Pharmacologic Targeting of Histone H3K27 Acetylation/BRD4-dependent Induction of ALDH1A3 for Early-phase Drug Tolerance of Gastric Cancer.

Anticancer drug-tolerant persister (DTP) cells at an early phase of chemotherapy reshape refractory tumors. Aldehyde dehydrogenase 1 family member A3 (ALDH1A3) is commonly upregulated by various anticancer drugs in gastric cancer patient-derived cells (PDC) and promotes tumor growth. However, the mechanism underlying the generation of ALDH1A3-positive DTP cells remains elusive. Here, we investigated the mechanism of ALDH1A3 expression and a combination therapy targeting gastric cancer DTP cells. We found that gastric cancer tissues treated with neoadjuvant chemotherapy showed high ALDH1A3 expression. Chromatin immunoprecipitation (ChIP)-PCR and ChIP sequencing analyses revealed that histone H3 lysine 27 acetylation was enriched in the ALDH1A3 promoter in 5-fluorouracil (5-FU)-tolerant persister PDCs. By chemical library screening, we found that the bromodomain and extraterminal (BET) inhibitors OTX015/birabresib and I-BET-762/molibresib suppressed DTP-related ALDH1A3 expression and preferentially inhibited DTP cell growth. In DTP cells, BRD4, but not BRD2/3, was recruited to the ALDH1A3 promoter and BRD4 knockdown decreased drug-induced ALDH1A3 upregulation. Combination therapy with 5-FU and OTX015 significantly suppressed in vivo tumor growth. These observations suggest that BET inhibitors are efficient DTP cell-targeting agents for gastric cancer treatment. SIGNIFICANCE: Drug resistance hampers the cure of patients with cancer. To prevent stable drug resistance, DTP cancer cells are rational therapeutic targets that emerge during the early phase of chemotherapy. This study proposes that the epigenetic regulation by BET inhibitors may be a rational therapeutic strategy to eliminate DTP cells.

S-nitrosoglutathione reductase maintains mitochondrial homeostasis by promoting clearance of damaged mitochondria in porcine preimplantation embryos.

OBJECTIVES: S-nitrosoglutathione reductase (GSNOR), a protein denitrosylase, protects the mitochondria from mitochondrial nitrosative stress. Mammalian preimplantation embryos are mitochondria-rich, but the effects of GSNOR on mitochondrial function in preimplantation embryos are not well-studied. In the present study, we investigate whether GSNOR plays a role in mitochondrial regulation during porcine preimplantation embryo development. MATERIALS AND METHODS: GSNOR dsRNA was employed to knock down the expression of GSNOR, and Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME), a pan-NOS inhibitor, was used to prevent protein S-nitrosylation. Mitochondrial amount and function in embryo development were assessed by performing immunofluorescence staining, Western blot, fluorescent probe and real-time reverse transcription PCR. RESULTS: GSNOR knock-down significantly impaired blastocyst formation and quality and markedly induced the increase in protein S-nitrosylation. Notably, GSNOR knock-down-induced overproduction of S-nitrosylation caused mitochondrial dysfunction, including mitochondrial membrane potential depolarization, mitochondria-derived reactive oxygen species (ROS) increase and ATP deficiency. Interestingly, GSNOR knock-down-induced total mitochondrial amount increase, but the ratio of active mitochondria reduction, suggesting that the damaged mitochondria were accumulated and mitochondrial clearance was inhibited. In addition, damaged mitochondria produced more ROS, and caused DNA damage and apoptosis. Importantly, supplementation with L-NAME reverses the increase in S-nitrosylation, accumulation of damaged mitochondria, and oxidative stress-induced cell death. Interestingly, autophagy was downregulated after GSNOR knock-down, but reversed by L-NAME treatment. Thus, GSNOR maintains mitochondrial homeostasis by promoting autophagy and the clearing of damaged mitochondria in porcine preimplantation embryos.

CRISPR-Cas9 Genome-Wide Knockout Screen Identifies Mechanism of Selective Activity of Dehydrofalcarinol in Mesenchymal Stem-like Triple-Negative Breast Cancer Cells.

There are no targeted therapies available for triple-negative breast cancers (TNBCs) in part because they represent a heterogeneous group of tumors with diverse oncogenic drivers. Our goal is to identify targeted therapies for subtypes of these cancers using a mechanism-blind screen of natural product extract libraries. An extract from Desmanthodium guatemalense was 4-fold more potent for cytotoxicity against MDA-MB-231 cells, which represent the mesenchymal stem-like (MSL) subtype, as compared to cells of other TNBC subtypes. Bioassay-guided fractionation led to the isolation of six polyacetylenes, and subsequent investigations of plant sources known to produce polyacetylenes yielded six additional structurally related compounds. A subset of these compounds retained selective cytotoxic effects in MSL subtype cells. Studies suggest that these selective effects do not appear to be due to PPARγ agonist activities that have previously been reported for polyacetylenes. A CRISPR-Cas9-mediated gene knockout screen was employed to identify the mechanism of selective cytotoxic activity of the most potent and selective compound, dehydrofalcarinol (1a). This genomic screen identified HSD17B11, the gene encoding the enzyme 17ß-hydroxysteroid dehydrogenase type 11, as a mediator of the selective cytotoxic effects of 1a in MDA-MB-231 cells that express high levels of this protein. The Project Achilles cancer dependency database further identified a subset of Ewing sarcoma cell lines as highly dependent on HSD17B11 expression, and it was found these were also highly sensitive to 1a. This report demonstrates the value of CRISPR-Cas9 genome-wide screens to identify the mechanisms underlying the selective activities of natural products.

Effects of Resveratrol and Mangiferin on PPARγ and FALDH Gene Expressions in Adipose Tissue of Streptozotocin-Nicotinamide-Induced Diabetes in Rats.

Type 2 diabetes (T2D) is characterized by insufficient insulin secretion by the pancreatic beta cells and insulin resistance in liver, skeletal muscle, and white adipose tissue. Adipose tissue plays a major role in glucose homeostasis and lipid metabolism. Dietary antioxidants such as resveratrol and mangiferin may offer some protection against the early stage of diabetes mellitus. Therefore, an attempt has been made to investigate the effects of resveratrol and mangiferin on biochemical parameters and molecular mechanism of PPARγ and FALDH gene expression in adipose tissue of streptozotocin- (STZ-) nicotinamide- (NA-) induced diabetic rats. Albino Wister rats were randomly divided into five groups: control rats (Group 1), diabetic control rats (Group 2), diabetic rats given resveratrol (40 mg/kg body weight per day; Group 3), diabetic rats given mangiferin (40 mg/kg body weight per day; Group 4), diabetic rats given glibenclamide (0.6 mg/kg body weight per day; Group 5). Serum biochemical parameters-total cholesterol (TC), total triglyceride (TG), low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, glycosylated hemoglobin (HbA1c), urea, and uric acid were analyzed. We found that the oral administration of resveratrol and mangiferin to STZ-NA-induced diabetic rats for 30 days showed the significant protective effect on all the biochemical parameters. A significant reduction in blood glucose and HbA1c levels was observed in rats treated with 40 mg/kg body weight per day of resveratrol or mangiferin. Moreover, both these antioxidants showed significant enhancement of PPARγ and FALDH gene expression in rat adipose tissue compared to control rats.

Normoergic NO-dependent changes, triggered by a SAR inducer in potato, create more potent defense responses to Phytophthora infestans.

In our experimental approach we examined how potato leaves exposed to a chemical agent might induce nitric oxide (NO) dependent biochemical modifications for future mobilization of an effective resistance to Phytophthora infestans. After potato leaf treatment with one of the following SAR inducers, i.e. ß-aminobutyric acid (BABA), 2,6-dichloroisonicotinic acid (INA) or Laminarin, we observed enhanced NO generation concomitant with biochemical changes related to a slight superoxide anion (O2(-)) and hydrogen peroxide (H2O2) accumulation dependent on minimal NADPH oxidase and peroxidase activities, respectively. These rather normoergic changes, linked to the NO message, were mediated by the temporary down-regulation of S-nitrosoglutathione reductase (GSNOR). In turn, after challenge inoculation signal amplification promoted potato resistance manifested in the up-regulation of GSNOR activity tuned with the depletion of the SNO pool, which was observed by our team earlier (Floryszak-Wieczorek et al., 2012). Moreover, hyperergic defense responses related to an early and rapid O2(-)and H2O2 overproduction together with a temporary increase in NADPH oxidase and peroxidase activities were noted. BABA treatment was the most effective against P. infestans resulting in the enhanced activity of ß-1,3-glucanase and callose deposition. Our results indicate that NO-mediated biochemical modifications might play an important role in creating more potent defense responses of potato to a subsequent P. infestans attack.

FK506 neuroprotection after cavernous nerve injury is mediated by thioredoxin and glutathione redox systems.

INTRODUCTION: Immunophilin ligands such as FK506 (FK) preserve erectile function (EF) following cavernous nerve injury (CNI), although the precise mechanisms are unclear. We examined whether the thioredoxin (Trx) and glutathione (GSH) redox systems mediate this effect after CNI. AIM: To investigate the roles of Trx reductase 2 (TrxR2) and S-Nitrosoglutathione reductase (GSNOR) as antioxidative/nitrosative and antiapoptotic mediators of the neuroprotective effect of FK in the penis after CNI. METHODS: Adult male rats, wild-type (WT) mice, and GSNOR deficient (GSNOR -/-) mice were divided into four groups: sham surgery (CN [cavernous nerves] exposure only) + vehicle; sham surgery + FK (5 mg/kg/day/rat or 2 mg/kg/day/mouse, for 2 days, subcutaneous); CNI + vehicle; and CNI + FK. At day 4 after injury, electrically stimulated changes in intracavernosal pressure (ICP) were measured. Penises were collected for Western blot analysis of TrxR2, GSNOR, and Bcl-2, and for immunolocalization of TrxR2 and GSNOR. MAIN OUTCOME MEASURES: EF assessment represented by maximal ICP and total ICP in response to electrical stimulation. Evaluation of protein expression levels and distribution patterns of antioxidative/nitrosative and antiapoptotic factors in penile tissue. RESULTS: EF decreased after CNI compared with sham surgery values in both rats (P < 0.01) and WT and GSNOR -/- mice (P < 0.05). FK treatment preserved EF after CNI compared with vehicle treatment in rats (P < 0.01) and WT mice (P < 0.05) but not in GSNOR -/- mice. In rats, GSNOR (P < 0.01) and Bcl-2 (P < 0.05) expressions were significantly decreased after CNI. FK treatment in CN-injured rats restored expression of GSNOR and upregulated TrxR2 (P < 0.001) and Bcl-2 (P < 0.001) expressions compared with vehicle treatment. Localizations of proteins in the penis were observed for TrxR2 (endothelium, smooth muscle) and for GSNOR (nerves, endothelium, smooth muscle). CONCLUSIONS: The neuroprotective effect of FK in preserving EF after CNI involves antioxidative/nitrosative and antiapoptotic mechanisms mediated, to some extent, by Trx and GSH systems.

[Administration of the omega-3 polyunsaturated fatty acid drug eiferol decreases alcohol motivation in albino rats by elevating the level of antibodies to alcohol dehydrogenase]. / Vvedenie preparata polinenasyshchennykh zhirnykh kislot semeistva omega-3 (éiferola) snizhaet alkogol'nuiu motivatsiiu u belykh krys, povyshaia uroven' antitel k alkogol'degidrogenaze.

The study experimentally assessed the approach proposed by the authors to lower alcohol motivation, which involves enhancement of a specific immunity at the stage of alcoholization when acetaldehydemodified ethanol exchange enzymes [alcohol dehydrogenase (ADH)] and acetaldehyde dehydrogenase may be expected to occur. Omega-3 polyunsaturated fatty acid (PUFA) drugs enhance the formation of autoantibodies to modified ADH and decrease the activity of ADH in the stomach and liver. At the same time, PUFA drugs can, under certain conditions, produce an anti-alcoholic activity and a positive effect on the psychoemotional status of animals after the ethanol deprivation period.

Preclinical toxicity and efficacy study of a 14-day schedule of oral 5-iodo-2-pyrimidinone-2'-deoxyribose as a prodrug for 5-iodo-2'-deoxyuridine radiosensitization in U251 human glioblastoma xenografts.

In anticipation of an initial clinical Phase I trial in patients with high-grade gliomas of p.o. administered 5-iodo2-pyrimidinone-2'-deoxyribose (IPdR) given daily for 14 days as a prodrug for 5-iodo-2'-deoxyuridine (IUdR)-mediated tumor radiosensitization, we determined the systemic toxicities and the percentage IUdR-DNA incorporation in normal athymic mouse tissues and a human glioblastoma xenograft (U251) after this dosing schedule of IPdR. Using a tumor regrowth assay of s.c. U251 xenografts, we also compared radiosensitization with this IPdR-dosing schedule to radiation therapy (XRT) alone (2 Gy/day for 4 days) or to XRT after continuous infusion IUdR for 14 days at the maximum tolerated dose in mice (100 mg/kg/day). Athymic mice with and without U251 s.c. xenografts tolerated 750 or 1500 mg/kg/day of p.o. IPdR (using gastric lavage) for 14 days without weight loss or activity level changes during treatment and for a 28-day posttreatment observation period. The percentage IUdR-DNA incorporation in U251 tumor cells was significantly higher after p.o. IPdR (750 and 1500 mg/kg/day) for 14 days (3.1 +/- 0.2% and 3.7 +/- 0.3%, respectively) than continuous infusion IUdR for 14 days (1.4 +/- 0.1%). Compared to XRT alone, a significant sensitizer enhancement ratio (SER) was found with the combination of p.o. IPdR (1500 mg/kg/d) + XRT (SER = 1.31; P = 0.05) but not for the combination of continuous infusion IUdR + XRT (SER = 1.07; P = 0.57) in the U251 xenografts. The percentage IUdR-DNA incorporation after IPdR at 1500 mg/kg/day for 14 days in normal bone marrow, normal small intestine, and normal liver were 1.2 +/-0.2%, 3.3 +/- 0.3%, and 0.2 +/- 0.1%, respectively. We conclude that a 14-day p.o. schedule of IPdR at up to 1500 mg/kg/day results in no significant systemic toxicity in athymic mice and is associated with significant radiosensitization using this human glioblastoma multiforme xenograft model. Based on these data and our previously published data using shorter IPdR dosing schedules, which also demonstrate an improved therapeutic index for IPdR compared to IUdR, an initial clinical Phase I and pharmacokinetic study of p.o. IPdR daily for 14 days is being designed.

Spectroscopic states of the CO oxidation/CO2 reduction active site of carbon monoxide dehydrogenase and mechanistic implications.

CO dehydrogenases catalyze the reversible oxidation of CO to CO2, at an active site (called the C-cluster) composed of an Fe4S4 cube with what appears to be a 5-coordinate Fe (called FCII), linked to a Ni (Hu, Z., Spangler, N. J., Anderson, M. E., Xia, J., Ludden, P. W., Lindahl, P. A., & Münck, E. (1996) J. Am. Chem. Soc. 118, 830-845). During catalysis, electrons are transferred from the C-cluster to an [Fe4S4]2+/1+ electron-transfer cluster called the B-cluster. An S = 1/2 form of the C-cluster (called Cred1) converts to another S = 1/2 form (called Cred2) upon reduction with CO, at a rate well within the turnover frequency of the enzyme (Kumar, M., Lu, W.-P., Liu, L., & Ragsdale, S. W. (1993) J. Am. Chem. Soc. 115, 11646-11647). This suggests that the conversion is part of the catalytic mechanism. Dithionite is reported in this paper to effect this conversion as well, but at a much slower rate (kso = 5.3 x 10(-2) M-1 s-1 for dithionite vs 4.4 x 10(6) M-1 s-1 for CO). By contrast, dithionite reduces the oxidized B-cluster much faster, possibly within the turnover frequency of the enzyme. Dithionite apparently effects the Cred1/Cred2 conversion directly, rather than through an intermediate. The conversion rate varies with dithionite concentration. The Cred1/Cred2 conversion occurs at least 10(2) times faster in the presence of CO2 than in its absence. CO2 alters the g values of the gav = 1.82 signal, indicating that CO2 binds to a C-cluster-sensitive site at mild potentials. CN- inhibits CO oxidation by binding to FCII (Hu et al., 1996), and CO, CO2 in the presence of dithionite, or CS2 in dithionite accelerate CN- dissociation from this site (Anderson, M. E., & Lindahl, P. A. (1994) Biochemistry 33, 8702-8711). The effect of CO, CO2, and CS2 on CN- dissociation suggested that these molecules bind at a site (called the modulator) other than that to which CN- binds. The effects of CO2, CS2, CO, and dithionite on the Cred1/Cred2 conversion rate followed a similar pattern, suggesting that this rate is also influenced by modulator binding. Some batches of enzyme cannot convert to the Cred2 form using dithionite, but pretreatment with CO or CO2/dithionite effectively "cures" such batches of this disability. The results presented suggest that the Ni of the C-cluster is the modulator and the substrate binding site for CO/CO2. The inhibitor CS2 in the presence of dithionite also accelerates the decline of Cred1, leading first to an EPR-silent state of the C-cluster, and eventually to a state yielding an EPR signal with gav = 1.66. CS2 binding thus shares some resemblance to CO2 binding. Approximately 90% of the absorbance changes at 420 nm that occur when oxidized CODHCt is reduced by dithionite occur within 2 min at 10 degrees C. This absorbance change occurs in concert with the gav = 1.94 signal development. The remaining 10% of the A420 changes occur over the course of approximately 50 min, apparently coincident with the Cred1/Cred2 conversion. One possibility is that the conversion involves reduction of an (unidentified) Fe-S cluster. A three-state model of catalysis is proposed in which Cred1 binds and oxidizes CO, Cred2 is two electrons more reduced than Cred1 and is the state that binds and reduces CO2, and Cint is a one-electron-reduced state that is proposed to exist because of constraints imposed by the nature of the CO/CO2 reaction and the properties of the clusters involved in catalysis.

Purification and kinetic characterization of gamma-aminobutyraldehyde dehydrogenase from rat liver.

Oxidative deamination of putrescine, the precursor of polyamines, gives rise to gamma-aminobutyraldehyde (ABAL). In this study an aldehyde dehydrogenase, active on ABAL, has been purified to electrophoretic homogeneity from rat liver cytoplasm and its kinetic behaviour investigated. The enzyme is a dimer with a subunit molecular weight of 51,000. It is NAD(+)-dependent, active only in the presence of sulphhydryl compounds and has a pH optimum in the range 7.3-8.4. Temperatures higher than 28 degrees C promote slow activation and the process is favoured by the presence of at least one substrate. Km for aliphatic aldehydes decreases from 110 microM for ABAL and acetaldehyde to 2-3 microM for capronaldehyde. The highest relative V-values have been observed with ABAL (100) and isobutyraldehyde (64), and the lowest with acetaldehyde (14). Affinity for NAD+ is affected by the aldehyde present at the active site: Km for NAD+ is approximately 70 microM with ABAL, approximately 200 microM with isobutyraldehyde and capronaldehyde, and > 800 microM with acetaldehyde. The kinetic behaviour at 37 degrees C is quite complex; according to enzymatic models, NAD+ activates the enzyme (Kact approximately 500 microM) while NADH competes for the regulatory site (Kin approximately 70 microM). In the presence of high NAD+ concentrations (4 mM), ABAL promotes further activation by binding to a low-affinity regulatory site (Kact approximately 10 mM). The data show that the enzyme is probably an E3 aldehyde dehydrogenase, and suggest that it can effectively metabolize aldehydes arising from biogenic amines.

Chemical modification of bacterial 4-aminobutyrate aminotransferase by phenylglyoxal.

4-Aminobutyrate aminotransferase (EC 2.6.1.19), obtained from Pseudomonas fluorescens, was irreversibly inhibited by phenylglyoxal, a reagent that specifically modifies arginyl residues. The inactivation appeared to be the result of a simple, bimolecular reaction since no evidence of a reversible complex between inhibitor and enzyme emerged. The second-order rate constant was 0.221 +/- 0.077 M-1 sec-1. The concentration of either substrate had no effect on the inhibition, but an increase in the concentration of pyridoxal 5'-phosphate reduced the rate of inactivation by phenylglyoxal. The data are consistent with the modification of amino acid residues at the cofactor binding site on the enzyme.

Metal requirements of the enzymes catalyzing conversion of glutamate to delta-aminolevulinic acid in extracts of Chlorella vulgaris and Synechocystis sp. PCC 6803.

In the biosynthetic conversion of glutamate to the tetrapyrrole precursor, delta-aminolevulinic acid (ALA), glutamate is activated at C-1 by glutamyl-tRNA synthetase-catalyzed ligation to tRNAGlu. Glutamyl-tRNA reductase next catalyzes reduction of the activated glutamate to glutamate-1-semialdehyde (GSA), which is then converted to ALA by GSA aminotransferase. Glutamyl-tRNA synthetase is known to require a divalent metal (usually Mg2+) for activity, but it has not been established whether Mg2+ or another metal ion is also required for glutamyl-tRNA reductase or GSA aminotransferase, because these enzymes have previously been assayed in combined incubations containing all factors required for conversion of glutamate to ALA. We now report the metal requirements individually for each of the three enzyme reactions. Glutamyl-tRNA reductase activity in extracts from both Chlorella vulgaris and Synechocystis sp. PCC 6803 was stimulated by Mg2+ and inhibited by EDTA. EDTA-pretreated Chlorella glutamyl-tRNA reductase-containing fraction had very little activity in the absence of added Mg2+, but recovered full activity in incubations containing added Mg2+. The divalent metal requirement could be met by Mg2+, Mn2+, or Ca2+. Maximum activity was reached at approximately 15 mM concentration of each of these metals, and higher concentrations were inhibitory. Zn2+ was inhibitory at micromolar concentrations. Chlorella glutamyl-tRNA synthetase showed a metal requirement that could be met by Mg2+ or Mn2+, but not Ca2+. Maximum activity was reached at approximately 15 mM Mg2+ or Mn2+. Although the presence of 10 mM Ca2+ did not affect the Mg2+ concentration optimum, Ca2+ increased the effectiveness of low concentrations of Mg2+. In contrast to glutamyl-tRNA synthetase and glutamyl-tRNA reductase, Chlorella GSA aminotransferase did not show a metal requirement or inhibition by EDTA. However, EDTA decreased nonenzymatic transformation of GSA to ALA.

Biochemical and histopathological changes in nasal epithelium of rats after 3-day intermittent exposure to formaldehyde and ozone alone or in combination.

To get a better insight into the pathophysiology of the nasal changes induced by formaldehyde-ozone mixtures, a 3-day inhalation study was carried out in rats, using intermittent exposure to formaldehyde (3.6 ppm) and ozone (0.4 ppm) alone or in combination and focusing on biochemical and histopathological changes in rat nasal respiratory epithelium. Formaldehyde dehydrogenase, glutathione S-transferase, glutathione reductase, and glucose-6-phosphate dehydrogenase activities in this epithelium were not affected by the individual compounds. However, combined exposure to formaldehyde and ozone resulted in slightly decreased activities of these enzymes. Formaldehyde was found to induce rhinitis, degeneration, frank necrosis, hyperplasia and squamous metaplasia of the ciliated and non-ciliated nasal respiratory epithelium, while ozone induced disarrangement, flattening and slight basal cell hyperplasia of the non-ciliated cuboidal epithelium accompanied by influx of neutrophils. Proliferating cell nuclear antigen (PCNA) expression was elevated not only in nasal areas showing ozone-induced histopathological changes but also in the otherwise normal-appearing epithelium of the nasal septum. No interactive effects were found with respect to proliferative response of the nasal respiratory epithelium after exposure to the formaldehyde-ozone mixture. The present study did not provide evidence of a major role of glutathione and glutathione-dependent enzymes in the pathogenesis of nasal lesions induced by formaldehyde and/or ozone, demonstrated the potential of ozone to affect the mucociliary epithelium lining the nasal septum, and suggested that PCNA expression is a sensitive tool for detection of early effects of respiratory irritants.

The role of tungstate and/or molybdate in the formation of aldehyde oxidoreductase in Clostridium thermoaceticum and other acetogens; immunological distances of such enzymes.

Besides Clostridium thermoaceticum and C. formicoaceticum other resting acetogenic clostridia such as C. aceticum and C. thermoautotrophicum and to a lesser extent non-clostridial acetogens such as Butyribacterium methylotrophicum and Eubacterium limosum were able to reduce propionate to propanol at the expense of carbon monoxide or formate. Methylviologen usually increased the reduction rate. Ten microM molybdate in the growth medium decreased this capability for C. thermoaceticum but increased it or had no effect for the other organisms. Ten microM tungstate in the growth medium increased the aldehyde oxidoreductase activity in all organisms. Crude extracts of C. thermoaceticum cells grown in the presence of 10 microM or 1 mM molybdate showed by ELISA the same or even a 4 fold concentration of aldehyde oxidoreductase in the latter case. However, the enzymic activity was very low in both cases. Omission of dithionite in the growth medium diminished the antigen by a factor of about 8. The immunological distance between the enzyme from C. thermoaceticum and C. thermoautotrophicum was rather low but very large to C. formicoaceticum and undeterminably large to the other organisms.

In vitro formation of retinoic acid from retinal in rat liver.

Enzymatic conversion of retinal to retinoic acid in rat liver cytosol was detected using a rapid and sensitive assay based on high pressure liquid chromatography (HPLC). This retinal oxidase assay system did not require extraction steps or any other manipulation of the sample mixture once the sample vial was sealed for incubation. The product (retinoic acid) and the reactant (retinal) were separated by HPLC in 14.0 min with a sensitivity of 15 and 40 pmol per injection for retinoic acid and retinal, respectively. Enzymatic activity was observed to be linear with protein concentration (0-2.4 mg/mL) and time (0-30 min) and displayed a broad pH maximum of 7.7-9.7. The enzyme exhibited Michaelis-Menten single-substrate kinetics with an apparent Km of 0.25 mM. The average specific activity in nine normal rats was 35.6 +/- 3.3 nmol retinoic acid formed/h per mg protein. Incubation of the enzyme with zinc did not affect the rate of retinoic acid synthesis. Dithiothreitol inhibited the reaction. Both NAD and NADH stimulated retinoic acid formation. Formation of retinol was also observed when these pyridine nucleotides were added to the reaction mixture, indicating the presence of retinal reductase activity. The results of kinetic studies suggest that NADH may act indirectly to stimulate retinoic acid formation.