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Biomedical subjects

K Ohta

Publications and source records attributed to K Ohta.

At least 433 records · Page 24Linked to original sources
PubMed1994

Changes in chromatin structure at recombination initiation sites during yeast meiosis.

Transient double-strand breaks (DSBs) occur during Saccharomyces cerevisiae meiosis at recombination hot spots and are thought to initiate most, if not all, homologous recombination between chromosomes. To uncover the regulatory mechanisms active in DSB formation, we have monitored the change in local chromatin structure at the ARG4 and CYS3 recombination hot spots over the course of meiosis. Micrococcal nuclease (MNase) digestion of isolated meiotic chromatin followed by indirect end-labeling revealed that the DSB sites in both loci are hypersensitive to MNase and that their sensitivity increases 2- to 4-fold prior to the appearance of meiotic DSBs and recombination products. Other sensitive sites are not significantly altered. The study of hyper- and hypo-recombinogenic constructs at the ARG4 locus, also revealed that the MNase sensitivity at the DSB site correlates with both the extent of DSBs and the rate of gene conversion. These results suggest that the local chromatin structure and its modification in early meiosis play an important role in the positioning and frequency of meiotic DSBs, leading to meiotic recombination.

Argininosuccinate Lyase↗
PubMed1994

Structure of the rat thyroid transcription factor-1 (TTF-1) gene.

TTF-1 is a homeodomain-containing thyroid transcription factor which activates the genes of thyroid specific protein, thyroglobulin, thyroid peroxidase and thyrotropin receptor. We have cloned the TTF-1 gene from rat liver genomic library, and the exon/intron organization and the structure of the 5' flanking region were determined. The clone contained the 5.2 kbp upstream sequence from translation initiation site, and we found that the gene has a single intron in the coding sequence. We found in the 5' flanking region the TTF-1 binding consensus sequence, CTCAAGC, at -175 to -169, which overlaps the consensus sequence of CAAT box, DNase I foot print analysis has revealed that the region is protected by nuclear extract from thyroid cells but not by the extract from the liver, suggesting that expression of the TTF-1 gene is autoregulated by TTF-1.

Animals↗
PubMed1994

Preparation of high-molecular-weight DNA: application to mycobacterial cells.

A method for isolating high-molecular-weight DNA from bacteria is described. A special feature of the method is the treatment of whole bacterial cells with an organic solvent (chloroform-methanol (2:1, v/v) or ethanol-ether (1:1, v/v)) prior to DNA extraction from the cells. The DNA preparations obtained from organic solvent-pretreated bacterial cells such as Mycobacterium smegmatis, M. phlei, and Escherichia coli contained highly polymerized DNA, as revealed by pulse-field gel electrophoresis. The size and yield of the DNA obtained from E. coli pretreated with the organic solvent were quite similar to that of the DNA obtained from protoplasts. The results strongly suggest that the organic solvent pretreatment is effective for extracting very large DNA from bacterial cells and especially from bacteria whose protoplasts cannot be easily formed.

DNA, Bacterial↗
PubMed1994

Site-directed mutagenesis of the histamine H1 receptor: roles of aspartic acid107, asparagine198 and threonine194.

Based on structural comparison with other biogenic amine receptors and the histamine H2 receptor, it has been suggested that in the human histamine H1 receptor, Asp107, Thr194, and Asn198 are the residues involved in binding of histamine. We therefore used site-directed mutagenesis to investigate the roles of these three amino acid residues. Asp107 was essential for both agonist and antagonist binding. Asn198 was necessary for agonist but not for antagonist binding. Thr194 was not important for either type of binding. A good correlation was found between agonist binding and receptor activation for all the wild-type and mutant receptors. The results show that the histamine H1 receptor recognizes and is activated by histamine through the interactions of Asp107 and the amino group, and Asn198 and the imidazole ring.

Amino Acid Sequence↗
PubMed1994

Role of angiotensin II in extracellular matrix and transforming growth factor-beta 1 expression in hypertensive rats.

The in vivo effects of alacepril (1-[(S)-3-acetylthio-2-methylpropanoyl]- L-prolyl-L-phenylalanine), an angiotensin converting enzyme inhibitor, and SC-52458 (5-[(3,5-dibutyl-1H-1,2,4-triazol-1- yl)methyl]-2-[2-(1H-tetrazol-5-ylphenyl)]pyridine), an angiotensin AT1 receptor antagonist, were examined on the cardiac and aortic gene expressions of extracellular matrices and TGF-beta 1 in young spontaneously hypertensive rats (SHR). In SHR, types I and III collagen mRNAs were increased in the left ventricle, and in contrast, fibronectin, collagen IV, and transforming growth factor-beta 1 (TGF-beta 1) mRNAs were increased in aorta, compared with those in Wistar-Kyoto rats. All the enhanced mRNAs in both organs in SHR were significantly inhibited by the short-term treatment with the above two drugs. Thus, angiotensin AT1 receptor may play an important role in the regulation of extracellular matrices and TGF-beta 1 expressions in SHR.

Angiotensin II↗
PubMed1994

Differential expression of bcl-2 and susceptibility to anti-Fas-mediated cell death in peripheral blood lymphocytes, monocytes, and neutrophils.

The recently identified Fas antigen (Ag) is a cell surface molecule that can mediate apoptosis. The cytoplasmic product of proto-oncogene bcl-2 has been shown to prolong the cellular survival by inhibiting apoptosis. To elucidate the physiologic significance of expression of both molecules, we examined the expression of Fas Ag and bcl-2 on blood leukocyte populations and evaluated their sensitivity to the cytolytic action of anti-Fas antibody. Although Fas Ag was expressed on a fraction of lymphocytes, both neutrophils and monocytes expressed Fas Ag constitutively. In contrast, there was marked difference among these leukocytes regarding bcl-2 expression. Lymphocytes expressed bcl-2 intensely, but monocytes showed weaker bcl-2 expression, and neutrophils were essentially absent for bcl-2 expression. Seemingly reflecting this lack of bcl-2-expression, neutrophils more easily underwent apoptotic cell death in vitro as compared with monocytes and lymphocytes. We showed that anti-Fas antibody affectively accelerated apoptotic cell death in neutrophils. However, the apoptosis-inducing effect of anti-Fas antibody was minimal on monocytes, and lymphocytes were resistant to this antibody. These results suggest that anti-Fas-mediated cell death may, in part, be determined by bcl-2 expression status in Fas+ lymphoid and hematopoietic cells.

Adult↗
PubMed1994

A novel in vivo assay system for consecutive measurement of brain nitric oxide production combined with the microdialysis technique.

A novel spectrophotometric nitrite (NO2-)/nitrate (NO3-) assay system for a small quantity (5 microliter) of dialysate sample obtained by in vivo brain microdialysis was developed based on the diazotization reaction. The system has the advantage of in vivo consecutive measurement, high precision, good reproducibility, technical simplicity, relatively short resolution time (2.5-20 min), and wide availability. The NO3- level in the rat striatum was found to be 3 times higher than the NO2- level. A nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine methyl ester, reduced striatal NO2-/citrulline formation in a dose-related manner and increased arginine, indicating that the tissue NO2- level detected by this assay system adequately reflects the striatal NO synthase activity.

Amino Acid Oxidoreductases↗
PubMed1994

The beta-anomeric and glucose preferences of glucose transport carrier for intestinal active absorption of monosaccharide conjugates.

The anomeric preference for intestinal absorption of glucosides and galactosides (1- and 2-naphthyl glycosides) was studied by the everted rat intestine method. After the absorption of beta-glucoside and beta- and alpha-galactosides, the glycosides itself appeared on the serosal side, whereas after the alpha-glucoside absorption, the glucoside itself was not detected on the serosal side, but a large amount of aglycone appeared instead. This indicates an alpha-anomeric preference of desglucosylation through the intestinal membrane. A significant decrease of the total (glycoside + aglycone + glucuronide metabolites) amount transported to the serosal side in the absence of Na+, a cosubstrate of the glucose transport carrier (GTC), was observed in alpha- and beta-glucosides and beta-galactoside, but not in alpha-galactoside. This indicates the poor contribution of GTC to the alpha-galactoside absorption. The Na(+)-dependent absorption of the glycosides by the GTC were beta-glucoside > alpha-glucoside > beta-galactoside (for 2-naphthyl glycosides), and beta-galactoside > alpha-galactoside (for 1-naphthhyl glycosides). These results and those of a previous study led to the conclusion that the intestinal glucose transport carrier prefers beta-anomer to alpha-anomer, and glucose to galactose for monosaccharide conjugates absorption.

Animals↗
PubMed1994

C-terminal topology of gastric H+,K(+)-ATPase.

An antibody was prepared against a peptide corresponding to residues 1024-1034 (the putative C-terminus) of the alpha-subunit of hog gastric H+,K(+)-ATPase. The antibody bound to a 95 kDa band of H+,K(+)-ATPase that was solubilized in SDS, but not to that of Na+,K(+)-ATPase. It also bound to products of tryptic digestion that included C-terminal fragments of the H+,K(+)-ATPase alpha-subunit. The same amount of the antibody bound to both intact (tight) and lyophilized (leaky) inside-out gastric vesicles, indicating that its epitope is present on the cytosolic side of the vesicles. This finding was further confirmed by using fluorescence-immunolocalization techniques and streptolysin-O to permeabilize newt oxyntic cells. Stimulation of isolated newt oxyntic cells with dibutyryl cyclic AMP induces fusion of tubulovesicles with the apical membrane, so that the luminal domains of the H+,K(+)-ATPase alpha-subunit directly face the cell-suspension medium. The antibody did not bind to the stimulated intact cell, but bound to cells permeabilized with streptolysin-O, indicating that it binds from the cytoplasmic side to the C-terminus of the H+,K(+)-ATPase alpha-subunit in apical and tubulovesicular membrane, and also that the H+,K(+)-ATPase alpha-subunit has an even number of transmembrane domains.

Amino Acid Sequence↗
PubMed1994

Modification of striatal arginine and citrulline metabolism by nitric oxide synthase inhibitors.

The effects of NG-substituted L-arginine (ARG) analogues on striatal ARG and citrulline (CIT) levels were investigated using in vivo microdialysis technique. A microdialysis probe was implanted into the striatum of anaesthesized Sprague-Dawley rats. Direct intrastriatal perfusion with 1 mM NG-nitro-L-arginine methyl ester (n = 8) increased striatal ARG release and decreased CIT release, suggesting suppressed NO synthase activity in the tissue. On the other hand, 1 mM NG-monomethyl-L-arginine (L-NMMA) (n = 6) evoked a persistent increase in both ARG and CIT. Considering that 4-320 microM L-ARG (n = 8) failed to increase CIT formation, CIT seems to be synthesized in the striatal tissue from L-NMMA by the enzyme that has been demonstrated in the kidney and aortic endothelium (NG,NG-dimethylarginine dimethyl-aminohydrolase).

Amino Acid Oxidoreductases↗
PubMed1994

Deficiency of the E1 beta subunit in the branched-chain alpha-keto acid dehydrogenase complex due to a single base substitution of the intron 5, resulting in two alternatively spliced mRNAs in a patient with maple syrup urine disease.

A patient with maple syrup urine disease (MSUD) associated with a E1 beta subunit deficiency of the branched-chain alpha-keto acid dehydrogenase (BCKDH) complex was investigated at the molecular level. The defect responsible for the deficiency of the E1 beta subunit protein was identified by analysis of cDNA and genomic DNA by polymerase chain reaction. Total RNA isolated from lymphoblastoid cells was transcribed into cDNA and amplified using a set of primers located within exon 3 and exon 9 of the E1 beta gene. Agarose gel electrophoresis of cDNA amplification products revealed two shortened bands as well as a faint band of normal size. Nucleotide sequencing of the shortened cDNA amplification products showed that sequences corresponding to exon 5 and both exons 5 and 6 were absent. Nucleotide sequencing of the proband's amplified genomic DNA corresponding to this region of the E1 beta gene revealed a single base substitution from G to T of the invariant GT dinucleotides at 5' splice site of the intron 5. Analysis of family members using primer-specified restriction map modification showed that the patient is homozygous for this mutation. We postulate that this mutation leads to the skipping of either exon 5 or both exons 5 and 6, thus producing two shortened E1 beta mRNA. The percentage of normal and two shortened transcripts was estimated to be 9, 71 and 20%, respectively. To our best knowledge, this is the first documented example of exon skipping in the E1 beta gene as the cause of MSUD and the novel mutation of the invariant G at the 5' splice site which results in two alternatively spliced mRNA due to the skipping of the preceding exon as well as both preceding and following exon.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1994

Ionizing radiation induces apoptotic cell death in human TcR-gamma/delta+ T and natural killer cells without detectable p53 protein.

The p53 tumor suppressor gene has been shown to be involved in programmed cell death, apoptosis, in murine immature thymocytes after treatment with ionizing radiation. Ionizing radiation also induces apoptosis in peripheral mature lymphocytes. In this work, we investigated the p53 participation in radiation-induced apoptosis in human peripheral blood lymphocytes (PBL) subpopulations. Exposure to gamma-irradiation resulted in an appreciable induction of apoptotic cell death in TcR-alpha/beta+ (CD4+ and CD8+) T cells, TcR-gamma/delta+ T cells, B cells and natural killer (NK) cells, as assessed by DNA fragmentation as well as the morphological characteristics. Importantly, it was found that there was a marked difference among PBL subpopulations as regards the induction of p53 protein by gamma-irradiation. Similar to previous observations for murine thymocytes, p53 induction in TcR-alpha/beta+ T cells and B cells after gamma-irradiation was evident by Western blot analysis. Radiation-induced apoptosis in TcR-alpha/beta+ T cells and B cells was efficiently inhibited by cycloheximide, indicating the requirement of de novo protein synthesis, including p53 protein, for radiation-induced apoptosis in both subpopulations. In marked contrast, no identifiable levels of p53 protein were induced in either TcR-gamma/delta+ T or NK cells after gamma-irradiation. In addition, it was demonstrated that radiation-induced cell death in TcR-gamma/delta+ T and NK cells could be prevented by interleukin-2, but not by cycloheximide. These results imply that radiation-induced lymphocytic apoptosis can be mediated by p53-dependent or -independent mechanisms.

Adult↗