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Biomedical subjects

K B Taylor

Publications and source records attributed to K B Taylor.

At least 19 recordsLinked to original sources
PubMed1992

Effect of the levels of dissolved oxygen on the expression of recombinant proteins in four recombinant Escherichia coli strains.

Four recombinant strains of Escherichia coli were examined for the effects of the dissolved oxygen level on the level of biomass, the plasmid content, and the level of recombinant protein at the stationary phase of batch growth. Strains JM101/pYEJ001, and TB-1/pYEJ001 (encoding chloramphenicol acetyltransferase), and strain TB-1/p1034, and TB-1/pUC19 (encoding beta-galactosidase) were grown at the constant dissolved oxygen levels of 0, 50, and 100% air saturation, as well as in the absence of dissolved oxygen control. The biomass of all strains under constant aerobic conditions was 12-36 times higher than that under anaerobic conditions, but was the same as or slightly higher than that without dissolved oxygen control. The plasmid content in all strains under aerobic conditions was 2.9-11.7 times higher than that under aerobic conditions. The optimal dissolved oxygen concentration for the specific activity of recombinant proteins was dependent upon the strain. In no strain were constant aerobic conditions optimal. However, because of the effect on biomass, controlled aerobic conditions were optimal for the volumetric activity of recombinant protein in all but one strain.

Aerobiosis↗
PubMed1989

Sanfilippo syndrome, type D: a spectrophotometric assay with prenatal diagnostic potential.

Sanfilippo syndrome, type D (MPS IIID), is characterized by moderate physical abnormalities, progressive mental deterioration, and deficient activity of N-acetylglucosamine 6-sulfate sulfatase, a lysosomal hydrolase involved in the degradation of heparin, keratan sulfate, and heparan sulfate. To date, demonstration of the enzyme deficiency typically relies on a radiolabeled trisaccharide substrate derived from heparan sulfate. In our study, we have developed a spectrophotometric assay for the determination of N-acetylglucosamine 6-sulfate sulfatase activity using the monosaccharide, N-acetylglucosamine 6-sulfate, as substrate. The reaction mixture was incubated for 6 h at 37 degrees C and, after Dowex chromatography, released N-acetylglucosamine was measured by a modification of the method of Reissig. Assay conditions were optimized for cultured skin fibroblasts and primary cultures of amniotic fluid cells. The pH optimum for each was 5.5. The assay was linear for 24 h and up to 0.1 absorbance units. Activities of the three known MPS IIID skin fibroblast cell lines were more than 4 SD below the skin fibroblast control mean and more than 5 SD below the control mean for amniotic fluid cells. An enzyme deficiency in cultured amniotic fluid cells of the same magnitude as the skin fibroblasts of the known patients would be detectable and, therefore, prenatal diagnosis by this method is feasible.

Amniotic Fluid↗
PubMed1989

Bile acid sulfotransferase I from rat liver sulfates bile acids and 3-hydroxy steroids: purification, N-terminal amino acid sequence, and kinetic properties.

A bile acid:3'phosphoadenosine-5'phosphosulfate:sulfotransferase (BAST I) from adult female rat liver cytosol has been purified 157-fold by a two-step isolation procedure. The N-terminal amino acid sequence of the 30,000 subunit has been determined for the first 35 residues. The Vmax of purified BAST I is 18.7 nmol/min per mg protein with N-(3-hydroxy-5 beta-cholanoyl)glycine (glycolithocholic acid) as substrate, comparable to that of the corresponding purified human BAST (Chen, L-J., and I. H. Segel, 1985. Arch. Biochem. Biophys. 241: 371-379). BAST I activity has a broad pH optimum from 5.5-7.5. Although maximum activity occurs with 5 mM MgCl2, Mg2+ is not essential for BAST I activity. The greatest sulfotransferase activity and the highest substrate affinity is observed with bile acids or steroids that have a steroid nucleus containing a 3 beta-hydroxy group and a 5-6 double bond or a trans A-B ring junction. These substrates have normal hyperbolic initial velocity curves with substrate inhibition occurring above 5 microM. Of the saturated 5 beta-bile acids, those with a single 3-hydroxy group are the most active. The addition of a second hydroxy group at the 6- or 7-position eliminates more than 99% of the activity. In contrast, 3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid (deoxycholic acid) is an excellent substrate. The initial velocity curves for glycolithocholic and deoxycholic acid conjugates are sigmoidal rather than hyperbolic, suggestive of an allosteric effect. Maximum activity is observed at 80 microM for glycolithocholic acid. All substrates, bile acids and steroids, are inhibited by the 5 beta-bile acid, 3-keto-5 beta-cholanoic acid. The data suggest that BAST I is the same protein as hydrosteroid sulfotransferase 2 (Marcus, C. J., et al. 1980. Anal. Biochem. 107: 296-304).

Amino Acid Sequence↗
PubMed1986

Evidence for an ordered reaction mechanism for bile salt: 3'phosphoadenosine-5'-phosphosulfate: sulfotransferase from rhesus monkey liver that catalyzes the sulfation of the hepatotoxin glycolithocholate.

The in vivo formation of the sulfate ester of glycolithocholate is a critical step in the elimination of this hepatotoxic bile salt. Rhesus monkeys fed chenodeoxycholate or ursodeoxycholate, the precursors of lithocholate, develop frank cirrhosis in association with accumulation of nonsulfated glycolithocholate in bile. An enzyme catalyzing the formation of glycolithocholate-3-sulfate has been isolated from hepatic cytosol of adult female rhesus monkeys and has been purified 146-fold. When reduced it appears as a 30 kD band on an SDS-polyacrylamide gradient gel. It has a pH optimum of 7.0 and is stimulated by low concentrations of Mg2+ (up to 2 mM), but does not have an absolute requirement for this metal ion. The kinetics of this enzyme have been investigated to ascertain whether its reaction mechanism can account for the poor in vivo rate of glycolithocholate sulfation. Inhibitor studies with an oxidized metabolite of lithocholate, 3-keto-5 beta-cholanoate, showed that the latter is a competitive inhibitor of glycolithocholate and is noncompetitive with the active form of sulfate, 3'phosphoadenosine-5'-phosphosulfate. The monophosphonucleotide 3'-AMP is a competitive inhibitor of 3'phosphoadenosine-5'-phosphosulfate, and is noncompetitive with glycolithocholate. These observations are consistent with a sequentially ordered Bi Bi reaction mechanism in which the bile salt is the first substrate to bind to the enzyme. Such a reaction mechanism for bile salt:3'phosphoadenosine-5'-phosphosulfate:sulfotransferase would be, therefore, the first time in which the sulfate acceptor (the bile salt) is the initial substrate to bind to a sulfotransferase. These studies have shown that although rhesus monkeys have a liver enzyme capable of forming the sulfate ester of glycolithocholate, its reaction mechanism and the potent inhibition caused by simple metabolites, such as 3-keto-5 beta-cholanoate, may serve to under-express the activity of the enzyme in vivo.

Animals↗
PubMed1984

Octopine dehydrogenase from Pecten maximus: steady-state mechanism.

The steady-state kinetic mechanism of the reaction catalyzed by octopine dehydrogenase [N2-(1-carboxyethyl)-L-arginine:NAD+ oxidoreductase] was investigated at pH 6.9 and 9.2 by studies of substrate inhibition, analogue inhibition, and product inhibition. In the direction of octopine synthesis, the inhibition patterns in the presence of delta- guanidinovalerate and propionate show that NADH binds to the enzyme first followed by L-arginine and pyruvate which bind randomly. In the direction of octopine oxidation, the substrate patterns show that NAD binds to the enzyme before octopine in a rapid equilibrium fashion, and the product inhibition patterns show that the products L-arginine and pyruvate are released in a random fashion. Double, synergistic, substrate inhibition by L-arginine and pyruvate was shown to be due to binding (hypothetically of the imine) to the free enzyme and the enzyme-NAD complex. Furthermore, an alternate minor pathway was demonstrated which includes an enzyme-NADH-octopine complex and an enzyme-octopine complex.

Amino Acid Oxidoreductases↗
PubMed1983

Solvent isotope effects on the reaction catalyzed by yeast hexokinase.

The pH dependence of the maximum velocity (V) for the phosphorylation of glucose, the V/Kglucose and the V/KMgATP have been obtained in H2O and 2H2O. In H2O, V decreases below a pK of 5.8, V/Kglucose decreases below a pK of 6.1 and V/KMgATP decreases below a pK of 6.7. In 2H2O, complex behavior is observed for these parameters as a function of pD. The ratios of the parameters in H2O and 2H2O above their respective pK values give solvent deuterium isotope effects of about 1.5-1.7 for all three parameters. When 1,5-anhydromannitol is used as an alternative substrate, an isotope effect different than unity is obtained only for V/K1,5-anhydromannitol which gives a value of about 0.7. Both the complex pH profiles and the relative magnitude of the isotope effects are interpreted in terms of a pH-dependent change in the E X glucose complex.

Catalysis↗
PubMed1983

A new opine derived from nopaline.

Nopaline (N-[4-[(aminoiminomethyl)amino-]-1S-carboxybutyl]-2R-aminopentanedioic acid and isonopaline (N-[4-[(aminoiminomethyl)amino-1S-carboxybutyl]- 2S-aminopentanedioic acid) have been synthesized and separated by crystallization. In addition, a derivative of each of these compounds that forms spontaneously from the parent compounds under the usual crystallization conditions was isolated and characterized. The chemical properties, elemental analysis, 1H-NMR spectrum, and electrophoretic behavior of the derivative from nopaline are consistent with N-[4-[ (aminoiminomethyl)amino]-1S-carboxybutyl]-2-pyrrolidone-5R-carboxylic acid, also called pyronopaline. The presence of pyronopaline in crown gall tumor tissue and the catabolism of it by the bacterium A. tumefaciens establish it as a new opine.

Arginine↗
PubMed1983

Fetal gastric and colonic implants in syngeneic and allogeneic mice developing typical inflammatory changes.

Implants of fetal stomach and colon under the kidney capsule of syngeneic, and H-2 compatible and H-2 incompatible allogeneic mice were examined histologically at different time intervals after the procedure. According to the time of implantation typical inflammatory changes were seen in syngeneic stomach and colon implants, which resembled changes seen in chronic atrophic gastritis and chronic ulcerative colitis. Immunofluorescence studies showed that the host developed antibodies against fetal antigens, while there was no evidence for cellular immune response to fetal syngeneic antigens with the direct leukocyte migration inhibition test. Possible explanations for these results are discussed.

Aging↗
PubMed1982

Octopine dehydrogenase from crown gall tumor and from Pecten maximus. Oxidation of (4R)- and (4S)-[4-3H]NADH.

The stereospecificity of octopine dehydrogenase from crown gall tumor and of octopine dehydrogenase from scallops (Pecten maximum) with respect to the oxidation of C-4 of the dihydronicotinamide ring of NADH was investigated by determination of the distribution of radioactivity after oxidation of (4R)- and (4S)-[4-3H]NADH. Octopine dehydrogenase from crown gall tumor and from scallops stereospecifically removes the pro-S hydrogen atom of the dihydronicotinamide ring with transfer of label to the solvent and to the product octopine (N-2-(1-carboxyethyl)-L-arginine). Although to a lesser extent, the exchange of label from (4S)-[4-3H]NADH with solvent was found to occur when octopine dehydrogenase from either crown gall tumor or from scallops was incubated in the absence of other substrates. Possible mechanisms to explain this exchange are discussed.

Amino Acid Oxidoreductases↗
PubMed1981

Immunity and nutrition in heroin addicts.

An immunological and nutritional survey was conducted on 16 heroin addicts and 16 control subjects matched for age and sex. Skin testing showed none of the addicts to be anergic. Three of four individuals who had positive purified protein derivative (PPD) skin tests failed to produce leucocyte migration inhibition factor (LMIF) to PPD in vitro; in contrast, four of five PPD skin test positive controls produced LMIF. A relative lymphocytosis was found in the heroin addicts although the percentage of 'T' lymphocytes (E-rosetting techniques) was similar to that of the control population. A significant elevation of serum IgG and IgM (p less than 0.0005) was present. Although none of the addicts studied were nutritionally deficient, the serum vitamin C, B6 and albumin levels were significantly lower than in the controls. Elevation of serum aminotransferase values occurred in half the patients, but there was no correlation with presence of serum markers of hepatitis B infection or absent LMIF production. The results suggest that heroin addicts are capable of responding to antigens when their nutritional status in normal, despite the presence of liver enzyme abnormalities.

Adult↗
PubMed1981

Subcellular localisation of vitamin B12 during absorption in the guinea-pig ileum.

The subcellular distribution of vitamin B12 was studied during its absorption in the guinea-pig ileum. Animals were fed either (57Co) or (58Co) cyanocobalamin and killed two or four hours later. At two hours labelled cyanocobalamin was concentrated in brush border and lysosomal fractions of ileal homogenates, with some remaining in the sample layer. In contrast, at four hours labelled cyanocobalamin was concentrated predominantly in the cytosol with much smaller peaks in the brush border and lysosomal fractions. These findings are consistent with vitamin B12 absorption by receptor-mediated endocytosis. It is suggested that, after binding at the brush border, vitamin B12 is first sequestrated within lysosomes, and then released into the cytosol, from where it leaves the cell to enter the portal blood.

Animals↗
PubMed1980

Inhibition of parietal cell function by human gammaglobulin containing gastric parietal cell antibodies.

Parietal cell antibodies (PCA) are found in up to 90% of sera from pernicious anaemia patients but it is often stated that they could represent an epiphenomenon without being directly responsible for the achlorhydria. In the present studies a direct effect of these antibodies on the secretory function of gastric acid-secreting cells has been demonstrated in two different experimental systems. In one set of experiments IgGs containing PCA activity were shown to inhibit acid secretion specifically in the living gastric mucosa of the bull frog suspended as a diaphragm between two chambers. The other system demonstrated their inhibition of carbonic anhydrase activity in a cytochemical bioassay for human G17-gastrin, suggesting a blocking effect on the gastrin receptors in the canalicular microvilli or the cell membrane. These experiments suggest a direct pathogenic role for PCA in autoimmune fundal gastritis and in pernicious anaemia.

Anemia, Pernicious↗
PubMed1979

Cell-mediated cytotoxicity and humoral immune response in ascorbic acid-deficient guinea pigs.

Guinea pigs were divided into three dietary groups: ascorbic-acid deficient, pair-fed, and ad libitum control. Two weeks later guinea pigs were immunized intradermally with 5 x 10(8) chicken erythrocytes in Freund's complete adjuvant. Hemagglutinating antibody titers to chicken erythrocytes 2 weeks after immunization were comparable in all three dietary groups. In vitro 51Cr release from labeled chicken erythrocyte target cells incubated with lymphoid cells from spleens of ascorbic acid-deficient guinea pigs was significantly less than with spleen cells from pair-fed and ad libitum control guinea pigs. The percentage of splenic lymphoid cells that formed rosettes with rabbit erythrocytes, a T cell marker, was the same in all three dietary groups. The defect of ascorbic acid deficiency may reflect an impairment of T lymphocytes function in cell-mediated cytotoxicity or a change in number or function of another cell type.

Animals↗
PubMed1979

Epidemic gastritis with hypochlorhydria.

Seventeen of 37 healthy volunteers participating in studies of acid secretion and 1 patient with Zollinger-Ellison syndrome became rapidly and profoundly hypochlorhydric. A mild illness with epigastric pain occurred in 9 subjects, usually several days before detection of hypochlorhydria. Gastric mucosal biopsy specimens taken from subjects during hypochlorhydria revealed severe fundal and antral gastritis; however, even when acid secretion was severely depressed, parietal cells were abundant and appeared normal histologically. During hypochlorhydria, gastric permeability to hydrogen, sodium, and lithium was normal in 4 subjects. Serum gastrin concentrations were usually normal, whereas serum pepsinogen concentrations were invariably elevated. Serum parietal cell antibodies were not present. Acid secretion returned to near baseline levels in 14 of 17 subjects after a mean of 126 days (range 53--235); severity of gastritis diminished concurrently in 7 of 10 subjects on whom biopsies were serially performed. An infectious etiology is suspected, although serologic studies and bacterial and conventional viral cultures of stool and gastric juice have not identified a candidate agent.

Achlorhydria↗