Enzymatic preparation of [1, 3-13C]dihydroxyacetone phosphate from [13C]methanol and hydroxypyruvate using the methanol-assimilating system of methylotrophic yeasts

Dihydroxyacetoone synthase (EC 2.2.1.3), which is a key enzyme of the C₁-compound-assimilating pathway in yeasts, catalyzes transketolation between formaldehyde and hydroxypyruvate, leading to the formation of dihydroxyacetone and CO₂. When [¹³C]formaldehyde was used as a substrate with dihydroxyacytone synthase from Candida boidinii 2201, ¹³C was confirmed to be incorporated to the C-1 and C-3 positions of dihydroxyacetone, and the ¹³C content of each carbon (atoms/100 atoms) was estimated to be 50%. [¹³C]Methanol was also useful for the enrichment of dihydroxyacetone with ¹³C, when alcohol oxidase from a methylotrophic yeast was added for the conversion of methanol to formaldehyde. A fed-batch reaction with periodic addition of the substrates was required for the accumalation of ¹³C-labelled dihydroxyacetone at a higher concentration, because the enzyme system was relatively susceptible to the C donor, formaldehyde or methanol. The optimum conditions for the production gave 160mM (14.4 mg/ml) dihydroxyacetone for 180 min; the molar yield relative to methanol added was 80%. Diyhdroxyacetone kinase (EC 2.7.1.29) from methanol-grown Hansenula polymorpha CBS 4732 was a suitable enzyme for the phosphorylation of dihydroxyacytone. The phosphorylation system, comprising of dihydroxyacetone kinase, adenylate kinase, and ATP, could be coupled with the system for dihydroxyacetone production. A fed-batch reaction afforded 185 mM [1, 3-¹³C]dihydroxyacetone phosphate from [¹³C]methanol; the molar yield of the ester relative to methanol added was 92.5%     

Molecular phylogeny based on the κ-casein and cytochrome b sequences in the mammalian suborder Ruminantia

Nucleotide sequences for the -casein precursor proteins have been determined from the genomic DNAs or hair roots of the Ruminantia. The coding regions, exons 2, 3, and 4, were amplified separately via the three kinds of PCRs and then directly sequenced. The primers were designed from the sequence of bovine -casein gene; they were applicable for the amplification of the -casein genes from the 13 species in the Ruminantia except exon 2 of the lesser mouse deer. These results permitted an easy phylogenetic analysis based on the sequences of an autosomal gene. A phylogenetic tree was constructed from the mature K-casein sequences and compared with the tree of the cytochrome b genes which were sequenced from the same individuals. The Cervidae (sika deer, Cervus nippon) were separated from the branch of the Bovidae on the tree of -casein genes with a relatively high confidence level of the bootstrap analysis, but included in the branch of the Bovidae on the tree of cytochrome b genes. The -casein tree indicated a monophyly of the subfamily Caprinae, although the internal branches were uncertain in the Caprinae. The tree based on the nucleotide sequences of cytochrome b genes clearly showed the relationships of the closely related species in the genus Capricornis consisting of serow (C. smatorensis), Japanese serow (C. crispus), and Formosan serow (C. swinhoei). These results would be explained by the difference of resolving power between the -casein and the cytochrome b sequences. Correspondence to: K. Chikuni     

Diverse Expression of β1,4-N-Acetylgalactosaminyltransferase Gene in the Adult Mouse Brain

Abstract: Among various tissues of mouse, β1,4- N -acetylgalactosaminyltransferase (GM2/GD2 synthase) gene is expressed predominantly in the brain. Further analysis of the gene expression in the mouse CNS was performed by northern blotting and by enzyme assays using extracts from various parts of the CNS. In situ hybridization was also done to investigate the distribution of cells generating GM2/GD2 synthase. In northern blots, diverse levels of the gene expression were observed, depending on the regions examined. By in situ hybridization, pyramidal cells in the hippocampus, granular cells in dentate gyrus and cerebral cortex, Purkinje cells in cerebellum, and mitral cells in the olfactory bulb expressed high levels of the mRNA; these results corresponded to the results obtained by northern blot. Enzyme levels in these sites were accordingly high. However, enzyme levels in certain areas with low mRNA intensities, such as thalamus and pons medulla, were higher than expected from the results of northern blotting. The significance of the high gene expression in certain areas for brain function and the reason for the discrepancy between mRNA level and enzyme activity in some regions are discussed.     

Control of the rate of phosphocreatine resynthesis after exercise in trained and untrained human quadriceps muscles

We examined the effect of differences in exercise intensity on the time constant (t _c) of phosphocreatine (PCr) resynthesis after exercise and the relationships betweent _c and maximal oxygen uptake (VO_2max) in endurance-trained runners (n = 5) and untrained controls (n = 7) (average VO_2max = 66.2 and 52.0 ml · min^–1 · kg^–1, respectively). To measure the metabolism of the quadriceps muscle using phosphorus nuclear magnetic resonance spectroscopy, we developed a device which allowed knee extension exercise inside a magnet. All the subjects performed four types of exercise: light, moderate, severe and exhausting. The end-exercise PCr: [PCr + inorganic phosphate (P_i)] ratio decreased significantly with the increase in the exercise intensity (P < 0.01). Although there was little difference in the end-exercise pH, adenosine diphosphate concentration ([ADP]) and the lowest intracellular pH during recovery between light and moderate exercise, significant changes were found at the two higher intensities (P < 0.01). These changes for runners were smaller than those for the controls (P < 0.05). The _c remained constant after light and moderate exercise and then lengthened in proportion to the increase in intensity (P < 0.05). The runners had a lowert _c at the same PCr and pH than the controls, particularly at the higher intensity (P < 0.05). There was a significant correlation betweent _c and [ADP] in light exercise and betweent _c and both end-exercise PCr and pH in severe and exhausting exercise (P < 0.05). The threshold of changes in pH andt _c was a PCr: (PCr + P_i) ratio of 0.5. There was a significant negative correlation between the VO_2max andt _c after all levels of exercise (P<0.05).However, in the controls a significant correlation was found in only light and moderate exercise (P < 0.05). These findings suggest the validity of the use oft _c at an end-exercise PCr:(PCr + P_i) ratio of more than 0.5 as a stable index of muscle oxidative capacity and the correlation between local and general aerobic capacity. Moreover, endurance-trained runners are characterized by the faster PCr resynthesis at the same PCr and intracellular pH.     

The interaction of RepC initiator with iterons in the replication of the broad host-range plasmid RSF1010.

The replication origin of the broad host-range plasmid RSF1010 contains 3.5 copies of a 20mer iteron sequence that bind specifically to the plasmid-encoded initiator, RepC. Here we demonstrated that even a single iteron was bent upon binding of RepC. Moreover, the bending angle seems to become larger along with the increment of the number of iterons. In a mutational analysis of the iteron sequence, we isolated seven kinds of base-substitution mutants of iterons, and estimated the replication activity of these mutants in vivo. We found that each of the subsections in the 20mer iteron sequence made a distinct contribution to the initiation of RSF1010 DNA replication. With the binding assay of RepC and mutated iterons in vitro, we found that the formation of a productive RepC-iteron complex was required for the initiation of plasmid DNA replication.     

Alcohol and aldehyde dehydrogenase genotypes and drinking behavior of Chinese living in Shanghai

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), the principal enzymes responsible for oxidative metabolism of ethanol, exist in multiple, genetically determined molecular forms. Widely different kinetic properties in some of these isozymes account for the individual differences in alcohol sensitivity. In this study we used the polymerase chain reaction/restriction fragment length polymorphism method to determine the genotypes of the ADH2 and ALDH2 loci of alcoholic and nonalcoholic Chinese living in Shanghai. We also investigated the subjects' drinking patterns by means of semistructured interviews. The alcoholics had significantly lower frequencies of the ADH2² and ALDH2² alleles than did the nonalcoholics, suggesting the inhibitory effects of these alleles for the development of alcoholism. In the nonalcoholic subjects, ADH2² had little, if any, effect, despite the significant effect of the ALDH2² allele in decreasing the alcohol consumption of the individual. Taken together, these results fit the proposed hypothesis for the development of alcoholism, i.e., drinking behavior is greatly influenced by the individual's gentoypes of alcohol-metabolizing enzymes, and the risk of becoming alcoholic is proportionate with the ethanol consumption of the individual.     

Characterization of Methanosarcina mazeii TMA isolated from a paddy field soil

We isolated a methanogenic strain, designated as strain TMA (=DSM 9195), from an enrichment culture inoculated with a Japanese paddy field soil. Strain TMA was Gram positive and strictly anaerobic. Cell shape was pseudosarcina-like, and cells were nonmotile. The strain was able to use methylamines, methanol, H₂–CO₂, and acetate as substrates for methanogenesis, but did not utilize formate. The optimum temperature and optimum pH were 30–37°C and 6.5–7.5 respectively. The G+C content of the DNA was 42.1 mol %. Strain TMA had DNA-DNA hybridization values of more than 80% with Methanosarcina mazeii S-6^T (T = type strain). On the basis of phenotypic and genotypic characteristics, we identified strain TMA as M. mazeii. This is the first methylotrophic methanogen isolated from a paddy field soil and identified to the species level.     

Differentiation of presumptive primordial germ cell (pPGC)-like cells in explants into PGCs in experimental tadpoles

A single blastomere containing the "germ plasm" of 32-cell stage Xenopus embryos was cultured with [3H]thymidine until the control embryos developed to the neurula stage. The explants, showing a spherical mass in which the nuclei of all cells were labeled, were implanted into the prospective place of presumptive primordial germ cells (pPGCs) in the endodermal cell mass of unlabeled host embryos of the neurula stage. Labeled PGCs as well as unlabeled, host PGCs were found in the genital ridges of experimental tadpoles. This indicates that the precursor of germ cells, corresponding to pPGCs in normal embryos of the neurula stage, in the explants migrated to genital ridges just at the right moment to become PGCs, and suggests that the developmental process progressed normally, even in the explants, as far as the differentiation of pPGCs is concerned.