6 h, with the solution becoming clear during the reaction time. The coupling product was isolated by ultrafiltration and freeze drying and analyzed by GPC and detection at 280 nm. A coupling yield of approximately 74% was found in this case.
Coupling of ox-HES-130 kDa to Glucagon-Like Peptide (GLP-1)
7.4 g of ox-HES-130 kDa were dissolved in a minimum volume of water by heating and gentle stirring. A solution of 10 mg of GLP-1 in the amide form (Bachem, Switzerland) in 50 mM phosphate buffer, pH 7.4, was added by pipette. The reaction was started by adding 35 mg of EDC and was cautiously stirred for 2 h. This was repeated 2× more because, after this time, a peptide peak was no longer evident in the GPC analysis at 280 nm, i.e. approximately complete conversion to the coupling product had taken place. This coupling product was diafiltered using a 30 kDa membrane and lyophilized from phosphate buffer solution. It was possible to conclude from the results of a MALDI mass spectroscopy that the stoichiometry between peptide and HES was 1:1.
Coupling of High Molecular Weight HES (HES-130 kDa) to Human Serum Albumin (HSA)
9.75 g of HES-130 KD were completely dissolved in water (6-7 ml), and then 50 mg of HSA, dissolved in 1 ml of 0.1 M phosphate buffer (pH 7.4) were added. The reaction mixture was stirred with a magnetic stirrer. The solution was then mixed with NaBH3CN (50-70 mg) and stirred gently for a few minutes. The solution was further stirred for 15 minutes every two hours. Then a further aliquot of NaBH3CN (about 50 mg) was added. At the end (after a reaction time of almost 36 h), a total amount of 285 mg of NaBH3CN had been employed. The solution was then dialyzed and lyophilized. Analysis took place as described in example 4. The coupling efficiency was about 65%.
Coupling of Low Molecular Weight HES (HES-10 kDa) to Human Serum Albumin (HSA)
4.5 g of HES were completely dissolved in water (4-5 ml) and 50 mg of HSA, dissolved in 1 ml of 0.1 M phosphate buffer (pH 7.4) were added. When the solution was clear, if necessary effected by stirring with a magnetic stirrer, NaBH4 (50-70 mg) was added and mixed in with gentle stirring. The solution was left to stand without stirring for two hours and then stirred for 15 minutes every two hours as for the reaction with high molecular weight HES. When the solution no longer showed any bubbles (H2 evolution), a further aliquot of NaBH4 (about 50 mg) was added. At the end, a total amount of 180 mg of NaBH4 had been employed. The solution was then dialyzed and lyophilized. Analysis took place by gel permeation chromatography (GPC), and the yield was about 15%.
Coupling of HES-40 kDa to Asparaginase
3.0 g of HES-40 kDa were completely dissolved in water (about 4 ml). A solution of 80 mg of asparaginase (Sigma, Taufkirchen) in 6 ml of 0.1 M borate buffer, pH 9.0, were added thereto and stirred until the reaction mixture was clear. The temperature was then raised to 37° C. and, after 2 h, about 50 mg of NaBH3CN were added. This reaction cycle was repeated 3× more. The product was worked up by dialyzing the reaction mixture against 0.1 M phosphate buffer, pH 7.4. The yield of coupling product was about 61%, and about 73% of the asparaginase activity was recoverable.
Coupling of HES-130 kDa to Human Interleukin-2 (IL-2)
50 mg of HES-130 kDa were completely dissolved in water (about 0.2 ml). A suspension of 0.25 mg of human IL-2 (Sigma, Taufkirchen) in 0.2 ml of 0.1 M borate buffer, pH 9.0, was added thereto and stirred until the reaction mixture was clear (4 h). 1 mg portions of NaBH3CN were added at intervals each of 4 h, and stirring was continued. After a further reaction time of 24 h, the mixture was dialyzed against 0.1 M phosphate buffer, pH 7.4 and lyophilized. The yield of coupling product was about 42% according to GPC analysis.
Coupling of HES-130 kDa to Insulin
4.0 g of HES-130 kDa were completely dissolved in water (about 6 ml). 55 mg of insulin from bovine pancreas (Sigma, Taufkirchen) in 7.5 ml of 0.1 M borate buffer (pH 9.0), were added thereto and stirred at 37° C. for about 24 h. The reducing agent NaBH3CN (60 mg in 30 ml) was slowly added dropwise over a period of 8 h. The reaction mixture was then stirred for a further 24 h and freed of faults and unreacted reagents by ultrafiltration (30 ). Lyophilization resulted in a stable coupling product. About 55% of the insulin employed was recovered as HES conjugate.
Coupling of ox-HES-130 kDa to Superoxide Dismutase (SOD)
130 mg of ox-HES-130 were completely dissolved in 6 ml of PBS pH 6, and then 10 mg of SOD (Roche, Mannheim) dissolved in 1 ml of PBS pH 6 were added. The coupling reaction was started by adding 10 mg of EDC. Addition of EDC was repeated every 3 h until 39 mg of the carbodiimide had been consumed. The reaction was monitored by GPC at 258 nm. After 24 h, 50% of the protein were found in the high molecular weight region of the separating column, and the reaction was stopped. The reaction mixture was dialyzed against 25 mM phosphate buffer pH 7.2 and lyophilized. The SOD activity was 95% of the initial activity. Determination of the mass distribution of HES protein samples by coupled GPC-light scattering revealed a molar ratio of HES to protein of 1:1.
Coupling of ox-HES 70 kDa to Glucagon
Glucagon (66×10−9 mol, 0.23 mg), oxHES 70 kDa (6.6×10−6 mol, 123 mg) were dissolved in phosphate buffer (1 ml, pH 5) in a round-bottom flask. 26 mg of EDC were added in 10 portions at intervals of 1 h. After a reaction time of 24 h, the reaction was stopped by adding 10 ml of water. The coupling product was purified by after dialysis against water by GPC and ion exchange chromatography. Freeze drying resulted in 88 mg of white coupling product (73%).
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Lately I've had an obsession with dust. Dust seems to be accumulating at a crazy rate in my house. I constantly see dust on tables, walls, picture frames, and blinds. My husband tells me that its normal, but I'm 99% sure he is lying to keep me from doing something radical. I swear to him that I've never experienced so much dust in my entire life and he always replies with, "Well you were raised by Debbie." Ummm I don't understand how my being raised by Debbie completely zeroes out my concerns, however it seems to be the end of every Dust conversation we've had this month. Yes, Debbie is clean. Yes, I grew up in a house that had vacuum marks in at least 3 rooms at all time. Yes, we scoured our bathrooms hardcore 2x a week. That does not mean though that dust is just something that happens! Argh. Seriously, this dust situation has me thinking of renting some sort of air filtration/humidifier to clean out the air in my home.
Anyway, today I spent a large portion of my day dusting my bedroom from ceiling to floor. Literally. With mop in hand I wiped down the ceiling, fan blades, walls, blinds and baseboards. I also washed every piece of textile in my room. (Washing my bedding is an obsession too.)
Dusting was going ok until I discovered that my drapes had fell victim to dust too. I guess I get so focused on surfaces that I fail to notice the dust accumulating on drapes. With a dust meltdown threatening to consume me, I put on a brave face and decided it was time to SAVE MY DRAPES!
Today I only had time to vacuum and lint-roll my drapes, however I'm hoping to find an easy tutorial for deep cleaning them. Any ideas? Do you steam your drapes? Spray? Help…
Yes this is a picture of my mattress and memory foam. Tacky, but I love it. Also, I am aware that there is a knob missing from my nightstand.
There is nothing like stripping off dirtied bedding and waiting for the fresh sheets, coverlet, and pillow cases to get out of the dryer!
The minute I completed making my bed Piper of course had to soil my perfectly cleaned space. Luckily I'm more obsessed with her then cleaning. What a vicious cycle.
This is me finally getting done with D-day around 5:30 pm. After all my hard work I was tempted to climb into bed and take a short nap… but realizing I hadn't showered today, I quickly gave up the notion and moved my dirty-self to the family room floor.
Tomorrow Dusting 2.0 will begin and I will be one room closer to a less dusty home.
If you have any tips that would ease my dusting/drape burdens, please tell me.

subset: commoncrawl


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Results: 1 - 5 of 5
17 things you go through when joining a golf club
A humorous look at the steps to joining a golf club
January 2015
- keywords:
- golf,
- humor,
- golf club,
- membership
Golf & Business
Jersey club to ex-member: You'll get your money when we're good and ready
By Peter Finch Many country clubs have promised "refundable" initiation fees in recent years as a way of attracting members. But when members leave, they often find it's not so easy getting their money...
Friday, 6/6/2014 at 3:32PM
Golf Courses & Golf Travel / Golf Courses
Nine Is Fine: Golf's New Lucky Number?
The game's leaders are ready to promote shorter rounds.
September 2013
- keywords:
- short golf courses,
- nine holes,
- usga,
- membership,
- time for nine
Magazines / Golf & Finance
Money Clip: Open Invitation
New ways to play hundreds of private-club courses.
June 2013
- keywords:
- golf digest,
- Peter Finch,
- finance,
- private courses,
- membership
My Shot: Lou Holtz
After missing out as cover boy to Jack Nicklaus, the coach (and Augusta National member) has his say
- keywords:
- Golf,
- Golf Digest,
- My Shot: Lou Holtz,
- Masters,
- Guy Yocom
Results: 1 - 5 of 5


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Wednesday, January 22, 2014
President of NAACP, Georgetown Chapter
Native of Georgetown
Lives in Santee section of Georgetown County
Retired as service operator from International Paper Co. after 41 years
Member of North Santee Action Club and Singleton A.M.E. Church
How long have you been involved with the NAACP?
I've been involved with the NAACP since back during the marches of Dr. Martin Luther King Jr.
This time of year brings back lot of fond memories.
I got to meet Dr. King and I marched with him several times.
He was a good man.
I'm glad we now celebrate his birthday as a national holiday.
The NAACP was represented in Saturday's MLK parade in Georgetown.
What things have changed since Dr. King's day?
Some of the people have changed, but we still have a lot of discrimination all over the country.
It is a good thing that President Obama and other black people have been elected to public office, but there is still bias.
Take all the programs that the President has tried to set forth.
People are steadily cutting him down based only on the color of his skin.
A lot of people didn't vote for him because he is black.
Why are you involved with the NAACP?
I am involved because people, especially black people, are still being discriminated against.
Some of the struggles we've gone through we are still going through.
A lot has changed and there are some good signs, but a lot of discrimination is still here and faces us daily.
I have traveled all over the U.S. and there is discrimination everywhere, although it is more prevalent in the South.
It shouldn't be like that today, but people will be people.
If we can get rid of discrimination, we can do away with the NAACP and a lot of other social groups.
What does the NAACP do for people?
People who have been discriminated against, either on the job or in public, are always calling us.
We try to be a mediator before filing a wrongful discrimination lawsuit.
We try to talk to both parties and find out what is happening.
Sometimes the person doesn't know he or she has discriminated against the other person. It's not always clear.
We file lawsuits with the federal government, either individual or class action.
We do whatever is necessary to correct the situation.
We work with people of all colors, not just black people.
If anyone has a concern or complaint about discrimination, they can call me at 546-9369,
What message would you like to share about discrimination?
We should all get past our prejudice.
All people are equal, regardless of race or sex.
As Dr. King said, "We shall overcome."
Can you tell me about your family?
I have six children, two boys and four girls.
I have seven grandchildren, four boys and three girls.
– As told to Clayton Stairs
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Students majoring in Sociology must select one of six concentrations. Each of the six concentrations has requirements in addition to the six required courses for the major.
The General Sociology concentration provides students with an opportunity to explore a wide range of subfields within the discipline. The other five concentrations offer students a focus on a specific subfield in Sociology or Anthropology. The cohesive sequence of courses in these subfields enables students to strengthen their knowledge. This knowledge will prove useful either for immediate employment opportunities upon graduation or for advanced post-graduate training.
The six concentrations from which Sociology majors may choose are:
- General Sociology
- Anthropology
- Business and Society
- Deviance and Social Control
- Human Services
- Social Justice
General Sociology (USOG)
The General Sociology concentration is designed for students who wish to gain a broad overview of the discipline. In addition to completing the six core requirements, students select eight Sociology electives. A minimum of two of these electives must be at the 300-level or higher.


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Drug Compare
1 2
- Basic Info
Generic: pyrazinamide
It is used for the prevention or treatment of tuberculosis (TB) infections
What is this medicine?PYRAZINAMIDE (peer a ZIN a mide) is used for the prevention or treatment of tuberculosis (TB) infections. This medicine is usually used in combination with at least one other agent.
What should I tell my health care provider before I take this medicine?They need to know if you have any of these conditions:
How should I use this medicine?Take this medicine by mouth with a glass of water. Follow the directions on the prescription label. Take your medicine at regular intervals. Do not take your medicine more often than directed. For your therapy to work as well as possible, take each dose exactly as prescribed. Do not skip doses or stop your medicine even if you feel better. Skipping doses may make the TB resistant to this medicine and other medicines. Do not stop taking except on your doctor's advice.
Talk to your pediatrician regarding the use of this medicine in children. Special care may be needed.
What if I miss a dose?If you miss a dose, take it as soon as you can. If it is almost time for your next dose, take only that dose. Do not take double or extra doses.
What may interact with this medicine?
What should I watch for while using this medicine?Visit your doctor or health care professional for regular check ups. You will need blood work done regularly.
Alcohol may interfere with the effect of this medicine. Avoid alcoholic drinks.
This medicine can make you more sensitive to the sun. Keep out of the sun. If you cannot avoid being in the sun, wear protective clothing and use sunscreen. Do not use sun lamps or tanning beds/ booths.
You may get a false- positive result for sugar in your urine while you are taking this medicine. Talk with your doctor.
1 2
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Coastal Bend Garden Calendar: 07.20.13
CORPUS CHRISTI - To control spider mites on indoor plants first try to spray the plant with a strong jet of water. If that does not work, spray with a soap/oil mixture. Mix 2 teaspoons insecticidal soap, ¼ teaspoon horticultural oil and one quart water in a spray bottle.
Pat Botkin
PLUMERIA SOCIETY: The Plumeria Society of South Texas will have a plumeria sale from 10 a.m. to 1:30 p.m. Saturday at the Garden Senior Center, 5325 Greely Drive. Information: 779-3181.
BROWN BAG: "Cacti and Succulents" brown bag is from noon to 1 p.m. Tuesday at the Texas A&M AgriLife Extension Service-Aransas County Office, 892 Airport Road, Rockport. Presented by Master Gardeners Lonnie Matthew and Ellen Reisinger. Learn how to identify various cactuses and succulents, growing and propagating techniques and how to use them in your home landscape. Overflow parking is next door at the Transfer Station. Information: 361-790-0103.
BONSAI SOCIETY: The Bonsai Society meets at 7 p.m. Thursday at the Garden Senior Center, 5325 Greely Drive. David Falcon will discuss "Jin & Shari — Deadwood." Free. Information: 992-0009.
PLANT SOCIETY: The Native Plant Society meets at 2 p.m. July 27 at the Janet F. Harte Public Library, 2629 Waldron Road. John Fucik who discuss "Native Ground Covers and the Butterflies They Attract." Free. Information: 776-3285.
BIRD AND BUTTERFLY WATCHING: Swarovski Optiks professional Clay Taylor will discuss selecting a spotting scope for bird and butterfly watching from 10 a.m. to noon July 27 at South Texas Botanical Gardens & Nature Center, 8545 S. Staples St. Free/members; nonmembers with paid general admission. Information: 852-2100.
FLAMINGO FANDANGO: The South Texas Botanical Gardens & Nature Center's Flamingo Fandango runs through July 31. Roughly 100 diversely-dressed, custom-costumed pink plastic flamingos will be on display along with a "Naked Flock" of about 200. The birds will be judged mid-July in 8 categories. The display can be viewed from 9 a.m. to 6 p.m. daily at the gardens, 8545 S. Staples St. Cost: $7/adults; $6/seniors, active duty military and college students; $3/ages 3-12. Information: 852-2100 or visit www.stxbot.org.
GARDENER INTERN CLASSES: Nueces Master Gardener Intern classes begin Aug. 7 with a required orientation at 9 a.m. July 24 at the Texas AgriLife Extension Service, 710 E. Main St., Suite 1, Robstown. Most classes will be from 8:15 a.m. to noon Wednesdays through Nov. 20. Aransas/San Patricio class starts Aug. 6 and ends Nov. 19 and will be held in Rockport. Class is limited and is on a first come, first served basis. Cost: $175. Information: 361-767-5217 or 361-790-0103.
NATIVE PLANT SOCIETY: The Native Plant Society meets at 2 p.m. July 27 at the Janet F. Harte Public Library, 2629 Waldron Road. John Fucik will discuss "Native Ground Covers and the Butterflies They Attract." Free. Information: 776-3285.
CACTUSES YOU CAN EAT: Learn which cactuses you can grow and eat in the Cultivating and Cooking Cactus seminar from 10 a.m. to noon Aug. 10 at South Texas Botanical Gardens & Nature Center, 8545 S. Staples St. Guest lecturer will be J.T. Garcia from the Texas Cactus Council. Cookbooks will be available for purchase. Free/members; nonmembers free with paid general admission. Information: 852-2100.
FALL TRAINING PROGRAM: South Texas Master Naturalists will start a fall training program Sept. 7. Classes will be at 9 a.m. Saturdays at South Texas Botanical Gardens & Nature Center through the fall. Cost: $85. Information: 361-563-2147 or email@example.com.
Compiled by Tina Vasquez
Q: Something is eating my bougainvillea, but I don't see any bugs. Do you know what might be causing the damage to the leaves?
A: Bougainvillea loopers are often seen in the summer. These tiny inchworms feed at night and hang out on the underside of leaves during the day. They often go unseen because they are small, only about one-inch long and blend in with leaves and stems due to green or brown coloration.
The best chemical control is any B.t. product since they are safe to use around pets and children, and they target only caterpillars. Caterpillars ingest the crystalline structures and die of terminal indigestion due to intestinal damage. There also are some contact insecticides like cararyl (Sevin) that can be used, but good coverage under leaves is particularly important for contact insecticides.
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I enjoy blogs that are both well written and where I feel part of the conversation.
Contacting me with questions, pointing out a typo/miskate, your thoughts about our industry or just to say hello, is encouraged. Regardless of the reason, you are more than welcome to send an email (firstname.lastname@example.com) to or bug me on Twitter.
Rest assure, I read every email I get. Due to my busy schedule though, I am not always able to respond quickly, or even at all. I understand that writing a long email can be a considerable effort. Don't take it personally if I don't write back. I am probably more apt to respond on Twitter so plan accordingly.
The best way to get me to react is blog the topic yourself. Moving the conversation forward in public is the best way for all of us to learn more.


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Rockwell Kent in Alaska, ca. 1925
Physical Details: Photographic print : 1 item : b&w ; 14 x 9 cm.
Creator: unidentified photographer
Description: Item is a postcard photograph.
Identification on verso (handwritten): In Seward this ?. Looks as if you letters had come!
Forms part of: Rockwell Kent papers, [circa 1840]-1993, bulk 1935-1961
Rights Statement: Current copyright status is undetermined
Citation: Rockwell Kent in Alaska, ca. 1925
Digital ID: 4540
- Kent, Rockwell


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Seattle Coffee Gear: Espresso Machine Parts
Seattle Coffee Gear offers a wide array of parts for both home customers and service centers. In general, we offer only external parts to home customers, but we have developed a series of Tune-Up Kits to assist with the regular care and maintenace of machines made by Ascaso, Rancilio and Saeco.
Additionally, we are the primary parts importer and distributor for Saeco equipment in the United States. To find the part you need and request it for your service center, please visit our Parts Request Form. You can also use this form to request parts from other manufacturers such as Rancilio or Rocket Espresso and our Parts team will let you know if we are able to source it for you.
Saeco Talea Water Tank LidYou lost it? We got it! The Saeco Talea Water Tank Lid is a replacement lid for the Saeco Talea Water Tank Assembly. Size: 13 inch. Learn More$15.95
Saeco Talea Bean Hopper LidThe Saeco Talea Bean Hopper Lid, is the cover to the container that holds whole coffee beans until ready to be dispensed and ground to make an espresso shot with Saeco Talea superautomatic espresso machine models. Learn More$17.95
Saeco Talea Giro Drip TrayHelp me Rhonda! The Saeco Drip Tray will help-help get you out of a sticky situation. The Talea Giro Drip Tray replacement makes a moat of liquid for easy one step disposal later on. Learn More$16.95
Saeco Talea Giro Coffee KnobThe Saeco Talea Giro Coffee Knob controls the SBS which controls how much pressure your machine will produce for different levels of crema on top of your espresso shots. Size: 2 inches Learn More$11.95
Saeco Talea Drip Tray Grate - Stainless SteelThe Saeco Talea Drip Tray Grate is stainless steel and lays on top of the drip tray to help prevent unwanted spills and messes when you're making that perfect espresso drink. Learn More$16.95
Saeco Talea Bean HopperThe Saeco Talea Bean Hopper is a replacement container that stores whole coffee beans before they are ready to grind inside Saeco Talea superautomatic espresso machine models. Size: 13 inch. Learn More$16.95
Out of Stock
Saeco Talea Ring Plus / Touch Drip TrayThe Saeco Talea Ring Plus / Talea Ring Touch Drip Tray keeps your cup high and dry whether you use a demitasse or a shot glass. It is easy to remove the drip tray from this superautomatic espresso machine for quick clean up. Learn More$14.95
Out of Stock
Saeco Talea Coffee Dispenser Rear CoverThe Saeco Talea Coffee Dispenser Rear Cover is the plastic chrome cover that supports the back of the Saeco Talea dispenser nozzle on this popular superautomatic espresso machine model. Learn More$16.95
Out of Stock


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http://Capricast.com - On the eve of the first mid-season finale of Caprica, actresses Luciana Carro and Magda Apanowicz paid Toronto Comic Con a visit and treated us to a panel that gave us some insight into their experience making the show, and dropped some hints about what we can expect from the back-9 episodes of the season! Hit up http://Capricast.com and leave a comment for your chance to win the Caprica Soundtrack! ~Matt + Nat
We're Matt + Nat, two big Geeks who happen to be lucky enough to get some sweet geeky content that's worth sharing. Keep an eye on http://YourGeekNews.com for Previews and Reviews of upcoming movies and video games with in-depth interviews and advanced clips!


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UNC RB coach Randy Jordan takes job with NFL's Redskins
Jan. 25, 2014 @ 10:36 PM
UNC running backs coach Randy Jordan is headed back to the NFL.
The Washington Redskins announced over Twitter Saturday night that they have hired Jordan as their new running backs coach.
Jordan, who played the position at UNC before graduating in 1993, had a nine-year NFL career and then began his coaching career as a special teams assistant with the Oakland Raiders in 1993.
Jordan had stints as running backs coach at Nebraska and Texas A&M before coming back to UNC when Larry Fedora took over as head coach. Jordan was also the special teams coordinator.


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Associate Bonus Watch 2011, Biglaw, Bonuses, Money
Yes, we know: you're all waiting eagerly for spring bonuses. We are too (because it's great for the news cycle and our traffic). But right now we have nothing to report on that front. As soon as you hear of spring bonus movement (cough cough, Sullivan & Cromwell), please email us or text us (646-820-8477 / 646-820-TIPS).
In the meantime, we'll catch up on regular bonus news. Even though it's already March, some firms are only just now getting around to announcing their 2011 year-end bonuses. We have various tips floating around for various firms, but we need additional corroboration for many of them. If you can help us out, you know where to reach us (see contact info, supra).
Today we have bonus news from Winston & Strawn, which announced its bonuses a while ago….
Winston announced its bonuses earlier this year (around late January or early February, we believe). Our understanding is that they — shocker! — largely followed the Cravath scale, provided that you made your hours. The hours cutoff was 2000 hours.
The firm also announced the standard pay raises for increased seniority. The pay scale is "essentially lockstep," as one source told us. As you may recall, last year Winston moved back to the standard scale ($160K, $170K, $185K, $210K, $230K, $250K, etc.).
If you have additional info about Winston, concerning bonuses or any other matter — congrats to the firm on the Motorola / Carl Icahn work, by the way — we welcome it. Thanks.
Winston & Strawn Advises Motorola Solutions on Icahn Stock Repurchase [Am Law Daily]
Earlier: Associate Bonus Watch: Winston & Strawn (And an additional issue about base salaries.)
Associate Salary Watch: Big (and Good) News from Winston & Strawn
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February 1, 2012
IT'S CARNIVAL TIME! Competitors in the 2009 Lake George Winter Carnival outhouse race drag their custom-built craft across the lake ice toward a finish line. This year's competition, to be held in the Fort William Henry parking lot due to lack of lake ice, is to be filmed by a national cable television channel.
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Crowd expected for Winter Carnival
Regardless of a lack of thick ice, the annual Lake George Winter Carnival is likely to present a fun-filled weekend, event officials said early this week.
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Unlocking the Key to Deep Sleep
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Making the Ferguson Commission Matter
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Obama Praises St. Louis' LaunchCode
By Lindsay Toler
By Lindsay Toler
"Tommie is a six-five whatever-he-wants-to-be," says Soderberg. "He's as good a guard as I've seen in twenty years of coaching. This young man has the ability to play beyond SLU."
Lofty prognostications are generally accepted as the routine currency of collegiate courtship. Nonetheless, they rankle North Carolina-based prep scout Dave Telep of TheInsiders.com.
"That statement is way out there," Telep says. "It does a disservice. The statistics are in no one's favor in the grand scheme of things. We use the words 'NBA' with so many kids; it creates false expectations. Why does the NBA have to get asked of any guy? The question with Tommie should be, 'Can he be an all-conference guy?' Anything beyond that is putting the cart before the horse."
If Soderberg is prone to bouts of hyperbole, he's done a bang-up job of insulating his staff against the long-held criticism that the Billikens do a lackluster job of recruiting locally. Besides Liddell, next year's freshman class will include Vashon High point guard Dwayne Polk and Borgia swingman Luke Meyer, two talented players whose games are more specialized and whose natural assets are more limited than those of the rangy, versatile Liddell.
"Saint Louis U. is convinced that one way to build a program is to keep some of the best players from leaving," says Floyd Irons, principal and coach at perennial St. Louis prep powerhouse Vashon. "Kids like to go places where they know people, where they fit in. You have to give Brad a lot of credit for what he's doing over there."
Another person who gives Soderberg credit is East Side baseball coach Maurice Scott, a steadfast community presence who has served as mentor to Miles, Liddell and other schoolboy studs.
"I think it was Soderberg's straightforwardness," Scott says of SLU's key to beating out the likes of Kansas, Marquette, Illinois and DePaul in the sweepstakes for Liddell's services. "Tommie's mom fell in love with him during their home visit."
Liddell's mom, Diane Rhodes, has fond memories of the visit. Tommie, having lost track of time while playing pickup ball at a nearby community center, showed up late, giving the Wisconsin-bred Soderberg time to bond with Rhodes over a televised Green Bay Packers game in the living room of the Liddell family's tiny apartment.
"He was like family," Rhodes says of the impression the SLU coach made.
Near the intersection of State and Tenth streets, on the outskirts of the rotting retail core of East St. Louis, sits an old farmer's market. In place of vendors hawking fresh plums, peas and peppers reside a hollowed-out Oldsmobile and a chalkboard bearing two telling words: "Third World." Around the corner on Sixth Street are the Samuel Gompers Apartments, a barracks-style compound of buildings erected in 1943 as East St. Louis' first public housing development.
In the kitchen of one of Building 27's spartan units, Tommie Liddell, fresh from practice, scoops taco meat into hard tortilla shells as his mother mops the linoleum. Plate prepped, the Flyer joins his littlest brother, nine-year-old James, in front of a small TV set showing a rerun of Everybody Loves Raymond. Liddell is clad in his standard leisure-time outfit: orange East Side practice jersey, pressed blue jeans and a black baseball cap pulled low over his braided black hair and inscribed with "ESTL" and the town's area code, 618. On his left shoulder is a tattoo of a basketball player carrying a rim, which Liddell co-opted from a picture in The Source magazine. Around his neck dangles a gold necklace with the symbols "T-Mac 25," a homage to his favorite player, Tracy McGrady of the Orlando Magic. McGrady wears a jersey numbered 1, though. Twenty-five is the exclusive domain of the talented Mr. Liddell.
No one in the Liddell family can figure out how Tommie grew to be so tall and lanky. Both of his parents are comparably compact, Tommie Sr. standing five-eleven and Mom about five-eight. Tommie's got his mama's lush eyes, while his demeanor, marked by a gentle speaking voice and delayed-reaction laugh, is more akin to his laid-back dad.
Tommie was born when Diane Rhodes was a junior at East Side. She and Tommie Sr. had another son, twelve-year-old Montrez, together. They never wed, though they remain on good terms. These days Tommie Sr. works the graveyard shift at Big River Zinc in nearby Sauget and shares a tidy apartment with his wife, Euronda, in Belleville, where his eldest son sometimes spends the weekend. Diane, who works at the President Casino, gets along fine with Euronda, to the point where both of Tommie's mommies attended the press conference at which the hotly recruited point guard, with an assist from close family friend Maurice Scott, declared his intent to accept the SLU scholarship.
Liddell's strong family support network enables him to buck the absentee-dad stereotype of the young, black, urban male. But if he intends to don number 25 in SLU blue next year, he'll need to hit the books. With a grade-point average hovering around 2.5, he must either dramatically improve his GPA during the balance of his senior year or ratchet up his score on the ACT standardized test, which he's already taken once, by two points. If he fails to achieve the minimum standard, Liddell will probably have to spend a year beefing up his grades and his game as best he can at a junior college or prep school -- and hope SLU stays interested.
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It Looks Like Market Turmoil Is Scaring Off Young Investors
By JLP | September 25, 2008
From today's Wall Street Journal:
The idea of saving for retirement always terrified Zack Teibloom. With the stock market's big drop this year, it seems even more daunting.
"I don't even have one K, let alone 401 Ks," says the 23-year-old Mr. Teibloom, a recent college grad who works as an editor for a small magazine in Chicago. "I'm worried that if I put money away, it won't even be safe the way the markets are going."
The saving and investing habits of young workers have long been dismal. Only 49% of eligible workers in their 20s participate in 401(k) plans offered through their employers, according to a 2007 study from Hewitt Associates Inc., a Lincolnshire, Ill., consulting firm. And less than 20% of this group is saving anything at all for retirement.
Source: WSJ – Market Turmoil Frightens Off Young Investors ($)
Isn't it crazy how we do the exact opposite of what we should be doing? If the stock market was going up, up, UP, people would be jumping in left and right—essentially buying over-priced stocks. Now that the market is on a downswing, people are sitting on the sidelines. The very next paragraph of the article even mentions this:
Declining stock prices actually favor young investors, because it means the shares they buy have more room to grow in the decades before they hit retirement. But anecdotal evidence suggests the rocky stock market is scaring off many young people.
My advice:
1. Remember this quote from Sam Stovall, chief investment strategist at Standard & Poor's:
"Since 1950 we have had 48 pullbacks – meaning declines of 5 – 10%. We've had 18 corrections – meaning 10- 20%, and 8 bear markets. At the worst on average we end up getting back to normal in about 3 1/2 years. But people just don't want to wait that long and they let fear overtake their emotions."
2. Take a look at these posts from my archives:
Why the Long-Run is so Important When Investing in Stocks
S&P 500 Rolling-Period Total Real Returns
3. Read Jeremy Siegel's Stocks for the Long Run*.
4. Finally, if you're young enough, tell yourself this: "I'm young. I have 30 years until retirement. I'm diversified. Why do I care what the market is doing today? The stock funds I'm buying today are on sale and I'm getting a good deal if I keep them for the long-run."
Sitting on the sidelines isn't a good option.
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Topics: Investing | 16 Comments »
16 Responses to "It Looks Like Market Turmoil Is Scaring Off Young Investors"
JC Says:
September 25th, 2008 at 12:13 pm
I totally agree with you. I think the root of the problem is that people don't understand the nature of the market and thus, they FEAR what they do not understand. This actually is a good instinctive defense mechanism. But when you combine that FEAR with LAZINESS (not educating yourself on the nature of the market), then it becomes a problem. the instinct is a good one, the response to it is not.
JLP Says:
September 25th, 2008 at 12:19 pm
That was an EXCELLENT comment. Thanks!
SP Says:
September 25th, 2008 at 12:34 pm
I understand what is going on. I understand that on many levels it is a good thing for me, as a young investor.
For me personally, it's not misunderstanding, it is emotions. It is difficult to watch balances drop, even if i know I shouldn't care today.
It's hard, but I'm staying the course.
Amanda Says:
September 25th, 2008 at 12:52 pm
I think that many young people also use the faltering economy as a justification for not saving. It's sad but true… A lot of people my age just do not want to save – they want to spend! Being able to blame it on the economy gives them an "easy out" from saving.
JLP Says:
September 25th, 2008 at 12:54 pm
You're right! I'm sure lots of younger people are doing just that.
Grumble Says:
September 25th, 2008 at 1:36 pm
So where are these "scared off" young investors going with their money?
JLP Says:
September 25th, 2008 at 1:38 pm
I'm pretty sure they're spending it!
JT Says:
September 25th, 2008 at 2:15 pm
This is more evidence that Social Security should
not be privatized. Most individuals are not ready to invest their retirement money in the market. If we add Social Security, many people will be in serious trouble when it is time to retire.
tom Says:
September 25th, 2008 at 3:18 pm
That is their problem. If you are too lazy to handle your own finances and pick up a few books, and are too scared to take your own money into your own hands, then that should be their problem. Don't you think if SS was privatized then we would have many, many firms helping people invest properly? Sure we'll have our fair share of shady firms making bad deals or running away with private SS money, but thats the free market. I, for one, know I could do a million times better with my SS money than the government. In fact, I'm not even counting on SS in my retirement projections.
Jeremy Says:
September 25th, 2008 at 3:59 pm
Sadly, I'm seeing a lot of this first hand. I gave a presentation on Monday to a group of new incoming employees on their retirement benefits, and I literally had some of the younger people laughing at the thought of enrolling in the 401k.
Even though there is a fixed account option with a 4.1% rate, and even though there is a company match, most younger people (under 30) were balking at the idea of even saving enough to get the 100% return on their money with the match. The only people taking advantage of this were the older ones who have investments already or were experienced with investing for retirement.
Sadly, if the 20-somethings use this as an excuse to put off starting their retirement savings, that generation is going to be no better off in 30+ years than the baby boomers who on average don't have more than one year's worth of retirement savings.
Waiting for the perfect time to start saving/investing is like waiting for the perfect time to have a baby. There will never be a perfect time, and you'll end up waiting until it's too late.
Don Says:
September 25th, 2008 at 7:35 pm
While I am myself looking at the current environment as more opportunity than trial (I'm still pretty young, under 40 at least), I don't scoff at people being afraid to invest at this moment.
Your quote about bear markets being short-lived is backward-looking, as all such things must be. It is not at all clear that we aren't on the brink of something like the decline in the Japanese market at the end of last century. If you had been heavily invested there, you'd be way down after a decade even.
I can understand a person who doesn't think they could stomach that personally. And when I invest, I well know that a decade like that is what I'm risking against. If you couldn't lose money, you wouldn't enjoy the premium that stocks generally afford over other investments.
Personally, I think Larry Swedroe has nailed the idea of risk better than pretty much any other author I have read. I found it in Wise Investing Made Simple, but the idea is about the same in all of his books. Risk means you could lose money, maybe more money than you are really comfortable with. And it might be gone an inconvenient period of time; longer than 3.5 years.
Lots of times it doesn't take that long. But we can't kid ourselves. We might be digging out of this for a very long time. It is entirely possible that in 2018 we'll be reading magazine articles about how all the rules we used to believe in were wrong. Stocks didn't hold against inflation. They didn't outperform every decade, etc.
If the stock market does any one thing well, it incorporates the truths that "everyone knows" and nullifies them. The fact that everyone knows that stocks are best for the long term, and that over a ten year period stocks never lose money, (add you favorite rule of thumb here), makes me particularly nervous. Houses always go up in value after all….
Richard Says:
September 25th, 2008 at 8:12 pm
I just got into the market this week. I just opened a Roth IRA and put my meager $600 initial deposit into an index fund.
I've got direct deposit set up for $200 per paycheck (bi-monthly) and will keep adding to that index fund till I start grad school in May. At that point I'll be leaving my good paying job and doing research part time…I still haven't figured out how much I'll be able to invest on that income.
I'm hoping that the market continues to sit low so I can get more money into it before it goes back up.
My wife and I are 24 and 26 respectively and we've got 2 kids.
Grumble Says:
September 25th, 2008 at 9:10 pm
JLP said:
I'm pretty sure they're spending it!
So it looks like they are keeping up out of recession.
EMilster Says:
September 26th, 2008 at 9:50 am
In this time, it might be a better bet for investors too look at safer places for their money like CDs no? Sure you won't get as high a return as you would on booming stocks, but in this market, any return is a good one.
Otis Says:
September 26th, 2008 at 1:42 pm
I'm 27 and I can tell you really simply why we are not investing.
1. Since the day we graduated we've seen nothing but bad news. Huge tech crash, current crash. We're not even back to where we were almost 10 years ago.
2. We have huge student loans to pay back.
3. Finally got out of college in 2004, saved as much as possible and am currently underwater…may as well have spent that extra money.
4. There are no solid investment vehicles that are even matching inflation right now.
5. We want to start families soon and need short-term funds, not long-term retirement funds.
6. We don't really have much trust in our country. Frankly, I don't think the US is really #1 anymore and our economy will only decline in the long run unless we are willing to tackle education's inadequacies, stop giving money for oil to countries that hate us, stop wasting money to blow up brown people, and truly address the issue of consumerism.
We don't see the market as a stable investment for the near future and would rather sit on cash and then get in either in the US once we balance out or elsewhere if my dollars are still worth anything.
Chad Says:
September 27th, 2008 at 4:29 am
How to handle a down market when investing…
1. Come up with a plan.
2. Stick to that plan.
3. TURN OFF THE TV and DONT READ THE PAPER.
and you will be fine over the long term.
short term news just scares the hell out of you and makes you divert from your original plan.


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