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Biomedical subjects

N Nakata

Publications and source records attributed to N Nakata.

At least 37 records · Page 2Linked to original sources
PubMed1996

Identification and characterization of ispA, a Shigella flexneri chromosomal gene essential for normal in vivo cell division and intracellular spreading.

The virulent phenotype of Shigella requires loci on the chromosome as well as on the large virulence plasmid, and is regulated via a complex web of interactions amongst various chromosomal and large plasmid genes. To further investigate the role of chromosomal loci in virulence, we performed random Tn10 mutagenesis in Shigella flexneri YSH6000T, and isolated an avirulent mutant (V3404) incapable of spreading throughout an epithelial cell monolayer. Although V3404 initially spread intercellularly at the same rate as the wild-type, it gradually slowed down and ceased spreading as a result of increasing defects in cell division, leading to the formation of long filamentous bacteria lacking septa, trapped within cells. In addition, the mutation affected the ability of V3404 to polymerize actin, a prerequisite for intra- and inter-cellular spreading ability. Sequencing of Tn10-flanking DNA revealed that the mutated gene, designated ispA (intracellular septation), was equivalent to a previously sequenced but uncharacterised gene of Escherichia coli located between trp and tonB. Using E. coli sequence data, we cloned the ispA gene from the YSH6000T chromosome and found that it complemented the V3404 mutation. Nucleotide sequencing and in vitro expression experiments revealed that ispA coded for a small (21 kDa), very hydrophobic protein. These results thus show that ispA is an essential virulence gene affecting several functions of the virulence process.

Actins↗
PubMed1995

Facilitated recovery from postischemic suppression of protein synthesis in the gerbil brain with ischemic tolerance.

Preconditioning of the gerbil brain with a 2-min period of sublethal ischemia followed by 4 days of reperfusion protects against neuronal damage following a subsequent 3-min period of ischemia, which normally destroys pyramidal neurons in the CA1 region of the hippocampus. To clarify the role of protein synthesis in this ischemic tolerance phenomenon, we performed an autoradiographic analysis with [14C]leucine at 4 h, 24 h, and 48 h after 3 min of ischemia with and without preconditioning. General protein synthesis in the CA1 region was severely suppressed after 4 h in both groups. The protein synthesis in CA1 partially recovered after 24 h and fully recovered after 48 h in animals with preconditioning, but never recovered in animals without preconditioning. Protein synthesis in the neocortex and the striatum was suppressed in the early reperfusion periods only in animals without preconditioning. The results show that the ischemic tolerance is closely related to the facilitated recovery from suppressed protein synthesis in the brain after ischemia.

Adaptation, Physiological↗
PubMed1995

[The ABO blood groups-dependent reference intervals for serum alkaline phosphatase isozymes and total activity in individuals 20-39 years of age].

Reference intervals of the activity of serum alkaline phosphatase isozymes (AP-IZ: high molecular-, liver-, bone- and intestinal-types) were determined according to the ABO (blood type) system in 200 healthy subjects aged 20-39 years. AP activity was determined according to the JSCC method. AP-IZ was stained by the formazan method after isolation by TITANIII-Lipo plate electrophoresis. For the electrophoresis, treated serum with neuraminidase and untreated one were concomitantly used for detecting liver AP and bone AP respectively. As a result of comparison of mean AP-IZ activity among the groups divided according to the ABO system, total AP, intestinal AP and liver AP activities in the type B and O persons were significantly higher than in the type A and AB persons. It is well known that the activities of total AP and intestinal AP in type B and O persons are higher than in type A and AB persons, but there have been no reports showing that the activity of liver AP in type B and O persons is higher than in type A and AB persons. Furthermore, in the type B and O persons there was a low correlation (r = 0.195, p < 0.05) between the activities of liver AP and intestinal AP. The present assessment included subjects in the age group (20-39 years) considered not to show fluctuations in the activity of bone AP, which is influenced by age. The above findings should be investigated in regard to other age groups.

ABO Blood-Group System↗
PubMed1994

Prostaglandin E2 protects cultured cortical neurons against N-methyl-D-aspartate receptor-mediated glutamate cytotoxicity.

The effects of prostaglandin (PG) E2 on glutamate-induced cytotoxicity were examined using primary cultures of rat cortical neurons. The cell viability was significantly reduced when cultures were briefly exposed to either glutamate or N-methyl-D-aspartate (NMDA) then incubated with normal medium for 1 h. Similar cytotoxicity was observed with the brief application of ionomycin, a calcium ionophore, and S-nitrosocysteine, a nitric oxide (NO)-generating agent. PGE2 at concentrations of 0.01-1 microM dose-dependently ameliorated the glutamate-induced cytotoxicity. PGE1, butaprost, an EP2 receptor agonist, and 8-bromo-cAMP were also effective in protecting cultures against glutamate cytotoxicity. By contrast, neither 17-phenyl-omega-trinor-PGE2, an EP1 receptor agonist, nor M&B 28767, an EP3 receptor agonist, affected glutamate-induced cytotoxicity. NMDA-induced cytotoxicity was ameliorated by PGE2, butaprost, MK-801, N-omega-nitro-L-arginine, a NO synthase inhibitor, and hemoglobin, which binds NO. These agents excluding MK-801 ameliorated the ionomycin-induced cytotoxicity. The cytotoxicity induced by S-nitrosocysteine was prevented only by hemoglobin but not by the other agents including PGE2. These findings indicate that PGE2 protects cultured cortical neurons against NMDA receptor-mediated glutamate neurotoxicity via EP2 receptors. EP2 receptor stimulation may suppress a step in NO formation triggered by Ca(2+)-influx through NMDA receptors.

Animals↗
PubMed1994

Molecular cloning and the COOH-terminal processing of gp64, a putative cell-cell adhesion protein of the cellular slime mold Polysphondylium pallidum.

The cellular slime mold Polysphondylium pallidum expresses a cell surface glycoprotein (referred to as gp64), which seems to be implicated in cell-cell adhesion. We identified a near full-length gp64 cDNA (1,104 base) upon screening a P. pallidum lambda gt11 library with a monoclonal antibody. The open reading frame encodes a protein of 320 amino acids with a molecular mass of 32,752 Da; the protein includes hydrophobic segments at both a NH2- and a COOH-terminal ends. By an Edman degradation analysis of S-pyridylethylated gp64 and its COOH-terminal peptide, it was found that the NH2- and COOH-terminal segments are both removed from the precursor protein of gp64. The COOH-terminal segment was isolated from a lysyl endopeptidase digest of gp64 by an affinity method. The COOH-terminal segment was identified at positions 266-279 in the primary sequence deduced from the cDNA sequence. The mature gp64 consisted of 279 amino acid residues and extremely rich in Cys residues (36 Cys/279 amino acids = 12.9%). Although there was already maximal accumulation of gp64 mRNA in vegetative cells, the protein reached a maximal level during aggregation stage, decreased, and then leveled off through the developmental cycle.

Amino Acid Sequence↗
PubMed1994

Ischemic tolerance and extracellular amino acid concentrations in gerbil hippocampus measured by intracerebral microdialysis.

Preconditioning of the brain with sublethal ischemia induces tolerance to subsequent longer periods of ischemia. To elucidate the role of excitatory and inhibitory amino acids in the induction of ischemic tolerance, we measured the extracellular concentrations of the amino acids in the gerbil hippocampus with intracerebral microdialysis. Mongolian gerbils were subjected to 3 min of forebrain ischemia 4 days after preconditioning with 2 min of ischemia or sham operation. Microdialysis probes were implanted into the hippocampus before the second ischemia and the amino acid concentrations in the dialysates were measured with HPLC. During and immediately after 3 min of ischemia without preconditioning, the concentrations of glutamate, glycine, gamma-aminobutyric acid, and taurine, but not glutamine, increased significantly. The increased amino acid levels rapidly returned to baseline after reperfusion. Preconditioning of the brain did not alter the amount of any amino acid released during and after the second ischemia. The excitotoxic index also unchanged in the preconditioned hippocampus. Thus, the results clearly show that ischemic tolerance is not induced through the alteration of the amounts of excitatory and inhibitory amino acids released during subsequent ischemia.

Amino Acids↗
PubMed1994

vacC, a virulence-associated chromosomal locus of Shigella flexneri, is homologous to tgt, a gene encoding tRNA-guanine transglycosylase (Tgt) of Escherichia coli K-12.

The genetic determinants required for invasion of epithelial cells by Shigella flexneri and for the subsequent bacterial spreading are encoded by the large virulence plasmid. Expression of the virulence genes is under the control of various genes on the large plasmid as well as on the chromosome. We previously identified one of the virulence-associated loci near phoBR in the NotI-C fragment of the chromosome of S. flexneri 2a YSH6000 and designated the locus vacC. The vacC mutant showed decreased levels of IpaC, and IpaD proteins as well as transcription of ipa, an operon essential for bacterial invasion (N. Okada, C. Sasakawa, T. Tobe, M. Yamada, S. Nagai, K. A. Talukder, K. Komatsu, S. Kanegasaki, and M. Yoshikawa, Mol. Microbiol. 5:187-195, 1991). To elucidate the molecular nature of the vacC locus, we cloned the vacC region from YSH6000 on a 1.8-kb SalI-BamHI DNA fragment. The nucleotide sequence of the 1,822-bp vacC clone was highly (> 98%) homologous to the tgt region of Escherichia coli K-12, which is located at 9.3 min on the linkage map. Complementation tests indicated that the vacC function was encoded by an open reading frame expressing a 42.5-kDa protein, which corresponded to the tgt gene of E. coli K-12, coding for tRNA-guanine transglycosylase (Tgt) (K. Reuter, R. Slany, F. Ullrich, and H. Kersten, J. Bacteriol. 173:2256-2264, 1991). The cloned tgt gene from E. coli K-12 restored the virulence phenotype to the vacC mutant of YSH6000. Characterization of the vacC mutant indicated that levels of VirG, a protein essential for bacterial spreading, and VirF, the positive regulator for the expression of the virG and ipaBCD operons, decreased significantly compared with those of the wild type. Similar phenotypic changes occurred in vacC mutants constructed by insertion of a neomycin resistance gene in shigellae and enteroinvasive E. coli strains, consistent with the hypothesis that the vacC (tgt) gene contributes to the pathogenicity of Shigella flexneri.

Amino Acid Sequence↗
PubMed1994

[Antiemetic efficacy of the repeated use of granisetron in multiple cycles of anti-cancer chemotherapy].

Twenty-three patients with gynecological cancer who were treated with 85 cycles of cytotoxic chemotherapy containing platinum received intravenous granisetron repeatedly. Granisetron (3 mg/body) was drip-infused twice for each cycle at a 24-hour interval. The antiemetic efficacy was evaluated and compared for each day and each cycle, and analysed using the chi-square and H tests. There were no significant differences between the first cycle and the subsequent second through fifth cycles in the severity of nausea and the frequency of vomiting. The latter tended to increase in the second day of each cycle. These results indicated that granisetron does not decrease in antiemetic efficacy by repeated administration during multiple cycles of anti-cancer chemotherapy.

Carboplatin↗
PubMed1994

[Serum IgD concentrations in normal individuals 20-39 years of age].

Immunoglobulin D(IgD) concentration in serum of normal individuals varies widely, and the reference interval for the concentrations remains to be defined. In the present study, IgD concentrations were determined in serum from 637 normal individuals 20-39 years of age by the latex nephelometric immunoassay. The IgD concentrations ranged from < 0.20 to 71.5mg per deciliter, and the median and the 95% range as reference intervals were 1.99 and < 0.20 to 17.3mg/dl, respectively. There was difference of serum IgD concentration between female 20-29 years of age and female 30-39 years of age (p < 0.05), but not on sex. The correlation between serum IgD concentration and serum IgA concentration was significant (p < 0.001).

Adult↗
PubMed1993

An immunohistochemical study of copper/zinc superoxide dismutase and manganese superoxide dismutase in rat hippocampus after transient cerebral ischemia.

We investigated the changes of copper/zinc superoxide dismutase (CuZn-SOD) and manganese superoxide dismutase (Mn-SOD) in the rat hippocampus after 10 min of cerebral ischemia induced by 4-vessel occlusion. The rats were allowed to survive for 4 h, 1 day, 3 days, and 7 days after ischemia. The distribution of SODs were determined by immunohistochemical staining with antibodies against rat CuZn-SOD and Mn-SOD. CA1 pyramidal neurons and granule cells of the dentate gyrus showed intense CuZn-SOD immunoreactivity, whereas CA3 and CA4 neurons showed weaker immunostaining than CA1 neurons in normal animals. The immunoreactivity was reduced by 4 h after ischemia in CA1, CA3, and CA4 neurons when no histological damage was observed. Mn-SOD immunostaining revealed more intense immunoreactivity in CA3 pyramidal neurons than in CA1 neurons in normal animals. Interneurons in the CA1 and CA3 regions and the dentate hilus also showed high Mn-SOD immunostaining. Although CA1 neurons lost Mn-SOD immunoreactivity by 1 day after ischemia, CA3 neurons and interneurons retained the immunoreactivity and preserved intact cell contour after ischemia. In addition, reactive glial cells, which were differentiated by immunocytochemical staining against glial fibrillary acidic protein for reactive astrocytes and histochemical staining for reactive microglial cells, were intensely stained for CuZn-SOD and Mn-SOD after ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Immunohistochemical localization of ubiquitin in gerbil hippocampus with induced tolerance to ischemia.

Using immunohistochemistry, we visualized the localization of ubiquitin in the gerbil hippocampus following 3 min of ischemia with or without pretreatment with 2 min of sublethal ischemia and 3 days of reperfusion. Ubiquitin immunoreactivity in the hippocampus disappeared 4 h after 3 min of ischemia both with and without pretreatment. The immunoreactivity in the CA3 and the dentate gyrus recovered by 24 h, but never recovered in the CA1, where delayed neuronal death takes place, without pretreatment. However, the pretreatment, which protects against CA1 neuronal damage, led to recovery of ubiquitin immunoreactivity in the CA1 by 48 h. Thus, recovery of ubiquitin may be a prerequisite to neuronal survival after ischemia and the role of ubiquitin in ischemic tolerance was suggested.

Animals↗
PubMed1993

Immunohistochemical visualization of heat shock protein-70 in the gerbil hippocampus following repeated brief cerebral ischemia.

Recent experiments have shown that neuronal damage following repeated cerebral ischemic insults is more extensive than the damage after a single equivalent period of ischemia. To clarify the mechanism of this cumulative neuronal damage after repeated ischemia, we visualized the localization of heat shock protein-70 (HSP70), a marker of neuronal stress, with immunohistochemistry using a monoclonal antibody. Mongolian gerbils were subjected to three 2-min forebrain ischemic insults spaced at 1-h intervals and to a single 6-min period of ischemia. The animals were killed 24 and 48 h after ischemia. Hippocampal CA1 pyramidal neurons, which are destined to die, showed no HSP70 staining 24 and 48 h after three 2-min ischemic insults, but showed a mild to moderate staining after 6 min of ischemia, suggesting more severe damage after repeated ischemia. CA3 neurons, which are resistant to ischemia, were intensely stained with HSP70 antibody following 6 min of ischemia but was stained only slightly after three 2-min ischemic insults, showing less stress after repeated ischemia. Thus, thresholds for cell damage are obviously different among different cell populations within the hippocampus; the different neuronal populations respond differently to single and repetitive ischemia. The result suggests that cumulative neuronal damage after repeated sublethal ischemic insults is produced by increased susceptibility to subsequent insults when ischemic stress reaches certain thresholds that are different among different neuronal populations.

Animals↗
PubMed1993

Structural study of the sugar chains of CD36 purified from bovine mammary epithelial cells: occurrence of novel hybrid-type sugar chains containing the Neu5Ac alpha 2-->6GalNAc beta 1-->4GlcNAc and the Man alpha 1-->2Man alpha 1-->3Man alpha 1-->6Man groups.

CD36 is a glycoprotein included in the bovine milk fat globule membrane derived from mammary secretory epithelial cells during lactation. Asparagine-linked sugar chains were quantitatively released from CD36 as oligosaccharides by hydrazinolysis. These sugar chains were converted to radioactive oligosaccharides by reduction with NaB3H4 and separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by sialidase digestion, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were fractionated by Bio-Gel P-4 column chromatography in combination with serial chromatography on immobilized lectin columns including a Wistaria floribunda agglutinin (WFA)-agarose column. WFA is known to bind oligosaccharides terminating with either an alpha- or beta-N-acetylgalactosamine residue. Structural studies of oligosaccharides in each fraction by sequential exoglycosidase digestion as well as methylation analysis revealed that CD36 contains high mannose-type, hybrid-type, and bi, tri-, and tetraantennary complex-type sugar chains. A portion of the hybrid-type and the complex-type sugar chains which bound to a WFA-agarose column (28% of all oligosaccharides) contained the GalNAc beta 1-->4GlcNAc group(s) instead of the Gal beta 1-->4GlcNAc group(s) in their outer chain moieties. Like oligosaccharides found in human luteinizing hormone [Weisshaar, G., Hiyama, J., Renwick, A. G., & Nimtz, M. (1991) Eur. J. Biochem. 195, 257-268], some of the GalNAc beta 1-->4GlcNAc groups found in the CD36 oligosaccharides were sialylated as the Neu5Ac alpha 2-->6GalNAc group. Furthermore, most of the hybrid-type sugar chains of CD36 with the Gal/GalNAc beta 1-->4GlcNAc beta 1-->2 outer chain on their Man alpha 1-->3 arm contained an unusual Man alpha 1-->2Man alpha 1-->3 group on their Man alpha 1-->6 arm.

Animals↗
PubMed1993

Tolerance and cross-tolerance to the antinociceptive effects of [D-Arg2]-dermorphin tetrapeptide analogue and morphine.

Rats were given repeated subcutaneous injections of [D-Arg2, Sar4]-dermorphin (1-4) [DAS-DER-(1-4)] and/or morphine over a period of 4 or 7 days. Antinociception was determined at 90 min for DAS-DER (1-4) and 30 min for morphine after each morning injection (9:00 a.m.), using the tail-flick and digit pinching tests in rats. Subcutaneous administration of DAS-DER (1-4) and morphine produced the development of antinociceptive tolerance, respectively. A marked tolerance to DAS-DER (1-4) and morphine was seen in rats made tolerant to morphine. However, administration of morphine produced no significant decrement in the antinociceptive activity in rats made tolerant to DAS-DER (1-4). These results suggest that the site of action of DAS-DER (1-4) may be more limited than that of morphine in the nociceptive pathways, for lack of its antinociceptive efficacy in morphine-tolerant rats.

Amino Acid Sequence↗
PubMed1993

Temporal profile of heat shock protein 70 synthesis in ischemic tolerance induced by preconditioning ischemia in rat hippocampus.

We investigated the temporal profile of heat shock protein 70 induction in the rat hippocampus using immunohistochemistry to clarify the mechanism of ischemic tolerance following preconditioning with sublethal ischemia. Although a 6-min period of forebrain ischemia produced severe neuronal damage to the hippocampal CA1 subfield, preconditioning with 3 min of ischemia followed by three days of reperfusion protected against the CA1 neuronal damage after 6 min of ischemia. Immunohistochemical staining against heat shock protein 70 showed that the protein is induced in CA1 pyramidal cells one, three and seven days after 3 min of ischemia, the immunostaining being most intense after three days. Heat shock protein synthesis was observed in CA1, CA3 and dentate hilar neurons one and three days after 6 min of ischemia, both with and without preconditioning. In addition, the heat shock protein was stained in the CA1 2 h and seven days after 6 min of ischemia with preconditioning, but the intensity of staining was relatively weak at these time points. The results suggest that stress response induced by sublethal ischemia protects against ischemic neuronal damage, and that the induced stress response, including heat shock protein 70 synthesis during and immediately after the second ischemic episode, is correlated with the protection because late induction of the heat shock protein did not prevent neuronal death.

Animals↗