George B. Harding

Microporous poly(caprolactam) hollow fibers for therapeutic affinity adsorption

Abstract Microporous nylon-6 hollow fibers were modified for use as affinity fibers. The initial amine end-group concentration of 21 μmoles/g fiber was amplified by reacting the polymer with lysine, using standard peptide synthetic methods. The carbohydrate side chain of a rabbit polyclonal anti-BSA IgG was oxidized to facilitate linkage of the Ab to the hollow fibers. The covalent links were either to terminal amine groups [from lysine of the poly(caprolactam)] or to hydrazide groups. The latter were produced by coupling adipic acid dihydrazide to the amine groups via a glutaraldehyde bridge. Coupling of the oxidized Ab to amine groups of required a pH⪢8.0, whereas the same Ab could react with the hydrazide groups at pH 5.5. The higher pH coupling conditions led to crosslinking of the IgG, presumably between side chain amine groups on the protein and the carbohydrate aldehyde groups. Although significant amounts of IgG could be coupled to the amine groups, the recognition of antigen by such Ab was markedly reduced. In contrast, the coupling of oxidized Ab to hydrazide groups, carried out at pH 5.5, gave bound IgG which exhibited the theoretical number of binding sites for its antigen. Equilibrium binding coefficients for BSA showed values ranging from 5,2×10 6 M −1 to 9.7×10 5 M −1 . The molar ratio of BSA to calculated anti-BSA binding sites was generally 2:1. Although stripping experiments with SDS at ⪢85°C indicated none of the BSA was covalently linked to either the IgG or the fiber substrate, less than 60% of the adsorbed BSA could be eluted with pH change, salt, chaotropic agent, etc. The same responses were observed with hydrazide modified Sepharose carried through the experiments for comparison.

Culture of Dialysis Fluids on Nutrient-Rich Media for Short Periods at Elevated Temperatures Underestimate Microbial Contamination

Recommended culture methods for monitoring bacterial contamination of H2O, dialysate and bicarbonate concentrate in dialysis centers in the USA involves culturing these fluids for 48 h at 37 degrees C. A variety of media and commercial culture methods are accepted for monitoring these fluids. Over a 3-month a comparison was made between an acceptable culture method, tryptic soy agar (TSA) employing the pour plate (PP) technique at 37 degrees C for 48 h, and PP cultures on standard methods agar (SMA) and R2A agar, incubated at ambient temperature (23 degrees C) for 48, 72, 168 h. Increases in the colony counts over time occurred for all three fluids. However, counts wee greater on SMA and R2A than on TSA. The increases over the standard 48-hour TSA cultures ranged as high as 10(4) times for 23 degrees C cultures at 7 days of incubation. Endotoxin levels even in the most contaminated samples were found to be below the acceptable 5 EU/ml recommended for reprocessor water. Bacterial colonies that appeared at 48, 72 and 168 h were isolated and identified. Pseudomonas, Moraxella, Acinetobacter and CDC group VI C-2 were among some of the common bacteria isolated. This study indicates that the media utilized, the time and temperature of incubation may result in a significant underestimation of the bacterial population of water and dialysis fluids, thus potentially placing the patient at a higher risk.

Steroid-protein interactions XXVII. Progesterone binding to polymers of human serum albumin☆

Abstract Human serum albumin was separated by Sephadex gel filtration into monomeric, dimeric, and tr meric-polymeric fractions. Electrophoresis in dodecyl sulfate polyacrylamide gel showed that dimerization was followed by trimer and tetramer formation. Mild reduction with mercaptoethanol did not eliminate the polymerized human serum albumin species. In analytical gel filtration, the fractions were less clearly defined by molecular size, presumably due to protein-protein interaction. The absorbance values, A 1 cm 1% at 278 nm, for the unfractionated, monomeric, and dimeric fractions, after correction for light scattering, were 5.14, 5.17, and 5.38, respectively. Binding of progesterone to the delipidated human serum albumin fractions was determined by equilibrium dialysis at 4°C. Computer analysis of Scatchard plots for two independent sets of binding sites gave n 1 = 1; k 1 = 3.6 · 10 5 M −1 , and n 2 = 8; k 2 = 6 · 10 3 M −1 for the human serum albumin molecule in both the monomeric and dimeric species. The trimer-polymer fraction had a significantly lower binding affinity.

Steroid-protein interactions. XXIII. Nonidentity of cortisol-binding globulin and progesterone-binding globulin in guinea pig serum

Abstract Exposure of pregnant guinea pig serum to heat and low pH decreases the activity of the cortisol-binding globulin (CBG) to a greater extent than the progesterone-binding activity. The inactivation is irreversible. Hydroxylapatite chromatography separates the two binding proteins from the bulk of other serum proteins; the progesterone binder emerges before CBG. Separation of the progesterone-binding protein from CBG is obtained by Sephadex G-200 filtration. The progesterone binder migrates in the electric field as a globulin, somewhat more slowly than CBG. The affinity constant of the progesterone complex with the progesterone-binding globulin (PBG) is approximately two orders of magnitude greater than that of the cortisol CBG complex. The molecular weights of the guinea pig PBG and CBG, assessed by gel filtration, are of the order of 100,000 and 50,000, respectively.

Dialysate contamination and back filtration may limit the use of high-flux dialysis membranes.

Endotoxins, or fragments thereof, can reach the blood stream of dialysis patients, transported by diffusion and connection across the intact high-flux membrane. This transfer depends upon the phenomenon of back filtration. Back filtration generally occurs under conventional high-flux dialysis conditions with membranes having an ultrafiltration coefficient in blood (UF-C) above 20 ml/hr/m2/mmHg. The clinical consequences of back filtration vary from center to center depending primarily on the quality of dialysate. We therefore surveyed the bacterial and endotoxin levels of purified water and effluent dialysate in a cross section of dialysis centers in the central United States. Using a high recovery medium, we found that 53% of the centers had bacterial counts above the Association for the Advancement of Medical Instruments standard in water (20% cfu/ml) and 35% above the standard in dialysate (2,100 cfu/ml). Endotoxin concentrations higher than 5.0 EU/ml in both water and dialysate were found in 4% and 11.8% of the centers, respectively. Since high-flux membranes are believed to be of benefit for long-term dialysis patients, manufacturers will have to offer dialysate preparation systems with additional safety features. The proper membrane design will be a key to the success of such systems.

TNF-alpha stimulates increased plasma membrane guanine nucleotide binding protein activity in polymorphonuclear leukocytes.

TNF‐α enhances the response of polymorphonuclear leukocytes (PMN) to chemoattractants: however, the mechanism by which this occurs is unclear. We addressed the hypothesis that TNF‐α enhances the PMN response to chemoattractants by increasing chemoattractant receptor transmembrane signaling, using fMLP as the model chemoattractant. fMLP‐stimulated guanine nucleotide binding (G) protein activation was significantly increased in plasma membranes isolated from PMNs exposed to TNF‐α 100 U/ml for 10 minutes (TNF‐M), compared to membranes from control cells (CM). Formyl peptide receptor number and affinity were not significantly different in CM and TNF‐M. Gi and G3 content were increased in TNF‐M as measured by pertussis toxin and cholera toxin (CT) catalyzed ADP‐ribosylation, respectively. The increased Gi was coupled to the formyl peptide receptor as shown by receptor‐specific CT labeling of Gi. Immunoblot analysis showed that both Gαi2 and Gα3 were increased in TNF‐M. The functional activity of the increased G protein content was demonstrated by increased NaF‐stimulated phospholipase D activity in TNF‐α‐treated PMNs. We conclude that TNF‐α rapidly stimulates increased PMN plasma membrane expression of G proteins that couple formyl peptide receptors to effector enzymes. Regulation of G protein expression may be a significant mechanism by which TNF regulates PMN function. J. Leukoc. Biol. 57: 500–506; 1995.

[8] Characterization of steroid-binding glycoproteins: Methodological comments

Publisher Summary The principal procedures used in a laboratory for the purification and binding analysis of the steroid-binding serum proteins have been previously described in Methods in Enzymology . For this reason, this chapter is limited to a number of unpublished observations made over the years, all relevant to preparative and analytical studies in the field of steroid–protein interactions. The chapter discusses charcoal treatment of serum or plasma, adsorption of steroids to various materials, elimination of binding inhibitors, anomalous mobility of glycoproteins in dodecyl sulfatc-polyacrylamidc electrophoresis, isoelectric focusing of steroid-binding glycoproteins, and determination of sedimentation coefficients at low protein concentrations.

Decomposition of cortisol-4-14C to 11β-hydroxyandrost-4-ene-3, 17-dione-4-14C

Abstract Cortisol-4- 14 C in aqueous or methanolic solution decomposed at 23° to products of lesser and higher polarity. Light accelerated the breakdown. Storage of cortisol-4- 14 C in methanoi at −85°C in the dark resulted in similarly altered material. The main decomposition product was identified as 11β-hydroxyandrost-4-ene-3, 17-dione by paper chromatography in four solvent systems; by CrO 3 oxidation to androst-4-ene-3, 11, 17-trione (adrenosterone) and its identification in three different chromatographic systems; and by co-crystallization with authentic 11β-hydroxyandrost-4-ene-3, 17-dione to constant specific activity.

Comparative metabolic effects of acetate and dichloroacetate infusion in the anesthetized dog

The comparative effects of acetate (10 mmol/h/kg) and dichloroacetate (DCA) (1 mmol/h/kg and 10 mmol/h/kg) on acid-base and intermediary metabolism were assessed using the fasted anesthetized dog, undergoing controlled ventilation, as a metabolic model. Infusion of acetate resulted in a marked metabolic alkalemia and a decline in PaO2, while DCA had minimal effects on acid-base state and oxygen consumption. Serum glucose decreased with both DCA and acetate infusion, although only significantly with the latter. At infusion rates of 10 mmol/h/kg, acetate caused marked decreases, while DCA caused marked increases, in serum potassium and phosphorus. Acetate and DCA also had opposing effects on lactate and citrate levels, the former caused increases and the latter decreases in both metabolites. Pyruvate levels decreased similarly in response to both infusates. Acetoacetate and beta-hydroxybutyrate levels increased significantly with both acetate and DCA infusions; however, the increases were much greater with acetate than with DCA infusion. Blood alanine levels decreased significantly during the infusion of both acetate and DCA, whereas, free fatty acids tended to increase with acetate infusion, remained unchanged with low dose DCA and fell significantly with high dose DCA. Plasma insulin levels were sustained during acetate infusion, but fell abruptly with termination of infusion. In contrast, insulin levels fell markedly with DCA infusion and remained depressed throughout the infusion and recovery periods. Blood levels of acetate and DCA rose markedly during infusion; however, while acetate levels decreased nearly to control values during the recovery period, DCA levels remained elevated.(ABSTRACT TRUNCATED AT 250 WORDS)

Steroid protein interactions. XXVIII. The isoelectric point and pH stability of the progesterone-binding globulin.

Abstract Isoelectric focusing of progesterone-binding globulin of pregnant guinea pig serum occured at pI = 2.8. This result was obtained when the binding activity of the focused protein fractions was measured by equilibrium dialysis. Reliance on localization of the radioprogesterone alone may lead to erroneous results presumably due to dissociation of the uncharged progesterone molecule from the protein during the electrophoretic migration into the acidic milieu. This assumption was corroborated by binding assays over the pH range from 2 to 11. Almost complete dissociation occurred at pH 4 and below; however, return to neutral pH restored over 80% of the binding activity. The pH gradient of the electrofocusing column was improved by adding aspartic and glutamic acid to the pH 3–5 Ampholines.