Purification and characterization of bile salt hydrolase from Clostridium perfringens.

Bile salt hydrolase (cholylglycine hydrolase, EC 3.5.1.

Regulation of bile acid synthesis. II. Effect of bile acid feeding on enzymes regulating hepatic cholesterol and bile acid synthesis in the rat.

Bile acid synthesis is believed to be regulated by bile salts returning to the liver via the portal vein and suppressing cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme in the bile acid biosynthesis pathway. In order to characterize the relative effectiveness of bile salts in regulating bile acid synthesis, seven different bile acids were administered (1% w/w in chow) to rats over a 14-day period. Biliary bile salt composition was determined from bile samples obtained prior to killing; in all cases, the fed bile acid became the predominant bile salt in bile.

Nucleotide sequence and regulation of a gene involved in bile acid 7-dehydroxylation by Eubacterium sp. strain VPI 12708.

Eubacterium sp. strain VPI 12708 is an anaerobic intestinal bacterium that has inducible bile acid 7-dehydroxylation activity. At least four new polypeptides were synthesized after addition of primary bile acids to the growth medium.

Regulation of bile acid synthesis. I. Effects of conjugated ursodeoxycholate and cholate on bile acid synthesis in chronic bile fistula rat.

Bile acid synthesis is thought to be regulated by a negative feedback mechanism which is presumably dependent upon the flux of bile acids in the enterohepatic circulation. To characterize further the role of bile acids in regulation of bile acid synthesis, we have administered pure taurine or glycine conjugates of ursodeoxycholic acid or cholic acid to chronic bile fistula rats by continuous intraduodenal infusion, thus simulating restoration of the enterohepatic circulation. The effects of these bile salt infusions on bile acid synthesis, biliary cholesterol and phospholipid secretion and on the activities of the hepatic microsomal enzymes cholesterol 7 alpha-hydroxylase and HMG-CoA reductase were evaluated.

Molecular cloning of a gene encoding a 45,000-dalton polypeptide associated with bile acid 7-dehydroxylation in Eubacterium sp. strain VPI 12708.

Eubacterium sp. strain VPI 12708 is an intestinal anaerobic bacterium which possesses an inducible bile acid 7-dehydroxylation activity. Two cholic acid-induced polypeptides with apparent molecular weights of 27,000 and 45,000, respectively, coeluted with bile acid 7-dehydroxylation activity upon anaerobic high-performance gel filtration chromatography of crude cellular protein extracts.

Development of a new shuttle plasmid system for Escherichia coli and Clostridium perfringens.

We constructed a 7.9-kilobase-pair recombinant shuttle plasmid, designated pHR106, by combining desired segments of three plasmids: an Escherichia coli plasmid (pSL100) which provides a multiple cloning site, a Clostridium perfringens plasmid (pJU122) which provides a clostridial origin of replication, and an E. coli plasmid (pJIR62) which provides an E.

Isolation of promoters from two anaerobic bacteria.

Promoters which function in Gram-positive organisms show, with few exceptions, remarkable conservation of sequences identical with those in Escherichia coli. An E. coli system was tested to select putative promoters of two anaerobes, the Gram-positive Clostridium absonum and the Gram-negative Bacteroides thetaiotaomicron.

Effects of ursodeoxycholic acid, analogues of ursodeoxycholic acid and combination of bile acids on bile acid synthesis in cultured rat hepatocytes.

The effect of individual 7 beta-hydroxy bile acids (ursodeoxycholic and ursocholic acid), bile acid analogues of ursodeoxycholic acid, combination of bile acids (taurochenodeoxycholate and taurocholate), and mixtures of bile acids, phospholipids and cholesterol in proportions found in rat bile, on bile acids synthesis was studied in cultured rat hepatocytes. Individual steroids tested included ursodeoxycholate (UDCA), ursocholate (UCA), glycoursodeoxycholate (GUDCA) and tauroursodeoxycholate (TUDCA). Analogues of UDCA (7-methylursodeoxycholate, sarcosylursodeoxycholate and ursooxazoline) and allochenodeoxycholate, a representative of 5 alpha-cholanoic bile acid were also tested in order to determine the specificity of the bile acid biofeedback.

Cofactor requirements of steroid-17-20-desmolase and 20 alpha-hydroxysteroid dehydrogenase activities in cell extracts of Clostridium scindens.

Two neutral steroid-transforming activities were demonstrated in cell extracts of Clostridium scindens. Steroid-17-20-desmolase and 20 alpha-hydroxysteroid dehydrogenase were found to be inducible in cells cultured in the presence of cortisol. Both activities required manganese ions and NAD+ or NADH for activity.

Biosynthesis of a novel bile acid nucleotide and mechanism of 7 alpha-dehydroxylation by an intestinal Eubacterium species.

Eubacterium species V.P.I.

Molecular cloning of bile acid 7-dehydroxylase from Eubacterium sp. strain VPI 12708.

Eubacterium sp. strain VPI 12708 is a human intestinal bacterium which contains an inducible bile acid 7-dehydroxylase. Two-dimensional polyacrylamide gel electrophoresis showed that at least four new polypeptides were synthesized after exposure of growing cells to sodium cholate.

Modified plasmid isolation method for Clostridium perfringens and Clostridium absonum.

A rapid plasmid isolation procedure for Clostridium perfringens and C. absonum is described. The ratio of culture volume to lysis buffer volume was found to be crucial for efficient plasmid isolation.

Absence of negative feedback control of bile acid biosynthesis in cultured rat hepatocytes.

The effect of individual bile acids on bile acid synthesis was studied in primary hepatocyte cultures. Relative rates of bile acid synthesis were measured as the conversion of lipoprotein [4-14C]cholesterol into 4-14C-labeled bile acids. Additions to the culture media of cholate, taurocholate, glycocholate, chenodeoxycholate, taurochenodeoxycholate, glycochenodeoxycholate, deoxycholate, and taurodeoxycholate (10-200 microM) did not inhibit bile acid synthesis.

Characterization of delta 4-3-ketosteroid-5 beta-reductase and 3 beta-hydroxysteroid dehydrogenase in cell extracts of Clostridium innocuum.

Cell extracts prepared anaerobically from Clostridium innocuum and Clostridium paraputrificum reduced delta 4-3-ketosteroids to 3 beta 5 beta and 3 alpha 5 beta derivatives, respectively. delta 4-3-Ketosteroid-5 beta-reductase (5 beta-reductase) from both organisms required NADH for activity. 5 beta-Reductase from C.

Suitability of primary monolayer cultures of adult rat hepatocytes for studies of cholesterol and bile acid metabolism.

Monolayer cultures of hepatocytes isolated from cholestyramine-fed rats and incubated in serum-free medium converted exogenous [4-14C]cholesterol into bile acids at a 3-fold greater rate than did cultures of hepatocytes prepared from untreated rats. Cholic acid and beta-muricholic acid identified and quantitated by gas-liquid chromatography and thin-layer chromatography were synthesized by cultured cells for at least 96 h following plating. The calculated synthesis rate of total bile acids by hepatocytes prepared from cholestyramine-fed animals was approximately 0.

HPLC purification and preparation of antibodies to cholic acid-inducible polypeptides from Eubacterium sp. V.P.I. 12708.

The role of bile acid-inducible polypeptides in 7-dehydroxylation was investigated in Eubacterium sp. V.P.

Effect of bile acid analogs on 7 alpha-dehydroxylase activity in Eubacterium sp. V.P.I. 12708.

7 beta-Methyl-chenodeoxycholic acid (7-MeCDC, 3 alpha, 7 alpha-dihydroxy-7 beta-methyl-5 beta-cholan-24-oic acid), 7 alpha-methyl-ursodeoxycholic acid (7-MeUDC, 3 alpha, 7 beta-dihydroxy-7 alpha-methyl-5 beta-cholan-24-oic acid), 7 xi-methyl-lithocholic acid (7-MeLC, 3 alpha-hydroxy-7 xi-methyl-5 beta-cholan-24-oic acid) and ursodeoxycholylsarcosine (UDCS) were tested as inhibitors of bacterial bile acid 7 alpha-dehydroxylase activity. At a concentration of 50 microM, 7-MeCDC and 7-MeUDC inhibited enzyme activity by 66% and 12%, respectively. 7 alpha-Dehydroxylase activity was not inhibited in the presence of 7-MeLC and UDCS.

Mode of action of steroid desmolase and reductases synthesized by Clostridium "scindens" (formerly Clostridium strain 19).

A recently isolated hitherto unknown Clostridium from human feces, designated Clostridium "scindens" (formerly strain 19), synthesizes at least two enzymes active on the side-chain of the steroid molecule and two enzymes active on the hydroxyl groups of the 7-position of bile acids. Steroid desmolase, responsible for side-chain cleavage of corticoids, and 20 alpha-hydroxysteroid dehydrogenase have not been detected in any other bacterial species of the resident colonic flora. Steroid desmolase is Eh-dependent (optimum ca.

Characterization of active and inactive forms of rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was purified to homogeneity using agarose-HMG-CoA affinity chromatography. Additional protein was isolated from the affinity column with 0.5 M KCl that demonstrated no HMG-CoA reductase activity, yet comigrated with purified HMG-CoA reductase on sodium dodecyl sulfate-polyacrylamide gels.

Separation of 7 alpha- and 7 beta-hydroxysteroid dehydrogenase activities from clostridium absonum ATCC# 27555 and cellular response of this organism to bile acid inducers.

Both 7 alpha- and 7 beta-hydroxysteroid dehydrogenases (HSDH) were induced by either chenodeoxy-(CDC) or deoxycholic (DC) acid in C. absonum. 7 beta-HSDH was partially purified 35-fold from CDC-induced cultures of C.

Metabolism of bile acid oxazoline derivatives by hepatocyte monolayer cultures and intestinal anaerobic bacteria.

Certain bile acid oxazoline derivatives (100 microM), but not corresponding unconjugated bile acids (100 microM), were found to inhibit the growth of Eubacterium sp. V.P.

Gamma scintigraphic localization of platelets labeled with indium 111 in a focus of infection.

To determine if autologous platelets would localize in a focus of infection, a pyogenic abscess was created in the left hind limb of dogs, using previously processed human stool, while an identical surgical procedure without bacterial inoculation was performed on the right hind limb. Autologous platelets labeled with indium 111 (500 microCi) were administered intravenously to five control dogs that had not undergone surgery, to eight dogs two hours following stool inoculation, and to five dogs 24 hours after stool inoculation. A statistically significant scintigraphic increase in tracer activity was apparent within 24 hours in each animal at the site of abscess creation.

Regulation of bile acid 7-dehydroxylase activity by NAD+ and NADH in cell extracts of Eubacterium species V.P.I. 12708.

The 7 alpha-dehydroxylation of primary bile acids by Eubacterium sp. V.P.

Antibacterial properties of bile acid oxazoline compounds.

Chenooxazoline (50-100 microM) inhibited (greater than 50%) both 7 alpha and 7 beta-dehydroxylase activities in whole cells and cell extracts of Eubacterium sp. V.P.

16 alpha-dehydration of corticoids by bacteria isolated from rat fecal flora.

Two strains (No. 144 and No. 146) of rat intestinal anaerobic bacteria, phenotypically similar to Eubacterium lentum, were isolated and found capable of 16 alpha-dehydrating corticoids.