Degradation of 4′-dimethylaminoazobenzene-2-carboxylic acid by Pseudomonas stutzeri

4-Dimethylaminoazobenzene-2-carboxylic acid (DMBC) was utilized as a necessary carbon and nitrogen source by Pseudomonas stutzeri IAM 12097. o-Aminobenzoic acid (o-ABA), N,N-dimethyl-p-phenylenediamine (DMPA) and cathecol were identified as intermediates of DMBC degradation. DMBC was degraded at a concentration below 70 mol dm^–3. The ability to utilize DMBC in P. stutzeri was lost spontaneously to some extent. When P. stutzeri was cured of plasmid DNA (approximately 8 MDal) by treatment with mitomycin C, acridine orange, and chloramphenicol, DMBC was not utilized by the resultant strain. These facts suggest that the degradative ability on DMBC in P. stutzeri is controlled by plasmid DNA. Correspondence to: C. Yatome     

Effect of age and sex on copper-induced toxicity in theMacular mutant mouse

It was determined if the sensitivity inmacular mutant mouse to copper-induced toxicity was affected by sex or age. The sensitivity in 6–8-d-old or 3–4-wk-oldmacular mutant mouse to copper-induced toxicity was not affected by sex. However, 8–9-wk-old mutant females were more sensitive to copper-induced toxicity than mutant males. Furthermore, 6–8-d-old or 3–4-wk-old mutant males were more sensitive to copper-induced toxicity than 8–9-wk-old mutant males. However, age-related differences in sensitivity to copper-induced toxicity did not occur significantly in mutant females. On the other hand, in the case of normal mice, the sensitivity in 6–8-d-old or 3–4-wk-old mice to copper-induced toxicity was not also affected by sex. In contrast to mutant, however, 8–9-wk-old normal males were more sensitive to copper-induced toxicity than 8–9-wk-old normal females. Adult males were also more sensitive to copper-induced toxicity than 6–8-d-old or 3–4-wk-old males. However, age-related differences in sensitivity to copper-induced toxicity did not occur significantly in normal females. These results indicate that sex- and age-related differences in the copper-induced toxicity exist inmacular mutant mice.     

Photosynthetic reaction center mutagenesis via chimeric rescue of a non-functional Rhodobacter capsulatus puf operon with sequences from Rhodobacter sphaeroides

Photosynthetically active chimeric reaction centers which utilize genetic information from both Rhodobacter capsulatus and Rb. sphaeroides puf operons were isolated using a novel method termed chimeric rescue. This method involves in vivo recombination repair of a Rb. capsulatus host operon harboring a deletion in pufM with a non-expressed Rb. sphaeroides donor puf operon. Following photosynthetic selection, three revertant classes were recovered: 1) those which used Rb. sphaeroides donor sequence to repair the Rb. capsulatus host operon without modification of Rb. sphaeroides puf operon sequences (conversions), 2) those which exchanged sequence between the two operons (inversions), and 3) those which modified plasmid or genomic sequences allowing expression of the Rb. sphaeroides donor operon. The distribution of recombination events across the Rb. capsulatus puf operon was decidedly non-random and could be the result of the intrinsic recombination systems or could be a reflection of some species-specific, functionally distinct characteristic(s). The minimum region required for chimeric rescue is the D-helix and half of the D/E-interhelix of M. When puf operon sequences 3 of nucleotide M882 are exchanged, significant impairment of excitation trapping is observed. This region includes both the 3 end of pufM and sequences past the end of pufM.     

Studies on the hemolytic and hydrolytic actions of phospholipases against mammalian erythrocyte membranes.

The hemolytic actions of three kinds of phospholipase C on horse and sheep erythrocytes were studied in relation to their hydrolytic activities on the phospholipid components of these red cells. Clostridium novyi (oedematiens) type A phospholipase C hemolyzed horse red cells by hydrolyzing phosphatidylcholine. However, the enzyme did not lyse sheep cells nor did it hydrolyze any phospholipid under the same conditions, although this enzyme hydrolyzed both sphingomyelin and phosphatidylethanolamine in the phospholipid mixture extracted from sheep red cells. Clostridium perfringens phospholipase C hemolyzed not only horse red cells by hydrolyzing phosphatidylcholine but also sheep red cells by hydrolyzing sphingomyelin. Sphingomyelin on sheep red cell membrane was hydrolyzed 10 times faster by this enzyme than that on horse red cell membrane. Pseudomonas aureofaciens phospholipase C hemolyzed horse red cells by attacking phosphatidylcholine and phosphatidylethanolamine. The enzyme did not attack sheep red cells but it did hydrolyze phosphatidylethanolamine in the extracted phospholipid mixture from sheep cells. The hemolytic activity of phospholipase C depends not only on the enzyme and the asymmetric distribution of phospholipids in the erythrocyte membrane but also on the accessibility of the enzymes to the phospholipids in the surface of the membranes. Hemolysis by phospholipase C belongs to a hot-cold type of lysis.     

D-Arg2-dermorphin tetrapeptide analogs: a potent and long-lasting analgesic activity after subcutaneous administration

To examine pharmacological properties of D-Arg2-dermorphin tetrapeptides, six tetrapeptide analogs based on the following formulas, H-Tyr-D-Arg-Phe-Gly-OX (X = H, ethyl, n-propyl), H-Tyr-D-Arg-Phe-Sar-OX (Sar = sarcosine; X = H, methyl, ethyl), were prepared. All these analogs exhibited highly potent and long-lasting analgesia as compared with that of morphine after subcutaneous administration in mice. Among analogs tested, H-Tyr-D-Arg-Phe-Sar-OH showed the highest activities, which were 21, 30 and 58 times more active than morphine in the tail pressure, tail flick and phenylbenzoquinone writhing tests, respectively, on a molar basis.     

Application of a statistical procedure for the control of yeast production

In many cases of fermentation processes, it is difficult to measure all of the state variables needed to indicate the culture state and to predict state changes. We propose the use of time-series data of measurable state variables instead of unmeasurable to describe the fermenatation process. We also employ a discrimination method of independent variables of regression analysis using statistical measures, PSS and AIC. These statistical procedure were applied to fed batch culture for yeast production under aerobic conditions, and the effectiveness was ascertained experimentally.     

L-Lactate dehydrogenase from Thermus caldophilus GK24, an extremely thermophilic bacterium. Desensitization to fructose 1,6-bisphosphate in the activated state by arginine-specific chemical modification and the N-terminal amino acid sequence

Heat-stable and fructose-1,6-bisphosphate-activated L-lactate dehydrogenase (EC 1.1.1.27) has been purified from an extremely thermophilic bacterium, Thermus caldophilus GK24 [Taguchi, H., Yamashita, M., Matsuzawa, H. and Ohta, T. (1982) J. Biochem. (Tokyo) 91, 1343-1348]. N-terminal sequence analysis of the first 34 amino acids of the enzyme indicates that the N-terminal arm region (first 1-20 residues) known for the vertebrate L-lactate dehydrogenases is completely missing in the T. caldophilus enzyme, while there is a high homology of sequence between the regions which are considered to be part of the NAD-binding domain. The C-terminal amino acid of the enzyme was phenylalanine. Analysis of the amino acid composition showed that T. caldophilus enzyme contained much more arginine and fewer lysine than other bacterial and vertebrate L-lactate dehydrogenases. On modification reaction with 2,3-butanedione in the presence of NADH and oxamate, an enhanced activity of the T. caldophilus L-lactate dehydrogenase was obtained independently of fructose 1,6-bisphosphate, and the modified enzyme was desensitized to fructose 1,6-bisphosphate. Amino acid analysis indicated that such a desensitization in the active state was caused by the modification of only one arginine residue per the enzyme subunit. Desensitization of the enzyme was inhibited in the presence of fructose 1,6-bisphosphate. A similar desensitization was observed using 1,2-cyclohexanedione instead of 2,3-butanedione. The enzyme was irreversibly modified with 2,3-butanedione and characterized. The irreversibly modified enzyme also showed an enhanced activity independently of fructose 1,6-bisphosphate, and its pyruvate saturation curve was similar to that of the native enzyme measured in the presence of fructose 1,6-bisphosphate. Fructose 1,6-bisphosphate, which increases the thermostability of the native enzyme, did not affect that of the modified enzyme, while thermostability of the modified enzyme slightly decreased. Amino acid analysis indicated that only the arginine content was decreased by the modification. These results show that arginine residue(s) exist in the binding site for fructose 1,6-bisphosphate on the enzyme, and that the arginine residue(s) play some important role in the allosteric regulation of the enzyme activity.     

Microbial production of chenodeoxycholic acid precursor, 12-ketochenodeoxycholic acid,from dehydrocholic acid

Summary Two kinds of bacteria (DC33 and DC1115) were isolated from soil as biotransformers of dehydrocholic acid to 12-ketochenodeoxycholic acid, and identified to be Brevibacterium fuscum and Lactobacillus xylosus, respectively. Dehydrocholic acid was converted via 7,12-diketolithocholic acid to 12-ketochenodeoxycholic acid by both strains, and the product and the intermediate were isolated and chemically identified. By using a jar fermentor, 12-ketochenodeoxycholic acid was produced with a more than 50% yield after 52 h by Brevibacterium fuscum with aerobic growth and anaerobic conversion, and after 24 h by Lactobacillus xylosus under anaerobic conditions, respectively.     

Studies on phosphatidylinositol phosphodiesterase (phospholipase C type) of Bacillus cereus. I. purification, properties and phosphatase-releasing activity.

A phosphatidylinositol phosphodiesterase from the culture broth of Bacillus cereus, was purified to a homogeneous state as indicated by polyacrylamide gel electrophoresis, by ammonium sulfate precipitation and chromatography with DEAE-cellulose and CM-Sephadex. The enzyme (molecular weight: 29000 +/- 1000) was maximally active at pH 7.2-7.5, AND NOT INFLUENCED BY EDTA, ophenanthroline, monoiodoacetate, p-chloromercuribenzoate or reduced glutathione. The enzyme specifically hydrolyzed phosphatidylinositol, but did not act on phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, under the conditions examined. The products from phosphatidylinositol of enzyme reaction were diacylglycerols and a mixture of myoinositol 1- and 1, 2-cyclic phosphates, suggesting that the enzyme was a phosphatidylinositol-specific phospholipase C. The enzyme released alkaline phosphatase quantitatively from rat kidney slices. A kinetic analysis was made on the release of alkaline phosphatase. The results suggest that phosphatidylinositol-specific phospholipase C can specifically act on plasma membrane of rat kidney slices.