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Biomedical subjects

N Nakamura

Publications and source records attributed to N Nakamura.

At least 145 records · Page 8Linked to original sources
PubMed2001

Differentiation of mouse hepatitis viruses in animal facilities in Japan by use of nucleotide analysis of the nucleocapsid gene.

The nucleotide sequences of the coding region of the nucleocapsid (N) gene of 12 mouse hepatitis virus (MHV) strains recently found in animal facilities in Japan were analyzed. Nucleotide sequencing was performed directly on polymerase chain reaction (PCR) products amplified by reverse transcription (RT) and polymerase chain reaction (RT-PCR) analysis from fecal samples or isolated viruses. Phylogenetic analysis of these MHV strains along with those reported previously indicated that sequence analysis of the N gene was a useful tool for differentiation of MHV strains,although most MHV strains in Japanese facilities were phylogenetically close. Results suggested that interchange of mice infected with MHV among facilities provided opportunities of introduction of MHV into otherwise MHV-free facilities and that the source of MHV infection could be traced by use of nucleotide analysis of the N gene.

Animals↗
PubMed2000

Pathogenesis of vascular dementia in stroke-prone spontaneously hypertensive rats.

Stroke-prone spontaneously hypertensive rats (SHRSP) are the best model for essential hypertension and stroke. In this study, one investigated whether SHRSP might be a useful animal model for vascular dementia. An impairment of learning-memory function was found in SHRSP. A disturbance in circadian rhythm after stroke in SHRSP was clarified. Desynchronization of light and dark alternation cycles and abnormal rhythm were also demonstrated. These observations point to the possibility that the decreased passive avoidance response observed in SHRSP might be similar to the phenomenon of memory impairment in patients with vascular dementia. The behavioral changes in ambulation in SHRSP, including the desynchronization between light and dark alternation cycles and the abnormal rhythm before death, might correspond to the behavioral changes associated with the delirium-state observed in patients with dementia. Cerebral cortex levels of acetylcholine and choline in SHRSP decreased significantly as compared with the Wistar Kyoto rats (WKY) control group. Hippocampal levels of acetylcholine and choline in SHRSP decreased significantly as compared with those in WKY. Moreover, a correlation between passive avoidance response latency and hippocampal acetylcholine levels was observed. These findings suggest that decreased acetylcholine levels in both the cerebral cortex and the hippocampus may be related to the impairment of learning-memory function and abnormal behavior. In SHRSP, increases in blood viscosity, hematocrit and fibrinogen might produce the formation of thrombus and induce cerebral infarction. Some histopathological findings caused by cerebrovascular disorder in human brain very similar to those observed in the SHRSP brain. On the other hand, so called 'senile changes' were detected only in the human case, and not observed in the SHRSP.

Acetylcholine↗
PubMed2000

Direct binding of hydroxylamine to the heme iron of Arthromyces ramosus peroxidase. Substrate analogue that inhibits compound I formation in a competetive manner.

The interaction of hydroxylamine (HA) with Arthromyces ramosus peroxidase (ARP) was investigated by kinetic, spectroscopic, and x-ray crystallographic techniques. HA inhibited the reaction of native ARP with H(2)O(2) in a competitive manner. Electron absorption and resonance Raman spectroscopic studies indicated that pentacoordinate high spin species of native ARP are converted to hexacoordinate low spin species upon the addition of HA, strongly suggesting the occurrence of a direct interaction of HA with ARP heme iron. Kinetic analysis exhibited that the apparent dissociation constant is 6.2 mm at pH 7.0 and that only one HA molecule likely binds to the vicinity of the heme. pH dependence of HA binding suggested that the nitrogen atom of HA could be involved in the interaction with the heme iron. X-ray crystallographic analysis of ARP in complex with HA at 2.0 A resolution revealed that the electron density ascribed to HA is located in the distal pocket between the heme iron and the distal His(56). HA seems to directly interact with the heme iron but is too far away to interact with Arg(52). In HA, it is likely that the nitrogen atom is coordinated to the heme iron and that hydroxyl group is hydrogen bonded to the distal His(56).

Binding Sites↗
PubMed2000

Complex structure and regulation of expression of the rat gene for inward rectifier potassium channel Kir7.1.

Genomic organization of the rat inward rectifier K(+) channel Kir7.1 was determined in an attempt to clarify how multiple species of its mRNA are generated in a tissue-specific manner and how its expression is regulated. The rat Kir7.1 gene spans >40 kilobases (kb) and consists of eight exons; the first four exons encode the 5'-untranslated region that is unusually long ( approximately 3 kb). The coding region is located in exons 5 and 6. In the testis, exon 4 is processed as four exons (4a-4d), whereas it is recognized as a single exon in the small intestine. The three major species of rat Kir7.1 mRNA (1.4, 2.2, and 3.2 kb) were found to arise from alternative usage of the two promoters and polyadenylation signals and by alternative splicing of the 5'-noncoding exons. The splicing pattern of the 5'-noncoding exons is quite complex and highly tissue-specific, suggesting that complex mechanisms may operate to regulate the Kir7.1 expression. Deletion and mutational analysis of the promoter activity indicated that the rat Kir7.1 gene is regulated by cAMP through a CCAAT element. The cAMP induction was also demonstrated using the rat follicular cell line FRTL-5 endogenously expressing Kir7.1.

5' Untranslated Regions↗
PubMed2000

Alteration of programmed cell death and gene expression by 5-bromodeoxyuridine during limb development in mice.

Some chemicals are known to induce limb malformations in mice. The occurrence of limb abnormality induced by chemical reagents is due to changes in the programmed cell death (PCD). 5-Bromodeoxyuridine (BrdU) is known as a potent teratogen and has been reported to induce polydactyly and other limb malformations in rodents (DiPaolo, Science 145, 501-503, 1964). Here, we undertook the morphological and genetic analyses of fetuses with limb malformations in BrdU-treated mice, in order to investigate an alteration of gene expression that resembles that of mutant mice with similar limb malformations. The fetuses of the BrdU-treated mice exhibited preaxial polydactyly and preaxial triphalangism of the hindlimb at a high incidence. Our observations showed that the PCD in the preaxial necrotic zone was found to be delayed or absent on day 11 of pregnancy. Histological analyses of these fetuses showed that the preaxial apical ectodermal ridge (AER) of the hindlimb was hyperplastic and consisted of several irregular layers. In observation of the whole-mount in situ hybridization, we detected the anterior-extended overexpression of Hoxd-11 and Hoxd-13 genes in the mesenchyme cells and the overexpression of Fgf4 and Fgf8 genes in the anterior region of the AER of hindlimbs of BrdU-treated fetuses. Our study shows that the injection of BrdU changed the PCD and gene expression during limb development and induced time-specific limb malformations during fetal development. This examination of the changes of the PCD and gene expression will be useful markers for the investigation of toxicities and teratologieties of other chemicals now present in the world environment.

Abnormalities, Drug-Induced↗
PubMed2000

A Di-leucine signal in the ubiquitin moiety. Possible involvement in ubiquitination-mediated endocytosis.

Some plasma membrane receptors in yeast are known to be internalized and degraded in lysosomes upon ligand-dependent ubiquitination. However, the role of ubiquitination in endocytosis and lysosomal degradation in higher eukaryotes has been controversial. In order to directly assess this question, we investigated the fate of chimeric molecules in which ubiquitin moiety was fused in-frame to the cytoplasmic region of membrane proteins. The chimeric proteins with the wild-type ubiquitin were endocytosed and delivered to lysosomes efficiently. Mutant ubiquitin with lysine-to-arginine substitution could still mediate endocytosis, suggesting that polyubiquitination is not required for the endocytosis. We next searched for the existence of an endocytosis signal(s) in the ubiquitin moiety, and identified a di-leucine signal, Leu(43)-Ile(44). The Leu(43)-Ile(44) sequence mediated endocytosis and lysosomal sorting in a Leu(43)-dependent manner. These results suggest that the di-leucine signal in ubiquitin can be involved in ubiquitination-mediated endocytosis and lysosomal targeting of membrane proteins.

Amino Acid Sequence↗
PubMed2000

Phosphorylation of collapsin response mediator protein-2 by Rho-kinase. Evidence for two separate signaling pathways for growth cone collapse.

We previously identified Rho-associated protein kinase (Rho-kinase) as a specific effector of Rho. In this study, we identified collapsin response mediator protein-2 (CRMP-2), as a novel Rho-kinase substrate in the brain. CRMP-2 is a neuronal protein whose expression is up-regulated during development. Rho-kinase phosphorylated CRMP-2 at Thr-555 in vitro. We produced an antibody that specifically recognizes CRMP-2 phosphorylated at Thr-555. Using this antibody, we found that Rho-kinase phosphorylated CRMP-2 downstream of Rho in COS7 cells. Phosphorylation of CRMP-2 was observed in chick dorsal root ganglion neurons during lysophosphatidic acid (LPA)-induced growth cone collapse, whereas the phosphorylation was not detected during semaphorin-3A-induced growth cone collapse. Both LPA-induced CRMP-2 phosphorylation and LPA-induced growth cone collapse were inhibited by Rho-kinase inhibitor HA1077 or Y-32885. LPA-induced growth cone collapse was also blocked by a dominant negative form of Rho-kinase. On the other hand, semaphorin-3A-induced growth cone collapse was not inhibited by a dominant negative form of Rho-kinase. Furthermore, overexpression of a mutant CRMP-2 in which Thr-555 was replaced by Ala significantly inhibited LPA-induced growth cone collapse. These results demonstrate the existence of Rho-kinase-dependent and -independent pathways for growth cone collapse and suggest that CRMP-2 phosphorylation by Rho-kinase is involved in the former pathway.

Amino Acid Sequence↗
PubMed2000

Analysis of the mechanism of discrepant nuclear morphometric results comparing preoperative biopsy and prostatectomy specimens.

OBJECTIVES: To explore the mechanism for the differing nuclear morphometric results between needle biopsy and surgical specimens of the prostate. METHODS: In experiment 1, a comparison of mean nuclear area (MNA), volume-weighted mean nuclear volume (MNV), and form factor (FF) for prostatic epithelial cells was performed between preoperative needle biopsy and prostatectomy specimens from 5 patients with benign prostatic hyperplasia (BPH). In experiment 2, a scheduled, sequential ex vivo needle sampling from the enucleated prostates (at 0, 2, 6, and 24 hours after surgical resection) was also performed for 7 patients with BPH. The prostatectomy specimens were left unfixed for 2 hours until the second needle sampling was done. Nuclear morphometric parameters were measured on the needle-sampled as well as on the prostatectomy specimens. RESULTS: MNA, MNV, and FF of BPH cells measured on preoperative biopsy specimens were smaller than those of surgical specimens in all 5 of the cases. The results of nuclear morphometry on the materials obtained by ex vivo needle sampling of prostates before and during fixation revealed that the MNA, MNV, and FF for BPH cells of 0-hour specimens were significantly smaller than those for needle samples at 2, 6, and 24 hours after surgical resection as well as those for prostatectomy specimens. CONCLUSIONS: The present study provided further evidence that the ischemic damage caused by delayed fixation could result in a substantial change of the nuclear morphology of prostate cells. An immediate start, as well as a rapid completion, of the fixation procedure seems critical for an accurate nuclear morphometry of prostatectomy specimens.

Biopsy, Needle↗
PubMed2000

Evaluation of anti-scratch properties of oxatomide and epinastine in mice.

Anti-scratch effects of oxatomide and epinastine were examined in mice. Scratching behavior and cutaneous reactions were induced in BALB/c, ICR and ddY mice by dinitrofluorobenzene painting, passive cutaneous anaphylaxis and substance P injection, respectively. Although oxatomide and epinastine failed to inhibit scratching behavior in BALB/c mice, they inhibited the cutaneous reaction significantly. The drugs potently inhibited both scratching behavior and cutaneous reaction in ICR mice. They also inhibited scratching behavior and cutaneous reaction in ddY mice, although cetirizine and terfenadine failed to affect them. Histamine did not induce frequent scratching behavior in BALB/c and ddY mice. These results indicate that oxatomide and epinastine inhibit the scratching behavior in ICR mice associated with passive cutaneous anaphylaxis mainly through an antagonistic action on histamine H(1) receptors. The results also indicate that these drugs inhibit substance P-induced scratching behavior in ddY mice through an action independent of the antagonistic action on histamine H(1) receptors.

Animals↗
PubMed2000

Induction of aldose reductase in cultured human microvascular endothelial cells by advanced glycation end products.

Accelerated formation and accumulation of advanced glycation end products, as well as increased flux of glucose through polyol pathway, have been implicated in the pathogenesis of diabetic vascular complications. We investigated effects of advanced glycation end products on the levels of aldose reductase mRNA, protein, and activity in human microvascular endothelial cells. When endothelial cells were cultured with highly glycated bovine serum albumin, aldose reductase mRNA in endothelial cells demonstrated concentration-dependent elevation. The increase in aldose reductase mRNA was accompanied by elevated protein expression and enzyme activity. Significant increase in the enzyme expression was also observed when endothelial cells were cultured with serum obtained from diabetic patients with end-stage renal disease. Pretreatment of the endothelial cells with probucol or vitamin E prevented the advanced glycation end products-induced increases in aldose reductase mRNA and protein. Electrophoretic mobility shift assays using the nuclear extracts of the endothelial cells treated with advanced glycation end products showed enhancement of specific DNA binding activity for AP-1 consensus sequence. These results indicate that accelerated formation of advanced glycation end products in vivo may elicit activation of the polyol pathway, possibly via augmented oxidative stress, and amplify endothelial cell damage leading to diabetic microvascular dysfunction.

Aldehyde Reductase↗
PubMed2000

Use of anion exchange resin-packed capillary column for rapid detection of anti-double-stranded DNA antibody in systemic lupus erythematosus serum.

An anti-double-stranded (ds) DNA antibodies in serum-detection system was developed based on flow immunoassay with packed-capillary column. Alkaline phosphatase conjugated DNA (ALP-DNA) and anti-dsDNA antibody were separated from unreacted ALP-DNA on the basis of the difference in isoelectric point. A linear anti-dsDNA antibody dose-response curve was obtained between luminescence intensity and concentration of anti-dsDNA antibody in the range 25-200 IU/mL. This simple technique permits the assay of anti-dsDNA autoimmune antibodies within 7 minutes. These results give reduced detection times compared with those previously reported (Lim et al., 1999) through the use of capillary columns.

Alkaline Phosphatase↗
PubMed2000

Heparin cofactor II inhibits thrombus formation in a rat thrombosis model.

Heparin cofactor II is postulated to be an extravascular thrombin inhibitor that is physiologically stimulated by dermatan sulfate. However, the role of heparin cofactor II has not yet been clearly demonstrated in vivo. In this study, we estimated the antithrombotic effect of heparin cofactor II administered exogenously in a rat model of thrombosis. Thrombus was induced in the rat femoral artery by endothelial damage due to the photochemical reaction between systemically injected rose bengal and transillumination with green light. Pretreatment with heparin cofactor II significantly prolonged the time required to occlude the femoral artery (occlusion time) in a dose-dependent manner. At an effective dose in this thrombosis model, heparin cofactor II did not prolong the activated partial thromboplastin time and the prothrombin time in normal rats. Argatroban, a selective synthetic thrombin inhibitor, significantly prolonged the occlusion time. However, argatroban also prolonged the activated partial thromboplastin time and prothrombin time at an effective dose. These results suggest that the administration of heparin cofactor II in vivo effectively inhibited thrombus formation on the vessel walls whose endothelium is damaged without a prolongation of the coagulation time while heparin cofactor II may also inhibit the thrombin activity in the subendothelial tissue in vivo.

Animals↗
PubMed2000

Secretory production of recombinant human chymase as an active form in Pichia pastoris.

We succeeded in expressing in a Pichia pastoris (P. pastoris) host a cDNA encoding a mature human chymase (h-chymase) which was secreted directly into the culture medium. Recombinant human heart chymase (rh-chymase) was purified from the culture medium via a single one-step heparin-agarose column chromatography tracing, using succinyl-Ala-Ala-Pro-Phe-para-nitroanilide (Suc-AAPF-pNA) hydrolysing activity. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the rh-chymase showed a diffused protein band with molecular weight of 32-37 kDa. After deglycosylation, however, rh-chymase changed to a sharp protein band with molecular weight 28 kDa, which is equal in size to deglycosylated h-chymase. The rh-chymase had an activity to convert one of the natural substrates, angiotensin I, to angiotensin II. Double reciprocal plot analysis revealed that the K(m) value ofrh-chymase against Suc-AAPF-pNA was approximately 5.1 mM, which is close to that of purified h-chymase.

Angiotensin I↗
PubMed2000

Syntaxin 7 mediates endocytic trafficking to late endosomes.

The lysosome functions are ensured by accurate membrane trafficking in the cell. We found that mouse syntaxin 7 could complement yeast vam3 and pep12 mutants defective in docking/fusion to vacuolar and prevacuolar membranes, respectively. Immunohistochemical studies showed that syntaxin 7 is localized to late endosomes, but not to early endosomes. Induced expression of mutant syntaxin 7 blocked endocytic transport from early to late endosomes but did not block the transport of cathepsin D and lamp-2 from the trans-Golgi network to lysosomes. Thus, syntaxin 7 mediates the endocytic trafficking from early endosomes to late endosomes and lysosomes. These results also suggest that the biosynthetic pathway utilizes a different machinery from that of the endocytic pathway in the docking/fusion to late endosomes.

Animals↗