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Membrane filter procedure for enumeration of Vibrio parahaemolyticus.

A membrane filtration procedure has been developed for the enumeration of Vibrio parahaemolyticus in marine waters. Background microbial growth on the primary medium was decreased through the use of sodium cholate and copper sulfate, high pH, 3% NaCl, and an elevated incubation temperature. A series of in situ tests was employed to obviate the picking of colonies for identification; thereby, the enumeration of V. parahaemolyticus was accomplished within 30 h. Confirmation of typical colonies approached 95%. Relative to immediate plating on brain heart infusion agar spread plates, the recovery of V. parahaemolyticus cells suspended in phosphate-buffered saline or in seawater held for 24 h at 4 to 6 degrees C was about 90%. Assay variability did not exceed that expected by chance. Recoveries of V. parahaemolyticus from coastal and estuarine surface waters exceeded those obtainable by other methods examined.

Culture Media↗

Effect of restricted aeration on catabolism of cholic acid by two Pseudomonas species.

Examination of some previously isolated bile acid-utilizing Pseudomonas strains showed that Pseudomonas sp. ATCC 31752, together with other fluorescent strains, can be assigned to Pseudomonas putida biotype B, whereas Pseudomonas sp. ATCC 31753, like most other nonfluorescent strains, is an unrecognized phenotype. A study was made of the growth of these two species at 25 degrees C and pH 7.0 in a fermentor with 2.5 g of sodium cholate liter-1 as sole carbon source, and the catabolism of the cholate and its products was followed by high-pressure liquid chromatographic and thin-layer chromatographic examination. At aeration rates of either 150 or 5 ml min-1 liter-1, growth of each species followed the same catabolic pathway. 7 alpha, 12 beta-Dihydroxy-1,4-androstadiene-3,17-dione was the major catabolite formed, with 0.3 g liter-1 being the maximum concentration that accumulated at the higher aeration rate, whereas 1.4 g liter-1 accumulated at the lower aeration rate, irrespective of the species used. The latter yield is sufficiently high to be of potential commercial value if such a catabolite were found to be economically useful for steroid drug manufacture. It is postulated that the rate-limiting step in cholic acid catabolism by these species at the lower aeration rate is 9 alpha-hydroxylation, a step requiring molecular oxygen, hence, the marked effect of oxygen limitation on catabolite accumulation. Another consequence of oxygen limitation is the production of a red pigment in the culture medium, which, however, does not affect catabolite recovery.

Aerobiosis↗

Resistance of Escherichia coli to penicillins. IX. Genetics and physiology of class II ampicillin-resistant mutants that are galactose negative or sensitive to bacteriophage C21, or both.

Ampicillin-resistant mutants of class II are determined by a doubling of chromosomally and episomally mediated ampicillin resistance on agar plates. Several mutants were isolated from a female as well as from an Hfr strain. The mutants differed from each other in various properties such as response to colicin E2 and sodium cholate, response to the phages T4 and C21, and fermentation of galactose. By conjugation and transduction experiments, it was shown that mutations in at least four loci gave the class II phenotype. The mutations were found to be in the galU gene, the ctr gene, and two new genes close to mtl denoted lpsA and lpsB. The carbohydrate compositions of the lipopolysaccharides of the mutants were investigated and found to be changed compared to the parent strains. GalU mutants lacked rhamnose and galactose and had 11% glucose compared to the parent strain. The lpsA mutant also lacked rhamnose and had only traces of galactose and 58% glucose, whereas the lpsB mutant contained 14% rhamnose, traces of galactose, and 81% glucose compared to the parent strain.

Ampicillin↗

Multiple forms of 7-alpha-hydroxysteroid dehydrogenase in selected strains of Bacteroides fragilis.

Multiple forms of 7-alpha-hydroxysteroid dehydrogenase were detected in six of nine strains of Bacteroides fragilis. The enzymes differed with respect to pyridine nucleotide specificity, thermal stability, divalent metal cation requirement, and elution profilies from Sephadex G-200 columns. The nicotinamide adenine dinucleotide phosphate (NADP)-dependent enzyme required divalent metal cations, preferentially Mn-2+ (Km, 57 muM), for maximum catalytic activity. The NADP-dependent enzyme was labile at 65 C for 10 min, whereas the nicotinamide adenine dinucleotide (NAD)-dependent enzyme was stable at 65 C for 10 min. The specific activity of both the NAD- and NADP-dependent enzymes in crude extracts increased markedly (15- and 7.5-fold, respectively) during the transition from exponential- to stationary-phase growth in glucose medium containing 0.5 mM sodium cholate. The time course of apparent enzyme induction correlated temporally with the transformation of the 7-alpha-hydroxy group of cholate in the culture supernatant fluid. Both NAD- and NADP-dependent 7-alpha-hydroxysteroid dehydrogenase activities were found to be widely, but not universally, distributed in different strains and subspecies of B. fragilis. No NAD- or NADP-dependent 7-alpha-hydroxysteroid dehydrogenase activity could be detected in B. fragilis subsp. vulgatus Virginia Polytechnic Institute (VPI) no. 4245, subsp. thetaiotaomicron VPI 0061-1, or subsp. distasonis VPI 4243.

Bacteroides↗

Protein-associated lipid of Bacillus stearothermophilus.

The composition and patterns of metabolism of phospholipids isolated as part of a lipid-depleted membrane fragment (LDM fragment) and associated with the membrane adenosine triphosphatase complex have been compared with those of the bulk membrane phospholipid. The bulk lipid was extracted from washed membranes with sodium cholate. The LDM fragments, which contained a portion of the electron transport system and the membrane adenosine triphosphatase complex, were purified by chromatography with Sepharose 6B. The LDM fragment preparations contained 0.10 +/- 0.02 mumol of lipid phosphorus per mg of protein, compared with 0.54 +/- 0.05 mumol of lipid phosphorus per mg of protein for washed membranes. The phospholipid associated with the LDM fragments consisted of 78 +/- 4% cardiolipin, 7 +/- 1% phosphatidylglycerol, and 15 +/- 3% phosphatidylethanolamine. Changes in the total membrane lipid composition (produced by culture conditions) did not alter the phospholipid composition of the LDM fragments. The adenosine triphosphate complex was separated from the other components of the LDM fragments by suspension of the fragments in 1% Triton X-100 and precipitation with antibody specific for the F(1) component of the adenosine triphosphatase complex. The phospholipid isolated with the adenosine triphosphatase complex consisted of 86% cardiolipin, 8% phosphatidylglycerol, and 6% phosphatidylethanolamine. In pulse-chase experiments with (32)P and [2-(3)H]glycerol, the labeling patterns of the phosphatididylglycerol and phosphatidylethanolamine associated with the LDM fragments were different from those of the bulk membrane phosphatidylglycerol and phosphatidylethanolamine. It was concluded that at least a portion of the phospholipid isolated with the LDM fragments was part of a native lipid-protein complex.

Adenosine Triphosphatases↗

Purification and characterization of membrane-bound fumarate reductase from anaerobically grown Escherichia coli.

Fumarate reductase has been purified 100-fold to 95% homogeneity from the cytoplasmic membrane of Escherichia coli, grown anaerobically on a defined medium containing glycerol plus fumarate. Optimal solubilization of total membrane protein and fumarate reductase activity occurred with nonionic detergents having a hydrophobic-lipophilic balance (HLB) number near 13 and we routinely solubilized the enzyme with Triton X-100 (HLB number = 13.5). Membrane enzyme extracts were fractionated by hydrophobic-exchange chromatography on phenyl Sepharose CL-4B to yield purified enzyme. The enzyme whether membrane bound, in Triton extracts, or purified, had an apparent Km near 0.42 mM. Two peptides with molecular weights of 70 000 and 24 000, predent in 1:1 molar ratios, were identified by sodium dodecyl sulfate polyacrylamide slab-gel electrophoresis to coincide with enzyme activity. A minimal native molecular weight of 100 000 was calculated for fumarate reductase by Stephacryl S-200 gel filtration in the presence of sodium cholate. This would indicate that the enzyme is a dimer. The purified enzyme has low, but measurable, succinate dehydrogenase activity.

Anaerobiosis↗

Influence of hepatic taurine concentration on bile acid conjugation with taurine.

In vitro, addition of taurine to liver homogenates increases the proportion of cholic acid conjugated with taurine. In the present study, the relation between hepatic taurine concentration and the proportion of infused sodium cholate conjugated with taurine was studied in the whole organ. The isolated perfused liver was studied to eliminate possible transfer of taurine to or from the large extrahepatic poosl present in vivo. During cholate infusion, the proportion of taurocholate excreted in bile decreased, and the proportion of glycocholate increased in a complementary fashion. Infusion of taurine with cholate prevented these changes. Hepatic taurine concentration, calculated from measured hepatic taurine concentrations before and at the end of cholate infusion, fell. Fall in proportion of total bile acid excreted as taurocholate was most rapid at low hepatic taurine concentrations between about 1.4 and 0.65 mumol/g liver. Hepatic taurine concentrations is a major determinant of the proportion of bile acid conjugated with taurine.

Animals↗

Reconstitution of solubilized V1 vasopressin receptors of human platelets.

We describe the reconstitution of solubilized human platelet arginine vasopressin (AVP) receptors into phospholipid vesicles. Purified platelet plasma membranes enriched in AVP receptors [binding equilibrium dissociation constant (Kd) = 1.87 +/- 0.14 nM, maximum number of binding sites (Bmax) = 261 +/- 10 fmol/mg protein] were solubilized with 20 mM sodium cholate. Phospholipid vesicles made of 10% cholesterol, 20% egg phosphatidylcholine, and 70% egg phosphatidylserine were formed by bath sonication. Solubilized AVP receptors were incorporated into the vesicles while the detergent was removed by filtration through Sephadex G-100. The reconstituted receptors retained a high affinity for [3H]AVP (Kd = 3.19 +/- 0.13 nM, Bmax = 257 +/- 9 fmol/mg). Competition experiments with different AVP analogues confirmed the V1 vascular nature of the reconstituted receptors. Saturation experiments carried out with the agonist [3H]AVP and the V1 antagonist [3H]d(CH2)5Tyr(Me)AVP revealed that agonist binding to the reconstituted receptors was divalent cation dependent, whereas antagonist binding was not. Moreover, the affinity of the agonist [3H]AVP for the reconstituted receptors was modulated by the nonhydrolyzable guanine nucleotide analogue guanosine 5'-[gamma-thio]triphosphate (GTP gamma S), whereas [3H]d(CH2)5Tyr(Me)AVP binding affinity was not. The phospholipid vesicles could be loaded with free fura-2 and displayed an enhanced fluorescence caused by calcium entry after addition of ionomycin. However, stimulation by AVP did not induce an increase of free calcium inside the vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Arginine Vasopressin↗

Lithocholyltaurine interacts with cholinergic receptors on dispersed chief cells from guinea pig stomach.

Although bile acids damage gastric mucosa, the mechanisms underlying tissue injury induced by these agents are not well understood. To determine whether bile acids alter gastric secretory function, we investigated the actions of sodium cholate, deoxycholate, lithocholate, and their taurine and glycine conjugates on a highly homogeneous population of gastric chief cells. Lithocholyltaurine (LCT), a particularly injurious bile acid, caused a threefold increase in pepsinogen secretion (detectable with 100 nM and maximal with 10 microM LCT). When combined with other secretagogues, increasing concentrations of LCT caused progressive inhibition of carbamylcholine (carbachol)-induced pepsinogen secretion but did not alter CCK- or 8-bromo-cAMP-induced secretion. Taurine and unconjugated lithocholate did not alter basal or carbachol-induced secretion. These observations suggested that LCT is a partial cholinergic agonist. To test this hypothesis, we examined the actions of the cholinergic antagonist atropine on LCT-induced pepsinogen secretion. Atropine (10 microM) abolished carbachol- and LCT-induced pepsinogen secretion. Likewise, carbachol (0.1 mM) and LCT (1 mM) induced an atropine-sensitive, two- to threefold increase in cellular levels of inositol 1,4,5-trisphosphate. We examined the actions of LCT on binding of the cholinergic radioligand [N-methyl-3H]scopolamine ([3H]NMS) to chief cells. Half-maximal inhibition of [3H]NMS binding was observed with approximately 0.5 mM carbachol and 1 mM LCT. These results indicate that the bile acid LCT is a partial agonist for muscarinic cholinergic receptors on gastric chief cells.

8-Bromo Cyclic Adenosine Monophosphate↗

Characteristic glomerular lesions in the ExHC rat: a unique model for lipid-induced glomerular injury.

ExHC rats were bred from the Sprague-Dawley strain and are highly susceptible to dietary hypercholesterolemic stimuli. Although chemical and histological examination of the aorta from ExHC rats fed a diet containing 3% cholesterol, 0.6% sodium cholate and 15% olive oil (high-cholesterol diet, HCD) has been reported, renal injury in this model has not been investigated to date. Therefore, we examined the renal pathological changes and proteinuria in this model. ExHC rats fed an HCD developed proteinuria and characteristic glomerular lesions within 4-8 weeks after initiation of the diet. The most striking glomerular change observed in this model was the marked accumulation of numerous lipid-filled foam cells, with a surface marker that identified them as macrophages, within the mesangial regions which could have been involved in focal and segmental glomerular sclerosis. Our study demonstrates that ExHC rats fed a cholesterol-supplemented diet develop glomerular injury that may be mediated by infiltrating macrophages. These results imply that ExHC rats may be well-suited for the use in investigations of the role of macrophages in the pathogenesis of lipid-induced glomerular injury.

Animals↗

Paradoxical reduction of fatty streak formation in mice lacking endothelial nitric oxide synthase.

BACKGROUND: The endothelial isoform of nitric oxide synthase (eNOS) has been considered to exert an antiatherosclerotic role through synthesis of NO. However, eNOS has been shown to generate superoxide, which could oxidize LDL and promote atherosclerosis. We sought to determine the role of eNOS in diet-induced fatty streak formation through the use of eNOS-deficient mice. METHODS AND RESULTS: Mice were fed an atherogenic diet containing 15% fat, 1.25% cholesterol, and 0.5% sodium cholate for 12 weeks, and atherosclerotic lesions at the aortic root were measured after oil-red O staining. Unexpectedly, eNOS-deficient mice developed much smaller aortic lesions than did wild-type control mice (2544+/-1107 versus 7023+/-1569 microm2/section; P=0.03). This reduction in lesion formation could not be explained by changes in plasma levels of lipids and susceptibility of lipoproteins to oxidation. To examine whether eNOS contributed to the oxidation of LDL within the arterial wall, endothelial cells were isolated from the aorta of mice and incubated with native LDL in the absence or presence of N-Omega-nitro-L-arginine methyl ester (L-NAME), a specific NOS inhibitor. L-NAME significantly inhibited LDL oxidation by endothelial cells from wild-type animals (P<0.05), but it had no effect on LDL oxidation by endothelial cells from eNOS-deficient mice. CONCLUSIONS: These data indicate that absence of eNOS-mediated LDL oxidation may contribute to the reduction of fatty-streak formation in eNOS-deficient mice.

Animals↗

Intramural hemorrhage and endothelial changes in atherosclerotic coronary artery after repetitive episodes of spasm in x-ray-irradiated hypercholesterolemic pigs.

To assess whether coronary spasm affects the progression of atherosclerosis and results in evolution of myocardial infarction, the role of coronary spasm on the fine structure of conduit coronary arteries was studied morphologically. Göttingen miniature pigs were fed a semisynthetic diet containing 2% cholesterol and 1.1% sodium cholate. One month after being on this diet, the pigs were anesthetized and the endothelium of a branch of the left coronary artery was denuded using a balloon catheter. X-ray irradiation in a dose of 1,500 rad was given twice selectively to the area denuded, after 4 and 5 months of cholesterol feeding. Five months after endothelial denudation, transient (group A) and repetitive episodes (group B) of coronary spasm were provoked by single and periodic (five times every 5 minutes) intracoronary injections of serotonin (10 micrograms/kg/injection), respectively. The extent of spasm by serotonin at the previously denuded site was 84 +/- 4% (n = 4) and 90 +/- 5% (n = 6) narrowing in groups A and B (p = NS between groups), respectively. Forty minutes after the final administration of serotonin, the left coronary artery was relaxed by nitroglycerin, and the heart was isolated and perfuse-fixed under physiological pressure. Intramural hemorrhage was noted at the spastic site in six pigs of group B but not in group A. The average percent luminal narrowing, on cross sections at the spastic site in group B, was significantly greater than in group A (56 +/- 7% vs. 23 +/- 5%, p less than 0.01). Scanning electron micrographs revealed that the endothelial lining was intact at the nonspastic site in both groups. In addition to the appearance of intercellular bridges at the spastic site in both groups, squeezing of endothelial cells and adhesion of white blood cells were present at the spastic site exclusively in group B. These findings are consistent with the hypothesis that repetitive spasm may have an important role in the progression of atherosclerosis and/or myocardial infarction.

Angiography↗

Reconstitution of steroid 17,20-lyase activity after separation and purification of cytochrome P-450 and its reductase from rat testis microsomes.

The testicular enzyme, 17,20-lyase, catalyzes the removal of the C-17 side chain from steroids in the synthesis of androgens. This activity employs cytochrome P-450 as an oxygen donor. Attempts to purify the cytochrome and its reductase from testis microsomes have previously been unsuccessful due to the low concentrations of these components (2--5% that of liver). The cytochrome and reductase were solubilized from rat testis microsomes using a mixture of sodium cholate and Emulgen 913. The components were then separated by DEAE chromatography. The cytochrome was further purified by chromatography using hydroxylapatite for an 8.5-fold enrichment. The reductase was further purified by hydroxylapatite and affinity chromatography. An 84-fold enrichment was achieved. 17,20-Lyase activity could be partially restored by mixing the cytochrome and reductase in the presence of phospholipid.

Aldehyde-Lyases↗

Effects of a dosing solution on the nasal absorption of non-glycosylated recombinant human granulocyte colony-stimulating factor in rats.

The nasal absorption of non-glycosylated recombinant human granulocyte colony-stimulating factor produced by Esherichia coli (E. coli-rhG-CSF) was studied in rats. We investigated the effects of the basic conditions of the dosing solution and the enhancing effects of various substances to find means to improve the nasal absorption of E. coli-rhG-CSF. Relative bioavailability following the nasal administration of E.coli-rhG-CSF solution to subcutaneous administration was 3.6%. Osmotic pressure of the dosing solution had virtually no effect on the nasal absorption of E. coli-rhG-CSF. Absorption was enhanced by a decrease in pH, an increase in buffer concentration at pH2 and an increase in drug concentration. Sodium cholate, ascorbic acid, tartaric acid, beta-cyclodextrin and dimethyl-beta-cyclodextrin in remarkably increased the nasal absorption of E. coli-rhG-CSF. Condition adjustments to the dosing solution and the use of several enhancers were significantly concluded to increase the intranasal absorption of E. coli-rhG-CSF.

Absorption↗

Anti-hyperliposis effect of maitake fruit body (Grifola frondosa). I.

Experimental rat models (5-week-old Sprague-Dawley rats) with hyperlipemia were prepared by feeding high-cholesterol feed containing sodium cholate and casein as a protein source. Dried maitake (Grifola frondosa) powder was mixed with the basic high-cholesterol feed and the serum lipids were periodically measured. Values of cholesterol, triglyceride and phospholipid in serum of rats in the maitake-feed group were suppressed by 0.3-0.8 times those in animals fed the basic feed, the latter values being close to those in rats given normal feed. The value of high density lipoprotein (HDL)-cholesterol in serum which is generally reduced by the ingestion of high-cholesterol feed remained the level it was at the beginning of the experiment. Weights of extirpated liver and epididymal fat-pads were significantly less (0.6-0.7 times) than those in the basic feed group, indicating that maitake inhibits lipid accumulation in the body. Liver lipids were also measured and the values were found to be decreased by maitake administration as true of serum lipid, suggesting maitake has an anti-liver lipid activity. Measurement of the amount of total cholesterol and bile acid in feces showed, the ratio of cholesterol-excretion had increased 1.8 times and bile acid-excretion 3 fold by maitake treatment. From these results, it is believed that maitake helps to improve the lipid metabolism as it inhibits both liver lipid and serum lipid which are increased by the ingestion of high-fat feed.

Animals↗

Mode of interaction between forskolin and manganese ion in activating catalytic unit of adenylate cyclase from rat brain.

Rat brain adenylate cyclase was solubilized with a combination of 0.7% sodium cholate and 0.6 M ammonium sulfate, and fractionated by addition of solid ammonium sulfate. The precipitate at 35% ammonium sulfate saturation contained neither guanine nucleotide-binding regulatory protein (G protein) nor calmodulin, and was used as the catalytic unit of the enzyme system. This catalytic unit was activated synergistically by forskolin and Mn2+. An apparent Km value for Mg-adenosine triphosphate (ATP) of the catalytic unit was about 80 microM in the basal state, while it increased in concurrence with the increase in the enzyme activity when forskolin was added to the assay system. The increase in the Km value depended on the forskolin concentration up to 1 microM, above which the value converged on ca. 200 microM. Furthermore, activation of the catalytic unit by forskolin was more marked at higher concentration of Mg-ATP. On the other hand, Mn2+ suppressed the increase in the Km value for Mg-ATP by forskolin, though the value in the basal state was not changed by Mn2+ alone. These findings indicate that the activation of the catalytic unit by forskolin is accompanied by the change in the affinity for Mg-ATP and Mn2+ modifies the change.

Adenylyl Cyclases↗

Promoting mechanism by bile salt related to water absorption in drug rectal absorption.

The promoting mechanism by bile salts in rat rectal absorption of antipyrine was studied by the in situ recirculating perfusion. The significant correlation between rectal absorption clearance (CLAP) of antipyrine (AP) and apparent water influx (influx') was found in the control without promoters, indicating the existence of solvent drag in the AP rectal absorption. Sieving coefficient of AP (bAP), i.e. the slope of the regression line between CLAP and influx', was 0.6 approximately equal to that in small intestine reported previously. The relation between the promoting effects and solvent drag was further studied, resulting that both CLAP and influx' were significantly enhanced by sodium taurocholate (TC-Na), sodium glycocholate (GC-Na) and sodium cholate (C-Na). Accordingly the promoting effects of bile salts might be due to the increase in solvent drag. However TC-Na did not produce the significant change in bAP. From these results, the enhancing mechanism in the epithelial cell membrane transport was discussed.

Animals↗

Differences in effects on drug absorption between dihydroxy and trihydroxy bile salts.

The effect of a dihydroxy bile salt, sodium taurodeoxycholate (STDC), on drug absorption from rat small intestine was investigated in comparison with that of trihydroxy one, sodium cholate (STC). The enhancement or inhibitory effects on the absorption of various model drugs by STDC bore similar tendency to those by STC, and the magnitude was greater than the latter. The absorption of sulfaguanidine was enhanced by STDC, which solubilized the phospholipids from the mucosal barrier membrane in addition to the STC-like, or ethylenediaminetetraacetate-like action. STDC inhibited the absorption of sulfadimethoxine contrary to the insignificant effect by STC. The direct action of STDC on the mucosal membrane reduced the absorption in addition to the physicochemical action, such as micellar complex formation. Quinine absorption was inhibited in the presence of STDC, which can be explained by the micellar complex formation. Possible mechanisms of the differences in the effect of STDC and STC were discussed in relation to their micellar properties.

Animals↗