Sulfoacetate generated by Rhodopseudomonas palustris from taurine

Genes thought to encode (a) the regulator of taurine catabolism under carbon-limiting or nitrogen-limiting conditions and (b) taurine dehydrogenase were found in the genome of Rhodopseudomonas palustris. The organism utilized taurine quantitatively as a sole source of nitrogen (but not of carbon) for aerobic and photoheterotrophic growth. No sulfate was released, and the C-sulfonate bond was recovered stoichiometrically as sulfoacetate, which was identified by mass spectrometry. An inducible sulfoacetaldehyde dehydrogenase was detected. R. palustris thus contains a pathway to generate a natural product that was previously believed to be formed solely from sulfoquinovose.The senior author (AMC) would like to express his thanks for the rewarding experience of doing postdoctoral research in the laboratory of Prof. H.-G. Schlegel.     

Taurocholate is more potent than cholate in suppression of bile salt synthesis in the rat

Synthesis of bile salts is regulated through negative feedback inhibition by bile salts returning to the liver. Individual bile salts have not been distinguished with regard to inhibitory potential. We assessed inhibition of bile salt synthesis by either cholate or its taurine conjugate in bile fistula rats. After allowing synthesis to maximize, baseline synthesis was determined by measuring bile salt output in four consecutive 6-hr periods. Next, sodium cholate (+[(14)C]cholate) or taurocholate (+[(14)C]taurocholate) was infused into the jugular vein for 36 hr and bile was collected in 6-hr aliquots. Hepatic flux of exogenous bile salt was determined by measuring output of radioactivity in bile divided by specific activity of the infusate. Synthesis was determined during the last four 6-hr periods of infusion by subtracting exogenous bile salt secretion from the total bile salt output. Thirteen studies using cholate and 13 using taurocholate were performed. Hepatic flux of infused bile salt varied from 1 to 12 micro mol/100 g per rat per hr. Percent suppression of synthesis varied directly with hepatic flux of exogenous bile salt for both cholate and taurocholate in a linear fashion (r = 0.66, P < 0.01 and r = 0.87, P < 0.0005, respectively). Slope of the taurocholate line was 7.82 (% suppression/ micro mol per 100 g per hr), while slope of the cholate line was 3.66 (P < 0.05), indicating that taurocholate was approximately twice as potent as cholate in suppression of synthesis. At fluxes of 10-12 micro mol/100 g per hr, taurocholate suppressed synthesis 84 +/- 8 (SEM) % while cholate suppressed synthesis only 42 +/- 12% (P < 0.02). The x-intercept of the taurocholate line was 0.65 ( micro mol/100 g per hr), while that of the cholate line was -1.01 (NS) suggesting that the threshold for initial suppression of synthesis did not differ for these two bile salts. We conclude that taurocholate is a more effective inhibitor of hepatic bile salt synthesis than cholate, and that intestinal deconjugation of bile salts may play a role in the regulation of synthesis.-Pries, J. M., A. Gustafson, D. Wiegand, and W. C. Duane. Taurocholate is more potent than cholate in suppression of bile salt synthesis in the rat.     

Degradation of and sensitivity to cholate in Pseudomonas sp. strain Chol1

A facultative anaerobic bacterium, Pseudomonas sp. strain Chol1, degrading cholate and other bile acids was isolated from soil. We investigated how strain Chol1 grew with cholate and whether growth was affected by the toxicity of this compound. Under anoxic conditions with nitrate as electron acceptor, strain Chol1 grew by transformation of cholate to 7,12-dihydroxy-1,4-androstadiene-3,17-dione (DHADD) as end product. Under oxic conditions, strain Chol1 grew by transformation of cholate to 3,7,12-trihydroxy-9,10-seco-1,3,5(10)-androstatriene-9,17-dione (THSATD), which accumulated in the culture supernatant before its further oxidation to CO₂. Strain Chol1 converted DHADD into THSATD by an oxygenase-dependent reaction. Addition of cholate (≥10 mM) to cell suspensions of strain Chol1 caused a decrease of optical density and viable counts but aerobic growth with these toxic cholate concentrations was possible. Addition of CCCP or EDTA strongly increased the sensitivity of the cells to 10 mM cholate. EDTA also increased the sensitivity of the cells to DHADD and THSATD (≤1.7 mM). The toxicity of cholate and its degradation intermediates with a steroid structure indicates that strain Chol1 requires a strategy to minimize these toxic effects during growth with cholate. Apparently, the proton motive force and the outer membrane are necessary for protection against these toxic effects.     

Vectorial transport of unconjugated and conjugated bile salts by monolayers of LLC-PK1 cells doubly transfected with human NTCP and BSEP or with rat Ntcp and Bsep

Na(+)-taurocholate-cotransporting peptide (NTCP)/SLC10A1 and bile salt export pump (BSEP)/ABCB11 synergistically play an important role in the transport of bile salts by the hepatocyte. In this study, we transfected human NTCP and BSEP or rat Ntcp and Bsep into LLC-PK1 cells, a cell line devoid of bile salts transporters. Transport by these cells was characterized with a focus on substrate specificity between rats and humans. The basal to apical flux of taurocholate across NTCP- and BSEP-expressing LLC-PK1 monolayers was 10 times higher than that in the opposite direction, whereas the flux across the monolayer of control and NTCP or BSEP single-expressing cells did not show any vectorial transport. The basal to apical flux of taurocholate was saturated with a K(m) value of 20 microM. Vectorial transcellular transport was also observed for cholate, chenodeoxycholate, ursodeoxycholate, their taurine and glycine conjugates, and taurodeoxycholate and glycodeoxycholate, whereas no transport of lithocholate was detected. To evaluate the respective functions of NTCP and BSEP and to compare them with those of rat Ntcp and Bsep, we calculated the clearance by each transporter in this system. A good correlation in the clearance of the examined bile salts (cholate, chenodeoxycholate, ursodeoxycholate, and their taurine or glycine conjugates) was observed between transport by human and that of rat transporters in terms of their rank order: for NTCP, taurine conjugates > glycine conjugates > unconjugated bile salts, and for BSEP, unconjugated bile salts and glycine conjugates > taurine conjugates. In conclusion, the substrate specificity of human and rat NTCP and BSEP appear to be very similar at least for monovalent bile salts under physiological conditions.     

Bile salts and taurine as chemical stimuli for glass eels,Anguilla anguilla: a behavioural study

Synopsis The behaviour of migrating glass eels towards different concentrations of seven bile salts and taurine was investigated by binary-choice experiments. All substances attracted glass eels when presented at concentrations below 10^–10M. Glycocholate, taurodeoxycholate and taurine remained attractive at higher concentrations, while taurocholate, cholate, deoxycholate, glycochenodeoxycholate and taurochenodeoxycholate became repellent. A role of bile salts in grouping and orientation behaviour of glass eels is discussed.     

Isethionate as a product from taurine during nitrogen-limited growth of <Emphasis Type="Italic">Klebsiella oxytoca</Emphasis> TauN1

Klebsiella oxytoca TauN1 represents a group of isolates which utilise taurine (2-aminoethanesulfonate) quantitatively as a sole source of combined nitrogen for aerobic growth. During growth, a compound is excreted, which has now been identified as isethionate (2-hydroxyethanesulfonate). An ion-chromatographic separation of isethionate was developed to quantify the putative isethionate, whose identity was confirmed by matrix-assisted, laser-desorption ionisation time-of-flight mass spectrometry. Strain TauN1 utilised taurine (and excreted isethionate) concomitantly with growth. Cell-free extracts contained inducible taurine transaminase, which yielded sulfoacetaldehyde. A soluble, NADP-dependent isethionate dehydrogenase converted sulfoacetaldehyde to isethionate. The enzyme was partially purified and it apparently belonged to the family of short-chain alcohol dehydrogenases.We hope that the Leader of the Sulfur Department, Norbert Pfennig LSD, will be amused by the biology involving some of the compounds from his domain.     

Cystine catabolism in mycelia of Microsporum gypseum,a dermatophytic fungus

The fate of ³⁵S label was studied during cystine degradation by mycelia of the dermatophytic fungus Microsporum gypseum. Excess free cystine in the medium was readily taken up and its sulfur moiety excreted as inorganic sulfate and sulfite. At intervals after 3–60 min of incubation with ³⁵S cystine the products of cystine catabolism were extracted from the mycelia by boiling water and separated by thin layer chromatography and electrophoresis. A total of 10 sulfur-containing compounds were identified, and their relative radioactivity was assessed. After 3 min the mycelia contained, in addition to cystine, labeled cysteine and particularly cysteine sulfinic acid which was accompanied by a smaller amount of cysteic acid. Later on, oxidized and reduced glutathione, inorganic sulfate and taurine appeared consecutively. In all extracts, small amounts of labeled S-sulfocysteine were found, not, however, sulfite.The results suggest that the intermediates of cysteine degradation in the fungal mycelia are cysteine, cysteine sulfinate, unstable sulfinylpyruvate, sulfite and sulfate, i.e., that the catabolic pattern is similar to that of higher organisms.The formation and the role of S-sulfocysteine, cysteic acid, and of taurine is not yet completely understood, although certainly autoxidative processes are involved in the formation of the latter two compounds, and sulfitolysis in that of the former compound.     

12alpha-Hydroxysteroid dehydrogenase from Clostridium group P strain C48-50 ATCC No. 29733: partial purification and characterization.

The growth of Clostridium group P strain C48-50 [an anaerobe that contains 12alpha-hydroxysteroid dehydrogenase (12alpha-HSDH) in the absence of other dehydrogenases active upon bile salts] is greatly enhanced by the addition of 2.0% d-fructose or d-glucose to the growth medium. Other sugars were less effective. The production of NADP-dependent 12alpha-HSDH paralleled the growth of the organism which was optimal at 72 hr. Growth (and enzyme production) were suppressed by the addition of bile salt to the medium; the order of suppression was deoxycholate > chenodeoxycholate > cholate; 1 mM of either of the dihydroxy-bile salts inhibited 96% of the growth and 100% of the enzyme production. Kinetic studies on cell-free preparations of 12alpha-HSDH revealed a pH optimum of 7.8 with greater linearity of NADP evolution with time occurring only at more alkaline pH values (9-10). Lineweaver-Burke plots revealed Michaelis constant (K(m)) values in the range of 3-5 x 10(-4) M for deoxycholate and its glycine and taurine conjugates, while higher values were found for cholate and conjugates (K(m) value for taurocholate was 3 x 10(-3) M). Although there was no activity with NAD, 12alpha-HSDH was shown to bind onto both NAD- and NADP-Sepharose columns, with stronger binding on the latter. The enzyme was purified 20-fold by NAD-Sepharose chromatography. The molecular weight was estimated at 100,000 by Sephadex G-200 and a series of molecular weight markers. Substrate specificity studies showed that a variety of bile salts containing 12alpha-OH groups reacted; notably, the 3alpha-sulfates of cholate and deoxycholate were nonsubstrates.-Macdonald, I. A., J. F. Jellett and D. E. Mahony. 12alpha-Hydroxysteroid dehydrogenase from Clostridium Group P strain C48-50 #29733: partial purification and characterization.     

Effects of oleic acid and bile salts on canine villous motility

The effects on canine villous motility of mucosal Tyrodes solution containing oleic acid (10 mM) and/or either taurocholic or cholic acid (15 mM) in the presence or absence of IV atropine (1 mg/kg) was used to assess the neural mediation of the effects of luminal nutrients. Villous motility was measured over 12 min periods by in vivo videomicroscopy of segments of jejunum. Neither bile salt had effects alone but villous motility increased after oleic acid was added to taurocholate and decreased after oleic acid was added to cholate. Villous motility increased when taurocholate and oleic acid were present initially and returned to control levels when removed. Villous motility was not affected by cholate and oleic acid but villous motility decreased when they were removed from the Tyrodes solution. Atropine blocked the increase in villous motility caused by taurocholate and oleic acid. Bile salts can modify the effect of oleic acid on villous motility and a cholinergic step is involved in the stimulation of motility.     

Comparison of Lactobacillus strains with respect to bile salt hydrolase activity, colonization of the gastrointestinal tract, and growth rate of the murine host.

The significance of bile salt hydrolase production by lactobacilli in the microecology of the murine intestinal tract has not been extensively studied previously. Assays of bile salt hydrolase (sodium taurocholate as substrate) associated with cell extracts of five Lactobacillus strains of murine origin gave a range of activities (from 915 nmol of cholate released per mg of protein per 30 min to none detected). All of the strains tested colonized the murine gastrointestinal tract equally well. The growth rates of mice were not affected by colonization of their intestinal tracts by lactobacilli whether or not the bacteria produced bile salt hydrolase.     

Terminal deoxynucleotidyl transferase in AKR leukemic cells and lack of relation of enzyme activity to cell cycle phase.

Cholate and taurocholate uptakes were studied in presence of albumin using isolated rat hepatocytes. Albumin decreased nonspecific binding of both bile acids and inhibited cholate uptake noncompetitively and taurocholate uptake competitively. Although different bile acids except dehydrocholate inhibited both cholate and taurocholate uptake, their relative inhibitory potency was not the same for both bile acids. Uptake of both bile acids was characterized by a saturable as well as an unsaturable process both in presence and in absence of albumin. The results suggest that both bile acids may be transported by more than one carrier and taurocholate is transported more efficiently than cholate by hepatocytes.     

Characterization and selection of <Emphasis Type="Italic">Lactobacillus</Emphasis> strains for their effect on bile tolerance,taurocholate deconjugation and cholesterol removal

Fourteen Lactobacillus strains of six species were investigated with their characteristics of bile salt tolerance, deconjugation of sodium taurocholate and cholesterol removal in the spent broth. Meanwhile, a co-precipitation curve of cholesterol with cholic acid at concentrations ranged 0.0–6.0 μM/ml was involved in the evaluation of cholesterol removal. Results demonstrated that both co-precipitation and assimilation effects contributed to cholesterol removal during the incubation of these Lactobacillus strains. It was also indicated that the supplementation of bile salts influenced the cholesterol removal, not only as an essential factor related to co-precipitation but also a critical condition for cholesterol assimilation. Out of all strains tested, four L. plantarum strains LS12, LS31, Lp501 and Lp529 exhibited a high ability of cholesterol assimilation (maximum 20.76 μg/ml), deconjugation of sodium taurocholate (maximum 5.00 μM/ml) and bile tolerance. They could be further studied and used as potential probiotics strains to reduce serum cholesterol in humans     

Sugar utilisation and conservation of the gal-lac gene cluster in Streptococcus thermophilus

The adaptation to utilise lactose as primary carbon and energy source is a characteristic for Streptococcus thermophilus. These organisms, however only utilise the glucose moiety of lactose while the galactose moiety is excreted into the growth medium. In this study we evaluated the diversity of sugar utilisation and the conservation of the gal-lac gene cluster in a collection of 18 S. thermophilus strains isolated from a variety of sources. For this purpose analysis was performed on DNA from these isolates and the results were compared with those obtained with a strain from which the complete genome sequence has been determined. The sequence, organisation and flanking regions of the S. thermophilus gal-lac gene cluster were found to be highly conserved among all strains. The vast majority of the S. thermophilus strains were able to utilize only glucose, lactose, and sucrose as carbon sources, some strains could also utilize fructose and two of these were able to grow on galactose. Molecular characterisation of these naturally occurring Gal+ strains revealed up-mutations in the galKTE promoter that were absent in all other strains. These data support the hypothesis that the loss of the ability to ferment galactose can be attributed to the low activity of the galKTE promoter, probably as a consequence of the adaptation to milk in which the lactose levels are in excess.     

Cultivation of a desulfurizing bacterium, Mycobacterium strain G3

Various carbon and sulfur sources on the growth and desulfurization activity of Mycobacterium strain G3, which is a dibenzothiophene (DBT)-degrading microorganism, were studied. Ethanol, glucose or glycerol as the sole carbon source and MgSO₄, taurine or dimethyl sulfoxide (DMSO) as the sole sulfur source were suitable for the growth. In addition, desulfurization activity was expressed in medium containing taurine, MgSO₄ or DMSO at 0.1 mM, when 217 mM ethanol was used as the sole carbon source. The highest desulfurization activity was in the stationary phase cells after 5 days' growth, rather than those harvested during active growth, when Mycobacterium G3 was cultivated in medium containing 217 mM ethanol and 0.1 mM MgSO₄. Thus alternative sulfur sources to DBT can be used for the cultivation of this desulfurizing microorganism.     

Comparative regulation of major enzymes in the bile acid biosynthesis pathway by cholesterol, cholate and taurine in mice and rats

These enzymes play important roles in the biosynthesis of bile acids. They are cholesterol 7alpha-hydroxylase (CYP7A1), the rate limiting enzyme in the classic pathway, sterol 12alpha-hydroxylase (CYP8B1), the key enzyme for synthesis of cholic acid (CA), and sterol 27-hydroxylase (CYP27), the initial enzyme in the alternative pathway. In the present study, the susceptibility of these three enzymes to dietary cholesterol and cholate, and the cholesterol lowering effect of taurine were determined in male C57BL/6 mice and Wistar rats. Both mice and rats were divided into 6 groups: control group (N), high cholesterol diet group (C), high cholesterol and cholate diet group (CB), and their 1% taurine-supplemented groups (NT, CT, CBT, respectively). After animals were fed with the respective diets for one week, the mRNA levels of CYP7A1 increased in the C-group compared with those of the N-group, and decreased in the CB-group compared with those of the C-group in both mice and rats. But the extent of decrease is different between the two species. CYP8B1 was also markedly repressed by cholate in mice, but not in rats. These results are consistent with the changes in serum and liver cholesterol concentrations. Taurine significantly increased CYP7A1 mRNA levels in the CBT-group compared with the CB-group in both animal models, with a subsequent decrease in serum and liver cholesterol levels and increase in fecal bile acid excretion. Up-regulated CYP8B1 was also observed after taurine supplementation in the CBT-group in mice. No increase in CYP7A1 was produced by taurine in the CT-group compared with that of the C-group in mice, although the changes of serum and liver cholesterol and fecal bile acids indicated taurine showed an efficient cholesterol lowering effect. In addition, CYP27 was induced in both C- and CB-groups of rats but not of mice, and no changes were produced by taurine. The overall results suggest that there are differences between mice and rats in susceptibility of the three enzymes to dietary cholesterol and cholate, and taurine induced CYP7A1 to produce its cholesterol-lowering effect only in the presence of cholate in the cholesterol diet.     

Hypoosmolarity Induces an Increase of Extracellular N-Acetylaspartate Concentration in the Rat Striatum

We previously showed that extracellular levels of N-acetylaspartate (NAA) increase when a medium with reduced NaCl concentration is perfused through a microdialysis probe, and proposed that NAA may be released during hypoosmotic swelling. Here, we demonstrate that this effect is due to hypoosmolarity of the perfusion medium, and not to low NaCl. NAA changes in the dialysate were compared with those of taurine as the osmoregulatory role of this amino acid is established. Reduction of the NaCl concentration in the perfusion medium increased the dialysate levels of NAA and taurine, but this effect was abolished when NaCl was replaced by sucrose to maintain isosmolarity. The NAA response to hypoosmolarity was smaller than that of taurine, but it may still be important to neurons as NAA is predominantly neuronal in the mammalian CNS.     

Effect of sodium taurocholate and sodium cholate on short-circuit current on amphibian membranes

The effects of the bile salts, sodium taurocholate (NaTc) and sodium cholate (NaCh), and toad bile gallbladder (bile) on short-circuit current (SCC) across isolated skin, and sodium taurocholate (NaTc) on isolated bladder of Bufo arenarum toads were tested. Sodium taurocholate (NaTc), sodium cholate (NaCh) and toad bile gallbladder (bile) promoted an increase in SCC, when added to the external side. The stimulatory effect was reversible after rinsing the preparation for 60 min. Implications on in vivo renal function of these results are discussed.