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The heat-shock response is a crucial system for survival of organisms under heat stress. During heat-shock stress, gene expression is globally suppressed, but expression of some genes, such as chaperone genes, is selectively promoted. These selectively activated genes have critical roles in the heat-shock response, so it is necessary to discover heat-inducible genes to reveal the overall heat-shock response picture. The expression profiling of heat-inducible protein-coding genes has been well-studied, but that of non-coding genes remains unclear in mammalian systems. Here, we used RNA-seq analysis of heat shock-treated A549 cells to identify seven novel long non-coding RNAs that responded to heat shock. We focussed on CTD-2377D24.6 RNA, which is most significantly induced by heat shock, and found that the promoter region of CTD-2377D24.6 contains the binding site for transcription factor HSF1 (heat shock factor 1), which plays a central role in the heat-shock response. We confirmed that HSF1 knockdown cancelled the induction of CTD-2377D24.6 RNA upon heat shock. These results suggest that CTD-2377D24.6 RNA is a novel heat shock-inducible transcript that is transcribed by HSF1., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Urinary bile acids of 39 healthy male undergraduates were analyzed by capillary gas chromatography and capillary gas chromatography-mass spectrometry. 3 alpha-Hydroxy-12-oxo-5 beta-cholanoic acid, 3 alpha, 12 beta-dihydroxy-5 beta-cholanoic acid, 3 beta, 7 alpha-dihydroxy-5 beta-cholanoic acid, and 3 alpha, 7 alpha, 12 beta-trihydroxy-5 beta-cholanoic acid, in addition to known bile acids, were identified and then quantified. The major part of the urinary bile acids was occupied by secondary bile acids. Every 7 beta-hydroxylated bile acid species was found in more than 80% of the subjects. The bile acid detected in the largest amount was 3 alpha-hydroxy-12-oxo-5 beta-cholanoic acid. The metabolites of cholic acid were quantitatively more predominant than those of chenodeoxycholic acid. These results indicate that bile acids with beta-hydroxyl and carbonyl groups at the C-3,7 and/or 12 positions are usual bile acids usually found in the urine of healthy humans. It is concluded that the occurrence of these bile acids is an effect of the intestinal bacterial flora and living conditions.

Under carefully controlled experimental conditions, the Michaelis constant for H2O2 was measured to be 1.39 and 1.29 M in the reactions of beef erythrocyte and liver catalases, respectively. These values remained unchanged at temperatures between 1 and 26 degrees C. The turnover number of the Michaelis complex was about 2.25 X 10(7) s-1 for either enzyme at 26 degrees C. The cyanide inhibition in the catalase reaction has been reported to be noncompetitive in spite of the fact that cyanide and H2O2 compete for the same site on the catalase molecule. At high concentrations of H2O2, however, the inhibition became clearly competitive. The existence of the Michaelis complex and the anomalous features of cyanide inhibition were clearly accounted for on the basis of simple kinetic models. At H2O2 concentrations below 100 mM, the catalase reaction obeyed first order kinetics with respect to H2O2 and its apparent second order rate constant was measured to be 7.6 X 10(6) and 7.9 X 10(6) M-1 . S-1 for erythrocyte and liver catalases, respectively.

Investigations were made of the multiplicity of the major C-nitrosoreductase in porcine liver cytosol catalyzing NADH-dependent reduction of p-nitrosophenol (p-NSP) to p-aminophenol (P-AmP). A partially purified preparation prepared by precipitation with ammonium sulfate, gel filtration with Sephadex G-100, and ion-exchange chromatography on DEAE-Sephadex A-50 showed two or more apparent Km values for p-NSP and also for NADH. This preparation could be resolved into at least four subfractions having different Km values by affinity chromatography on 5'-AMP-Sepharose, Even a more purified preparation, which was obtained from the Sephadex G-100 gel filtrate by affinity chromatography followed by ion-exchange chromatography, could be resolved into multiple subfractions by isoelectric focusing in polyacrylamide gel. All nitrosoreductase subfractions extracted from the polyacrylamide gel showed NADH-aldehyde reductase (alcohol dehydrogenase [EC 1.1.1.1]) activity and, except for a few minor subfractions, the two activities were in parallel. A commercially supplied, crystalline preparation of equine liver alcohol dehydrogenase also showed a similar multiplicity and the multiple subfractions had the both enzymatic activities, although the pattern of isoelectric focusing was different from those of the porcine liver preparations. Different heat inactivation curves were observed with various preparations, but in each preparation the curve for nitrosoreductase activity always agreed with that for aldehyde reductase activity. The nitrosoreductase preparations and the alcohol dehydrogenase preparation showed very similar pH-activity curves in catalyzing NADH-dependent reduction of p-NSP. Furthermore, ethanol inhibited the NADH-p-NSP reductase reaction competitively and p-AmP inhibited the NADH-aldehyde reductase reaction competitively. These results clearly indicate that the major C-nitrosoreductase in porcine liver was present in multiple forms having different Km values and the enzyme was identical with liver alcohol dehydrogenase.

By a mild alkaline treatment, pyocin R1 was disassembled into its structural parts, a contracted sheath (and its fragments), a core, and fibers. An alkaline sucrose density gradient centrifugation after this treatment was effective in obtaining fiber-density fractions. The pooled fractions were treated with IgGs against isolated sheaths and isolated cores, simultaneously, and then chromatographed on DEAE-Sepharose CL-6B. The final preparation of fibers purified in this way was confirmed to be homogeneous by electron microscopic observation and an immuno-precipitation reaction. The isolated fiber was found to consist of two major subunit proteins, No. 2 and No. 9, with molecular weights of 71,000 and 31,000, respectively. The fiber exhibited the ability to be adsorbed on sensitive bacterial cells (pseudomonas aeruginosa P14), and to protect against the inactivation of pyocin R1 by a lipopolysaccharide preparation from the bacteria.

The effects of bile duct ligation on bile acid and cholesterol metabolism were examined in male Wistar strain rats. Quantitative and qualitative changes of bile acids and cholesterol in serum and urine occurred; beta-muricholic acid predominantly increased in serum and urine and the ratio of urinary cholic acid and beta-muricholic acid changed from about 5:3 on day 1 to about 1:8 on day 5 under biliary obstruction. The form of the increased urinary bile acids was mainly taurine-conjugated and partly sulfated. Under conditions of bile duct ligation on day 5, 14C-labeled 3 beta-hydroxy-5-cholenoic, lithocholic, and chenodeoxycholic acids were intragastrically administered to the rats after pretreatment with antibiotics and the metabolites of these three acids were investigated. 3 beta-Hydroxy-5-cholenoic acid was most efficiently converted to beta-muricholic acid. The present study strongly suggested the presence of an alternative metabolic pathway induced by bile duct ligation, which caused the change in composition of urinary bile acids, and especially the marked increase in beta-muricholic acid formation. A possible alternative pathway for bile acid biosynthesis under biliary obstruction in rats is postulated.

The subcellular distribution of NADH-p-nitrosophenol (p-NSP) reductase activity at pH 6.0 in porcine liver was studied by spectrophotometric assay. About two-thirds of the activity was found in the cytosol fraction and the pH optimum of this fraction was about 5.5. The activity at pH 5.8 of cytosol fractions from various tissues of rats, quails, frogs, carp, and scallops was also studied. All these fractions showed more or less NADH-p-NSP reductase activity but the activity of NADH-aldehyde reductase (alcohol dehydrogenase [EC 1.1.1.1]) was detected only in these from liver and a few other tissues. Supernatants from sonicated cells of Bacillus subtilis and Escherichia coli also showed C-nitrosoreductase activity but were devoid of aldehyde reductase activity. The major C-nitrosoreductase of porcine liver cytosol was purified 20- to 30-fold by fractionation with ammonium sulfate, gel filtration, and ion-exchange chromatography. The pH optimum of this preparation was 5.5 and activity was strongly inhibited by p-chloromercuribenzoate (p-CMB). The enzyme preparation was stable at 5 degrees C for at least a week in the presence of NADH at pH 8.4. High concentrations of ammonium sulfate also stabilized the enzyme. An equilibrium between monomeric and dimeric forms of the enzyme was found and the molecular weight was estimated to be about 83,000 and 160,000 daltons for the monomeric and dimeric forms, respecively. The enzyme utilized NADH almost specifically and 2 mol of NADH were consumed per mol of p-NSP reduced to p-aminophenol. Nitrosobenzene and aldehydes could also serve as the electron acceptor. The aldehyde reductase activity became concentrated roughly in parallel with the C-nitrosoreductase activity during the course of the purification and these two activities could not be separated even after further purification by 5'-AMP-Sepharose affinity chromatography. N-Nitroso compounds were not affected by this enzyme.

The reaction kinetics of the major cytoplasmic NADH-nitrosoreductase, which is the identical enzyme to alcohol dehydrogenase [EC 1.1.1.1], was studied with a partially purified preparation from porcine liver. On the basis of the data obtained, the following scheme is proposed as the mechanism of this enzyme reaction: [Formula: see text], where E and E' are the enzyme unit (one subunit of alcohol dehydrogenase) and an intermediate form of the enzyme unit-substrate compound which appears by two-electron reduction of the enzyme unit-substrate compound, respectively, S is p-nitrosophenol (p-NSP), N is NADH, and P1 and P2 are NAD+ and p-aminophenol (p-AmP), respectively. In this case, it is assumed that k1 less than K2. Para-aminophenol, the reaction product, showed an inhibition competitive to p-NSP at fixed concentrations of NADH and also showed a mixed-type inhibition to NADH at fixed concentrations of p-NSP. NAD+ inhibited the reaction in a competitive manner to NADH at fixed concentrations of p-NSP and in a non-competitive manner to p-NSP at fixed concentrations of NADH. These results can also be accounted for by the proposed mechanism.

An NAD(P)H-dependent C-nitrosoreductase was purified 90 fold from porcine heart cytosol fraction by ammonium sulfate fractionation, gel filtration with Sephadex G-100, ion-exchange chromatography on DEAE-Sephadex A-50, and affinity chromatography on 5'-AMP-Sepharose. The enzyme had no aldehyde reductase activity and showed a pH optimum of 5.5. Its molecular weight estimated by gel filtration was about 65,000 and 70,000 daltons. In the reaction catalyzed by this enzyme, 2 mol of NADH were consumed per mol p-nitrosophenol (p-NSP) reduced to p-aminophenol (p-AmP). Nitrosobenzene and other aryl nitroso compounds were also reduced but neither phenylhydroxylamine nor hydroxylamine could serve as the electron acceptor. Kinetic measurements were also carried out and, based on the data obtained, the following scheme is proposed for the mechanism of the reaction: [Formula: see text], where E, E', and E" represent the active enzyme unit, the enzyme unit after two-electron reduction, and the enzyme unit after four-electron reduction, respectively, N in NADH, S is p-NSP, and P1 and P2 are NAD+ and p-AmP, respectively. Para-aminophenol showed an inhibition noncompetitive wih NADH and also one apparently noncompetitive with p-NSP. NAD+ showed an inhibition competitive with NADH and one uncompetitive with p-NSP. These results can also be accounted for by the proposed mechanism.