Isolation and identification of β-citryl-L-glutamic acid from newborn rat brain

Ab unknwon compound containing glutamic acid residue was found in newborn rat brain. The compound occurred predominantly in brain. Its concentration was approx. 1 μmol/g tissue at birth and decreased to one-tenth 24 days after birth.The compound was isolated from newborn rat brains, and subjected to elementary analysis and to infrared and mass spectrometric analysis. Glutamic acid and citric acid were formed from the compound on acid hydrolysis. The compound was presumed to be a citryglutamic acid.Two isomers, α- and β-citrylglutamic acid, were sunthesized. The unknown compound was identified as β-citryl-L-glutamic acid. The occurrence of this compound has not been reported in nature.     

Identification of three pertussis toxin substrates (41, 40 and 39 kDa proteins) in mammalian brain Comparison of predicted amino acid sequences from G-protein α-subunit genes and cDNAs with partial amino acid sequences from purified proteins

We have determined the partial amino acid sequences of the 40 kDa protein, one of the three pertussis toxin substrates in porcine brain. Purified 40 kDa protein from porcine brain was completely digested with TPCK-trypsin. Digested peptides were separated by reverse-phase HPLC and subjected to analysis by gas-phase protein sequencing. Several sequences of porcine brain 40 kDa protein completely matched with those which were deduced from the nucleotide sequences of the human G_i2α gene and rat G_i2α cDNA. On the other hand, the previously determined sequences of the rat brain 41 and 39 kDa proteins were in complete agreement with the predicted amino acid sequences of rat G_i1α and G_oα cDNAs, respectively.     

Regional distribution of arachidonic acid metabolites in rat brain following convulsive stimuli

Seizures were induced in female Wistar rats by electroconvulsive shock (ECS) or administration of pentetrazole (PTZ). Brain content of various prostanoids measured by radioimmunoassay showed time-dependent changes after the induction of convulsions; highest levels were found for PGD₂ followed by PGF_2α, PGE₂, TXB₂ and 6-keto-PGF_1α. Analysis of the various arachidonic acid metabolites in seven parts of the rat brain dissected according to the method of Glowinski and Iversen revealed the largest increases in hippocampus and cerebral cortex and smaller ones also in hypothalamus and corpus striatum both after ECS and PTZ. The ratios of the different cyclo-oxygenase products remained virtually the same in whole brain as well as in those regions where the formation of prostaglandins was markedly elevated. 15-keto-13,14-dihydro-PGF_2α also increased simultaneously in parallel to its parent compound, PGF_2α and was detected in significant amounts only in hippocampus and cerebral cortex. However, concentrations of 15-keto-13,14-dihydro-PGF_2α in these brain regions as well as in whole brain represented only 3–10% of the amounts found for PGF_2α. Thus, the metabolizing enzymes 15-hydroxy-PG-dehydrogenase and Δ13-PG-reductase seem to be of minor importance for the inactivation of prostanoids in brain tissue.     

A γ-aminobutyrate pathway in mammalian kidney cortex

Rat kidney cortex converts l-glutamate to γ-aminobutyrate by a decarboxylation reaction which differs from the corresponding reaction in brain. Renal l-glutamate decarboxylase has two apparent K_m values for glutamate in homogenates (0.4 and 2.5 mM). γ-Aminobutyrate is converted by a transaminase whose capacity appears to exceed the decarboxylase. γ-Aminobutyrate is converted ultimately to succinate and CO₂.γ-Aminobutyrate stimulates respiration of kidney cortex slices in vitro and the compound crosses cell membranes in kidney by a respiration-linked, mediated process.Chronic acidosis lowers renal γ-aminobutyrate in the rat; brain γ-aminobutyrate is unaffected by acidosis. Glutamic acid decarboxylase and γ-aminobutyrate transaminase activities are unchanged in acidosis. α-Methylglutamate, an inhibitor of renal glutaminase, lowers the γ-aminobutyrate and glutamate content of rat kidney in normal and acidotic states. Aminooxyacetic acid in vivo, an inhibitor of γ-aminobutyrate transaminase, causes a striking increase in renal γ-aminobutyrate during chronic acidosis.At concentrations of glutamate in vitro, which are similar to the tissue glutamate content in vivo, the γ-aminobutyrate pathway accounts for approximately one-fourth of glutamate disposal in rat kidney cortex slices.     

Immunoreactivity of subunits of the (Na + K)-ATPase Cross-reactivity of the α,α+ and β forms in different organs and species

The immunologic cross-reactivity of the α and α+ forms of the large subunit and the β subunit of the (Na⁺ + K⁺)-ATPase from brain and kidney preparations was examined using rabbit antiserum prepared against the purified holo lamb kidney enzyme. As previously reported by Sweadner ((1979) J. Biol. Chem. 254, 6060–6067) phosphorylation of the large subunit of the (Na⁺ + K⁺)-ATPase in the presence of Na⁺, Mg²⁺, and [γ-³²P]ATP revealed that dog and, very likely, rat brain contain two forms of the large subunit (designated α and α+) while dog, rat, and lamb kidney contain only one form (α). The cross-reactivity of the α and α+ forms in these preparations was investigated by resolving the subunits by SDS-polyacrylamide gel electrophoresis. The separated polypeptides were transferred to unmodified nitrocellulose paper, and reacted with rabbit anti-lamb kidney serum, followed by detection of the antigen-antibody complex with ¹²⁵I-labeled protein A and autoradiography. By this method, the α and α+ forms of rat and dog brain, as well as the α form found in kidney, were shown to cross-react. In addition, membranes from human cerebral cortex were shown to contain two immunoreactive bands corresponding to the α and α+ forms of dog brain. In contrast, the brain of the insect Manduca sexta contains only one immunoreactive polypeptide with a molecular weight intermediate to the α and α+ forms of dog brain. The β subunit from lamb, dog and rat kidney and from dog and rat brain cross-reacts with anti-lamb kidney (Na⁺ + K⁺)-ATPase serum. The mobility of the β subunit from dog and rat brain on SDS-polyacrylamide electrophoresis gels is greater than the mobility of the β subunit from lamb, rat or dog kidney.     

γ-Secretase Dependent Production of Intracellular Domains Is Reduced in Adult Compared to Embryonic Rat Brain Membranes

Background

γ-Secretase is an intramembrane aspartyl protease whose cleavage of the amyloid precursor protein (APP) generates the amyloid β-peptide (Aβ) and the APP intracellular domain. Aβ is widely believed to have a causative role in Alzheimer''s disease pathogenesis, and therefore modulation of γ-secretase activity has become a therapeutic goal. Besides APP, more than 50 substrates of γ-secretase with different cellular functions during embryogenesis as well as adulthood have been revealed. Prior to γ-secretase cleavage, substrates are ectodomain shedded, producing membrane bound C-terminal fragments (CTFs).

Principal Findings

Here, we investigated γ-secretase cleavage of five substrates; APP, Notch1, N-cadherin, ephrinB and p75 neurotrophin receptor (p75-NTR) in membranes isolated from embryonic, young or old adult rat brain by analyzing the release of the corresponding intracellular domains (ICDs) or Aβ40 by western blot analysis and ELISA respectively. The highest levels of all ICDs and Aβ were produced by embryonic membranes. In adult rat brain only cleavage of APP and Notch1 could be detected and the Aβ40 and ICD production from these substrates was similar in young and old adult rat brain. The CTF levels of Notch1, N-cadherin, ephrinB and p75-NTR were also clearly decreased in the adult brain compared to embryonic brain, whereas the APP CTF levels were only slightly decreased.

Conclusions

In summary our data suggests that γ-secretase dependent ICD production is down-regulated in the adult brain compared to embryonic brain. In addition, the present approach may be useful for evaluating the specificity of γ-secretase inhibitors.     

Steroid 5α-Reductase: Comparative Study of Mechanism of Inhibition by Nonsteroids ONO-3805 and LY191704

Two nonsteroids, ONO-3805 and LY191704, were evaluated as inhibitors of the human and rat 5α-reductases (5αR). ONO-3805 was prepared in a 12-step convergent synthesis. This compound is a potent inhibitor of the human and rat 5αRs, with more potent inhibition seen against the rat enzymes. The inhibition patterns of this compound were best fit to an uncompetitive model which suggests binding in a ternary complex with enzyme and NADP⁺. ApparentK_ivalues of 27, 31, 1, and 0.5 n$of5$M versus testosterone were obtained with human type 1, human type 2, rat type 1, and rat type 2 5αR, respectively. Multiple inhibition studies with ONO-3805 and NADP⁺support synergistic binding of these two inhibitors with all isozymes. LY191704 was also evaluated as an inhibitor of the human and rat 5αRs. This compound is a selective, competitive inhibitor of human type 1 5αR. Poor inhibition was observed with human type 2 and rat types 1 and 2 5αR.     

Validation of an analytical procedure to measure trace amounts of neurosteroids in brain tissue by gas chromatography–mass spectrometry

A selective and extremely sensitive procedure has been developed and optimized, using high-performance liquid chromatography (HPLC), specific derivatization and gas chromatography–mass spectrometry (GC–MS), to simultaneously quantify very small amounts of different neurosteroids from rat brain. Unconjugated and sulfated steroids in brain extracts were separated by solid-phase extraction. The unconjugated fraction was further purified by HPLC, the steroids being collected in a single fraction, and the sulfated fraction was solvolyzed. All steroids were derivatized with heptafluorobutyric acid anhydride and analyzed by GC–MS (electron impact ionization) using selected-ion monitoring. High sensitivity and accuracy were obtained for all steroids. The detection limits were 1 pg for pregnenolone (PREG), dehydroepiandrosterone (DHEA) and their sulfate esters PREG-S and DHEA-S, 2 pg for progesterone (PROG) and 5 pg for 3α,5α-tetrahydroprogesterone (3α,5α-THP). In a pilot study on a rat brain, the concentrations of PREG-S and DHEA-S were 8.26±0.80 and 2.47±0.27 ng/g, respectively. Those of PREG, DHEA and PROG were 4.17±0.22, 0.45±0.02 and 1.95±0.10 ng/g, respectively. Good linearity and accuracy were observed for each steroid. The methodology validated here, allows femtomoles of neurosteroids, including the sulfates, found in small brain samples (at least equal to 10 mg) to be quantified simultaneously.     

Specific stimulation of α2-6 sialyltransferase activity by a novel cytosolic factor from rat colon

A factor present in the 100 000 g supernatant from the homogenate of rat colon stimulated the activity of purified GaIβ1-4GlcNAc α2,6 sialyltransferase [α2-6ST(N)] from rat liver and α2-6ST(N) from either liver microsomes or Golgi membrane. The stimulation of α2-6ST(N) activity by the colon factor using protein acceptors was about four-fold and highly reproducible when the reaction product of the α2-6ST(N) was assayed by either precipitation or affinity chromatography. In contrast, the colon factor did not stimulate the GaIβ1-4GlcNAc α2,3 sialyltransferase [α2-3ST (N)], from rat jejunum microsomes or purified Galβ1-3GalNAc α2,3 sialyltransferase [α2-3ST (O)] from porcine liver, or purified β1,4 galactosyltransferase (GT) from bovine milk. In addition to rat colon, the 100 000 g supernatant from the homogenates of rat brain and kidney also stimulated the α2-6ST(N) activity. The stimulation of α2-6ST(N) by the colon factor resulted in a decrease in the K_m (by about two-fold) and an increase in V_max (about 2- to 3-fold) for desialylated α₁ acid glycoprotein and CMP-[¹⁴C]N-acetylneuraminic acid. The stimulation of α2-6ST(N) activity by the colon factor was temperature dependent, protease sensitive and was inhibited by CTP, but did not need the presence of either metal ions or detergent. The cytosolic factor was partially purified by ion-exchange chromatography with the retention of the activator activity in the peaks containing low molecular weight proteins, but the activity was lost on attempts to further purification. A specific marked stimulation of the α2-6ST(N) activity by cytosolic factors in certain tissues might suggest a physiological role for these factors in the regulation of α2-6ST(N) activity.     

Prostanoid synthesis and vascular responses to exogenous arachidonic acid following cerebral ischemia in piglets

In newborn pigs, cerebral ischemia abolishes both increased cerebral prostanoid production and cerebral vasodilation in response to hypercapnia and hypotension. Attenuation of prostaglandin endoperoxide synthase activity could account for the failure to increase prostanoid systhesis and loss of responses to these stimuli. To test this possibility, arachidonic acid (3,6, or 30μg/ml) was placed under cranial windows in newborn pigs that been exposed to 20 min of cerebral ischemia. The conversion to prostanoids and pial arteriolar responses to the arachidonic acid were measured. At all three concentration, arachidonic acid caused similar increases in pial arteriolar diameter in sham control piglets and piglets 1 hr postischemia. Topical arachidonic acid caused dosedependent increases of PGE₂ in cortical periarachnoid cerebral spinal fluid. 6-keto-PGF_1α and TXB₂ only increased at the highest concentration of arachidonic acid (30 μg/ml). Cerebral ischemia did not decrease the conservation of any concentration of arachidonic acid to PGE₂, 6-keto-PGF_1α, or TXB₂. We conclude that ischemia and subsequent reperfusion do not result in inhibition of prostaglandin endoperoxide synthase in the newborn pig brain. Therefore, the mechanism for the impaired prostanoid production in response to hypercapnia and hypotension following cerebral ischemia appears to involve reduction in release of free arachidonic acid.     

Stimulation of brain adenylate cyclase activity by the undecapeptide substance P and its modulation by the calcium ion

Synthetic substance P stimulated adenylate cyclase activity in particulate preparations from rat and human brain.The concentration of substance P for half maximal stimulation in rat brain was 1.8 · 10⁻⁷ M.The stimulatory effect of substance P on the rat brain adenylate cyclase activity was 88% compared with 48% by noradrenalin, 163% by prostaglandin E₁ and 184% by prostaglandin E₂.Both the basal and substance P-stimulated adenylate cyclase activity in rat brain were inhibited by concentration of Ca²⁺ above 10⁻⁶ M.The chelating agent ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid at a concentration of 0.1 mM reduced the basal adenylate cyclase activity by 64% and eliminated the substance P-stimulated activity.The inhibition by ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid was completely reversed by increasing concentrations of Ca²⁺.     

On the phospholipid metabolism of glial cell primary cultures

Primary cultures prepared from newborn rat brain, consisted after 16 or 17 days mainly of astrocytes and of oligodendrocytes. 1-Alkenyl-sn-glycero-3-phosphoethanolamine (lysoplasmalogen) was used as substrate for studies on the metabolism of ethanolamine-glycerophospholipids. After 3 hr incubation two main products were observed: a) 1-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine (=ethanolamine plasmalogen) and b) 1-alkenyl-2-acyl-sn-glycero-3-phosphocholine (=choline plasmalogen). The acylation rate reached saturation at about 10 nmol substrate/mg cell protein with aV _max of 30 nmol×mg cell protein^–1×3 hr^–1. This acylated compound amounted to almost 60% of all radioactivity internalized, whereas the second product, choline plasmalogen, came to 20%. Unchanged substrate was found within the cells only in small amounts, even at maximum substrate internalization. These results were discussed in comparison with those obtained with 1-alkyl-sn-glycero-3-phosphoethanolamine under the same conditions (25).     

Determination of 9α,11β-prostaglandin F2 by stereospecific antibody in various rat tissues

In view of the recent finding that prostaglandin D₂ is stereospecifically converted to 9α,11β-prostaglandin F₂, an isomer of prostaglandin F₂α, a highly specific and sensitive radioimmunoassay for 9α,11β-prostaglandin F₂ was developed and applied to determine the content of this prostaglandin in various rat tissues. Antisera against 9α-11β-prostaglandin F₂ were raised in rabbits immunized with the bovine serum albumin conjugate, and [³H]9α,11β-prostaglandin F₂ was enzymatically prepared from [³H]prostaglandin D₂. The assay detected 9α,11β-prostaglandin F₂ over the range of 20 pg to 1 ng, and the antiserum showed less than 0.04% cross-section with prostaglandin F₂α, prostaglandin F₂β and 9β,11β-prostaglandin F₂. To avoid postmortem changes, tissues were frozen in liquid nitrogen immediately after removal. The basal level of 9α,11β-prostaglandin F₂ was hardly detectable in various tissues of the rat examined, including spleen, lung, liver and brain; although it was found to be 0.31 ± 0.06 ng/g wet weight in the small intestine. During convulsion induced by pentylenetetrazole, enormous amounts of prostaglandin D₂ (ca. 180 ng/g wet weight) and prostaglandin F₂α (ca. 70 ng/g) were produced in the brain; however, 9α,11β-prostaglandin F₂ was detected neither there nor in the blood. This result demonstrates that the conversion to 9α,11β-prostaglandin F₂ is a minor pathway, if one at all, of prostaglandin D₂ metabolism in the rat brain.     

Changes in n-6 and n-3 polyunsaturated fatty acids during lipid-peroxidation of mitochondria obtained from rat liver and several brain regions: effect of α-tocopherol

The effect of intraperitoneal administration of α-tocopherol (100 mg/kg weight/24 h) on ascorbate (0–0.4 mM) induced lipid peroxidation of mitochondria isolated from rat liver, cerebral hemispheres, brain stem and cerebellum was examined. The ascorbate induced light emission in hepatic mitochondria was nearly completely inhibited by α-tocopherol (control-group: 114.32±14.4; vitamin E-group: 17.45±2.84, c.p.m.×10⁻⁴). In brain mitochondria, 0.2 mM ascorbate produced the maximal chemiluminescence and significant differences among both groups were not observed. No significant differences in the chemiluminescence values between control and vitamin E treated groups were observed when the three brain regions were compared. The light emission produced by mitochondrial preparations was much higher in cerebral hemispheres than in brain stem and cerebellum. In liver and brain mitochondria from control group, the level of arachidonic acid (C20:4n6) and docosahexaenoic acid (C22:6n3) was profoundly affected. Docosahexaenoic in liver mitochondria from vitamin E group decreased by 30% upon treatment with ascorbic acid when compared with mitochondria lacking ascorbic acid. As a consequence of vitamin E treatment, a significant increase of C22:6n3 was detected in rat liver mitochondria (control-group: 6.42 ±0.12; vitamin E-group: 10.52 ±0.46). Ratios of the α-tocopherol concentrations in mitochondria from rats receiving vitamin E to those of control rats were as follows: liver, 7.79; cerebral hemispheres, 0.81; brain stem, 0.95; cerebellum, 1.05. In liver mitochondria, vitamin E shows a protector effect on oxidative damage. In addition, vitamin E concentration can be increased in hepatic but not in brain mitochondria. Lipid peroxidation mainly affected, arachidonic (C20:4n6) and docosahexaenoic (C22:6n3) acids.     

Functional expression and sites of gene transcription of a novel α subunit of the GABAA receptor in rat brain

Two α subunits of the gabaa receptor in rat brain have been identified by molecular cloning. The deduced polypeptide sequences share major characteristics with other chemically gated ion channel proteins. One polypeptide represents the rat homologue of the α3 subunit previously cloned from bovine brain [14], while the other polypeptide is a yet unknown subunit, termed α5. When coexpressed with the β1 subunit in Xenopus oocytes the receptors containing the α5 subunit revealed a higher sensitivity to GABA than receptors expressed from α1 + β1 subunits or α3 + β1 subunits (K_a = 1 μM, 13 μM and 14 μM, respectively). The α5 subunit was expressed only in a few brain areas such as cerebral cortex, hippocampal formation and olfactory bulb granular layer as shown by in situ hybridization histochemistry. Since the mRNA of the α5 subunit was colocalized with the αl and α3 subunits only in cerebral cortex and in the hippocampal formation the α5 subunit may be part of distinct GABA_A receptors in neuronal populations within the olfactory bulb.     

Distribution of 3α-hydroxysteroid dehydrogenase in rat brain and molecular cloning of multiple cDNAs encoding structurally related proteins in humans

3α-Hydroxysteroid dehydrogenase in the brain is responsible for production of neuroactive tetrahydrosteroids that interact with the major inhibitory gamma-aminobutyric acid receptor complexes. Distribution of 3α-hydroxysteroid dehydrogenase in different regions of the brain in rats was evaluated by activity assay and by Western immunoblotting using a monoclonal antibody against liver 3α-hydroxysteroid dehydrogenase as the probe. The olfactory bulb was found to contain the highest level of 3α-hydroxysteroid dehydrogenase activity, while moderate levels of the enzyme activity were found in other regions such as cerebellum, cerebral cortex, hypothalamus and pituitary. Some activity was found in the rest of the brain such as amygdala, brain stem, caudate putamen, cingulate cortex, hippocampus, midbrain, and thalamus. The protein levels of 3α-hydroxysteroid dehydrogenase in different regions of the brain as detected by Western immunoblotting are comparable to those of the enzyme activity. We used the rat cDNA as the probe to screen a human liver λ gt11 cDNA library. A total of four different cDNAs were identified and sequenced. One of the cDNAs is identical to that of the human chlordecone reductase cDNA except that our clone contains a much longer 5′-coding sequence than previously reported. The other three cDNAs display high degrees of sequence homology to those of both rat 3α-hydroxysteroid dehydrogenase and human chlordecone reductase. We are currently investigating the functional relationship between the enzymes encoded by these human cDNAs and 3α-hydroxysteroid dehydrogenase.     

Effects of acute and chronic ethanol administration on thromboxane and prostacyclin levels and release in rat brain cortex

The effects of acute (3 g/kg i.p. two jours before sacrifice) and chronic (6% in drinking water and libitum for 15 days) ethanol administration to male rats (200 g body weight) on basal levels and release of TxB_2n2 and 6-keto-PGF_1α in brain cortex were studied. Also the effects of chronic ethanol (30 days) on the fatty acid composition of brain cortical tissue and liver phospholipids were investigated. Acute treatment reduced basal levels of 6-keto-PGF_1α in brain cortical tissue (rats sacrificed by microwave radiation) and decreased the accumulation of 6-keto-PGF_1α in brain cortex after post-decapitation ischemia (PDI). Basal TxB₂ levels were also reduced in brain cortex, but TxB₂ release during PDI was enhanced. Chronic treatment (15 days) induced changes of TxB₂ and 6-ketoPGF_1α levels and release during PDI in brain cortex less pronounced than those observed after acute treatment. The reduced effectiveness of chronic ethanol on brain vasoactive eicosanoids suggest adaptation processes. After chronic treatment (30 days), the fatty acid composition of brain cortex total phospholipids were not significantly modified. Changes of eicosanoid production after ethanol were thus independent from modifications of the fatty acid precursor pool(s). Ethanol-induced changes in the production of vascular eicosanoids in the CNS may be of relevance to the action of the compound on the CNS and may also have implications for the clinic.     

Biosynthesis of prostaglandins and thromboxane B2 by fetal lung homogenates

The conversion of arachidonic acid to prostaglandins (PG's) and thromboxane B₂ (TXB₂) was investigated in homogenates from fetal and adult bovine and rabbit lungs. Adult bovine lungs were very active in converting arachidonic acid (100 μg/g tissue) to both PGE₂ (10.7 μg/g tissue) and TXB₂ (6.2 μ/g tissue). Smaller amounts of PGF_2α (0.9 μ/g) and 6-oxoPGF_1α were formed. Homogenates from fetal calf lungs during the third trimester of pregnancy were quite active in converting arachidonic acid to PGE₂, but formed very little TXB₂, PGF_2α or 6-oxoPGF_1α. Homogenates from rabbit lungs converted arachidonic acid (100 μg/g) mainly to PGE₂, both before and after birth. The amount of PGE₂ formed increased during gestation to a maximum of about 6 μg/g tissue at 28 days of gestation. It then decreased to a minimum (1.5 μg/g) which was observed 8 days after birth, followed by an increase to about 4 μg/g in older rabbits.     

Formation of Hyodeoxycholic Acid from Muricholic Acid and Hyocholic Acid by an Unidentified Gram-Positive Rod Termed HDCA-1 Isolated from Rat Intestinal Microflora

From the rat intestinal microflora we isolated a gram-positive rod, termed HDCA-1, that is a member of a not previously described genomic species and that is able to transform the 3α,6β,7β-trihydroxy bile acid β-muricholic acid into hyodeoxycholic acid (3α,6α-dihydroxy acid) by dehydroxylation of the 7β-hydroxy group and epimerization of the 6β-hydroxy group into a 6α-hydroxy group. Other bile acids that were also transformed into hyodeoxycholic acid were hyocholic acid (3α,6α,7α-trihydroxy acid), α-muricholic acid (3α,6β,7α-trihydroxy acid), and ω-muricholic acid (3α,6α,7β-trihydroxy acid). The strain HDCA-1 could not be grown unless a nonconjugated 7-hydroxylated bile acid and an unidentified growth factor produced by a Ruminococcus productus strain that was also isolated from the intestinal microflora were added to the culture medium. Germfree rats selectively associated with the strain HDCA-1 plus a bile acid-deconjugating strain and the growth factor-producing R. productus strain converted β-muricholic acid almost completely into hyodeoxycholic acid.