Molecular analysis of five independent Japanese mutant genes responsible for hypoxanthine guanine phosphoribosyltransferase (HPRT) deficiency

Five independent mutations in the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene were identified in a partially HPRT deficient patient with gout and in four Lesch-Nyhan patients. Using the polymerase chain reaction (PCR) technique coupled with direct sequencing, the nucleotide sequences of the entire HPRT coding region amplified from the cDNA and also of each exon amplified form the genomic DNA were analyzed. Three independent point mutations in the coding region were detected in the partially HPRT deficient patient (Case 1) and in two Lesch-Nyhan patients (Case 2 and 3), resulting in single amino acid substitutions. The family study of Case 3, utilizing a PvuII restriction site created in the mutant gene, indicated that the mother was a heterozygote, and a sister and a fetal brother had inherited the normal HPRT gene from the mother. In two other mutants causing Lesch-Nyhan syndrome, a portion of the HPRT gene was deleted, and RNA splicing was missing in both mutants. A 4-bp deletion at the 5 end of exon 4 resulted in formation of three different types of abnormal mRNA (Case 4). The other mutant (Case 5) produced abnormal mRNA including 26bp of intron 8 instead of the deleted 58bp at the 5 end of exon 9, because of a 74-bp deletion from intron 8 to exon 9.     

Complete nucleotide sequence of the mitochondrial DNA from a liverwort,Marchantia polymorpha

Libraries of cosmid and plasmid clones covering the entire region of mtDNA from the liverwortMarchantia polymorpha were constructed. These clones were used for the determination of the complete nucleotide sequence of the liverwort mtDNA totally 186,608 bp (GenBank no. M68929) and including genes for 3 species of ribosomal RNAs, 29 genes for 27 species of transfer RNAs, and 30 genes for functionally known proteins (16 ribosomal proteins, 3 subunits of cytochromec oxidase, apocytochromeb protein, 3 subunits of H⁺-ATPase, and 7 subunits of NADH ubiquinone oxidoreductase). The genome also contains 32 unidentified open reading frames. Thus the complete nucleotide sequences from both chloroplast and mitochondrial genomes have been determined in the same organism. Plasmid clones are available upon the request. Gene names are represented according to Lonsdale and Leaver (1988) with modifications recommended by Lonsdale (personal communication).     

Application of the tryptophanase promoter to high expression of the tryptophan synthase gene inEscherichia coli

Summary The application of an inducible regulation system using the trytophanase operon promoter (TPase promoter; P_tna) was examined for its high expression of the tryptophan synthase (TS) gene in Escherichia coli. The main problem in the application of P_tna for industrial purposes is catabolite repression by glucose, since glucose is the most abundant carbon source. However, this problem could be avoided by changing glucose to an organic acid, such as succinate, fumarate, malate and acetate, in the course of cultivation after glucose initially added was completely consumed. Under these conditions, l-tryptophan was also used to induce tryptophan synthase. Thus, the specific activity of TS in E. coli strain no. 168 harbouring pBR322F-P_tnaTS was increased 500-fold compared to that of the cultured host strain. About 1 mol l-tryptophan/l reaction mixture was formed from indole and l-serine at 37° C for 3.5 h. Offprint requests to: H. Yukawa     

Induction of interleukins by mycoplasma in culture of human mononuclear leukocytes

Mycoplasma shows a variety of effects on immune system, including the activation of macrophage, the increase in T cell cytotoxicity, and the enhancement of the proliferation and maturation of B cells, etc. As it is well known that many cytokines regulate the immune system, it is interesting to examine whether or not human peripheral blood mononuclear cells (PBMC) produce interleukin (IL) in response to mycoplasmas. In the present study, human PMBC were incubated with 7 species of mycoplasmas for 48 hours, and IL-1 beta, IL-2 and IL-6 activities in the supernatants were determined by ELISA. All the species of mycoplasmas were able to induce IL-1 beta and IL-6, although IL-2 was induced only by M. pneumoniae. These results suggest that the influence of mycoplasma infection on immune system may be partly due to the interleukins induced by mycoplasmas.     

Seasonal changes in the spermatogenic epithelium of adult Japanese macaques (Macaca fuscata fuscata)

A histological study was undertaken to clarify seasonal changes in the spermatogenic epithelium of Japanese macaques. Testicular tissue samples were excised by biopsies from five adult laboratory-maintained males in mating and non-mating seasons. The samples were fixed with Bouin's solution, embedded in paraffin, and stained with PAS and hematoxylin. Microscopic observations on cross-sections of seminiferous tubules revealed that the seminiferous epithelium in the mating season was thicker than in the non-mating season. PAS-stained granules were found in some of the dark A-type spermatogonia, which significantly increased in the non-mating season. Spermatids of the steps preceding the appearance of the acrosomic cap in stages I to III were observed significantly more often than those in the step coinciding with the formation of the acrosomic cap in stage IV. In stage I, the ratio of mature spermatids or spermatozoa to immature spermatids in the mating season was higher than that in the non-mating season. These findings suggest that spermiogenesis, as well as spermatocytogenesis, is inhibited in the non-mating season.     

The synergistic effects of interleukin 2 and interleukin 7 on the proliferation and autologous tumor cell lysis of tumor-associated lymphocytes

Interleukin-7 (IL-7) has an ability to stimulate the proliferation of pre-B cells. It has been shown that IL-7 can also activate T lymphocytes. We here demonstrate that IL-7 in combination with interleukin-2 (IL-2) can drive cell proliferation and enhance the autologous tumor cell lysis by peripheral blood mononuclear cells (PBMC) and autologous mixed lymphocyte tumor cell culture (MLTC)-derived effector cells (MLTC cells). These synergistic effects of IL-2 and IL-7 on the proliferation and the augmentation of autologous tumor cell lysis were found for both effector cells. These effects were inhibited by neutralizing antibodies to IL-2 or IL-7, and by a combination of both antibodies, significantly. In terms of phenotypical expression, CD3 positive cells comprised the vast majority of MLTC cells after culture in medium containing IL-2 and IL-7 with an increase of IL-2 receptor positive cells.Abbreviations CD cluster differentiation - IFN interferon - IL interleukin - JRU Japanese Reference Unit - LAK lymphokine activated killer - mAb monoclonal antibody - MLTC mixed lymphocyte tumor cell culture - PBMC peripheral blood mononuclear cells - TILs tumor infiltrating lymphocytes     

Age-related alterations in the size of human hepatocytes

Age-related alterations in the size of human hepatocytes (both mononuclear and binucleate forms), were studied in histological sections and in separated cells and nuclei using cytophotometrical and microspectrophotometrical methods. The following results were obtained: 1. The volume of nuclear DNA increased in proportion to nuclear size. The increase occurred in a group pattern reflecting nuclear polyploidization. 2. Cell size increased in proportion to nuclear size. Tetraploid cells (4C) were roughly two times greater than diploid cells (2C). 3. In most of the binucleate cells examined, the ploidy class of the two nuclei in a binucleate cell was observed to be equal. Heterogeneity of the ploidy class among the nuclei of a binucleate cell was present in less than 1% of total binucleate cells examined. The nuclear DNA volume of individual nuclei in binucleate cells appeared to be the same as that of mononuclear cells. 4. The cell size of binucleate cells corresponded with that of mononuclear cells whose ploidy class was the same as the sum of the ploidy classes of two nuclei of a binucleate cell. 5. The incidence of binucleate cells in the lobular periphery was about 4 to 6% in the third decade, and increased slightly with age up to 5 to 7% in the tenth decade. 6. The incidence of binucleate cells in the liver at different ages followed a similar pattern to that observed in mononuclear cells whose ploidy class was half of the sum of ploidy classes of the two nuclei of the binucleate cell.     

Distribution of antimicrobial‐resistant lactic acid bacteria in natural cheese in Japan

To determine and compare the extent of contamination caused by antimicrobial‐resistant lactic acid bacteria (LAB) in imported and domestic natural cheeses on the Japanese market, LAB were isolated using deMan, Rogosa and Sharpe (MRS) agar and MRS agar supplemented with six antimicrobials. From 38 imported and 24 Japanese cheeses, 409 LAB isolates were obtained and their antimicrobial resistance was tested. The percentage of LAB resistant to dihydrostreptomycin, erythromycin, and/or oxytetracycline isolated from imported cheeses (42.1%) was significantly higher than that of LAB resistant to dihydrostreptomycin or oxytetracycline from cheeses produced in Japan (16.7%; P = 0.04). Antimicrobial resistance genes were detected in Enterococcus faecalis (tetL, tetM, and ermB; tetL and ermB; tetM) E. faecium (tetM), Lactococcus lactis (tetS), Lactobacillus (Lb.), casei/paracasei (tetM or tetW), and Lb. rhamnosus (ermB) isolated from seven imported cheeses. Moreover, these E. faecalis isolates were able to transfer antimicrobial resistance gene(s). Although antimicrobial resistance genes were not detected in any LAB isolates from Japanese cheeses, Lb. casei/paracasei and Lb. coryniformis isolates from a Japanese farm‐made cheese were resistant to oxytetracycline (minimal inhibitory concentration [MIC], 32 µg/mL). Leuconostoc isolates from three Japanese farm‐made cheeses were also resistant to dihydrostreptomycin (MIC, 32 to > 512 µg/mL). In conclusion, the present study demonstrated contamination with antimicrobial‐resistant LAB in imported and Japanese farm‐made cheeses on the Japanese market, but not in Japanese commercial cheeses.     

Screening for Antioxidants of Microbial Origin

Asymmetric hydrolysis of (dl)-1-acyloxy-2-halo-1-phenylethanes by lipoprotein lipase Amano P from Pseudomonas fluorescens and the lipase from Chromobacterium viscosum afforded the optically active (R) residual substrates and (S)-2-halo-1-hydroxy-1-phenylethanes in 100% enantiomeric excess (e.e.). The length of acyl residues from acetyl to octanoyl in the substrates did not influence the enantioselectivity.

Both enantiomers of optically active styrene oxides were synthesized from the enzymatic products.     

Modulatory effects of oligodendrocytes on the conduction velocity of action potentials along axons in the alveus of the rat hippocampal CA1 region

Like neurons and astrocytes, oligodendrocytes have a variety of neurotransmitter receptors and ion channels. However, except for facilitating the rapid conduction of action potentials by forming myelin and buffering extracellular K(+), little is known about the direct involvement of oligodendrocytes in neuronal activities. To investigate their physiological roles, we focused on oligodendrocytes in the alveus of the rat hippocampal CA1 region. These cells were found to respond to exogenously applied glutamate by depolarization through N-methyl-D-aspartate (NMDA) receptors and non-NMDA receptors. Electrical stimulation of the border between the alveus and stratum oriens evoked inward currents through several routes involving glutamate receptors and inward rectifier K(+) channels. Moreover, electrical stimulation resembling in vivo activity evoked long-lasting depolarization. To examine the modulatory effects of oligodendrocytes on neuronal activities, we performed dual, whole-cell recording on CA1 pyramidal neurons and oligodendrocytes. Direct depolarization of oligodendrocytes shortened the latencies of action potentials evoked by antidromic stimulation. These results indicate that oligodendrocytes increase the conduction velocity of action potentials by a mechanism additional to saltatory conduction, and that they have active roles in information processing in the brain.