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Biomedical subjects

K Miyazaki

Publications and source records attributed to K Miyazaki.

At least 109 records · Page 6Linked to original sources
PubMed2001

[A case of fundus albipunctatus with a retinol dehydrogenase 5 gene mutation in a child].

BACKGROUND: We examined a family with fundus albipunctatus in which mutation of the retinol dehydrogenase 5(RDH 5) gene was suspected to be the cause of this disease. CASE: An 8-year-old girl had diffuse multiple white dots in her fundus except for the macula. She had good central vision. The amplitude of her electroretinogram wave was low, but it recovered after three hours of dark adaptation. Dark adaptometry showed an elevated threshold for rod adaptation. No visual field loss was observed. A homozygous missense mutation was found in exon 5 of the RDH 5 gene that substituted histidine for arginine at codon 280(Arg 280 His). Her mother had a normal fundus but was heterozygous for the same mutation. CONCLUSION: A missense mutation of RDH 5(Arg 280 His) was found in a Japanese family with fundus albipunctatus.

Alcohol Oxidoreductases↗
PubMed2001

Popliteal artery entrapment syndrome. Reconstruction of the transected medial head of the gastrocnemius muscle.

Popliteal artery entrapment syndrome (PAES) is an uncommon cause of arterial insufficiency, especially in younger, athletic patients. Surgical treatment should be performed in all cases of PAES whether the artery is occluded or not. Although transection of the compressing muscle or fascial band is required to obtain good results, controversy exists about reconstruction of the transected muscle or fascial band. We present a case of a young athlete with PAES for whom rapid recovery of the athletic activity was provided by reconstruction of the divided medial head of the gastrocnemius muscle.

Adolescent↗
PubMed2000

Molecular characterization and nuclear localization of rat timeless-like gene product.

Among three period genes (per1, per2, per3) in mammals, only per2 gene was shown to be involved in the core clock mechanism. To elucidate the molecular function of rat PERIOD2 (rPER2), we searched for binding proteins to the PAS domain of rPER2. We isolated a binding protein to this domain and identified it as a TIMELESS-like protein (TLP) on the basis of mass analyses. Then, we isolated a rat TLP cDNA from the rat hypothalamus library. RNA blot analysis and in situ hybridization indicates that rTLP mRNA was expressed in all rat tissues from whole brain, the suprachiasmatic nucleus, eye, lung, heart, liver, kidney, placenta, and testis. When rTLP gene product was expressed in COS-1 cells, nuclear localization of rTLP was detected in 99.6% of transfected cells. These results suggest that the interaction of rPER2 with rTLP may influence the regulation of circadian clock components in nucleus after rPER2 is translocated into the nucleus.

Amino Acid Sequence↗
PubMed2000

Gene expression for dihydropyrimidine dehydrogenase and thymidine phosphorylase influences outcome in epithelial ovarian cancer.

PURPOSE: Dihydropyrimidine dehydrogenase (DPD) is a pyrimidine salvage enzyme responsible for degradation of thymine, which is produced from thymidine by thymidine phosphorylase (TP). Our purpose was to determine whether DPD affects prognosis in patients with epithelial ovarian cancer and how the two enzymes may interact in such effects. PATIENTS AND METHODS: DPD gene expression was analyzed by reverse transcription-polymerase chain reaction in 27 samples from normal ovaries and the 85 epithelial ovarian cancers previously studied with regard to TP gene expression. RESULTS: DPD gene expression was significantly lower in epithelial ovarian cancers than in normal ovaries (P: <.0001), whereas TP gene expression and the ratio of TP to DPD gene expression (TP:DPD) were significantly higher in epithelial ovarian cancer (P: <.0001 for both). In patients with epithelial ovarian cancer, DPD gene expression and the TP:DPD ratio did not significantly correlate with any clinicopathologic factors. Patients with a high TP:DPD ratio (higher than the median) had significantly poorer outcomes than those with lower ratios (P: =.0002). The difference in survival between groups with high and low TP:DPD ratios was greater than the difference between groups with high and low TP gene expression. Multivariate analysis showed the TP:DPD ratio to be the independent prognostic factor (P: =.0002). In tumors with high TP gene expression, low DPD gene expression significantly correlated with poor survival (P: =. 04). CONCLUSION: Downregulation of DPD gene expression may enhance the negative prognostic effect of high TP gene expression in patients with epithelial ovarian cancer. Certain newly available chemotherapeutic choices may take the TP:DPD ratio into consideration.

Adult↗
PubMed2000

Isolation and activity of proteolytic fragment of laminin-5 alpha3 chain.

Laminin-5 (alpha3beta3gamma2) is an important component of epithelial basement membranes. The 190-kDa alpha3 chain undergoes extracellular cleavage within the carboxyl (C) terminus consisting of five globular domains (G1 to G5), producing the mature laminin-5 with the 160-kDa alpha3 chain. To understand the physiological meaning of this processing, we isolated the C-terminal fragments of the alpha3 chain from the conditioned media of two kinds of human cell lines. The amino-terminal sequence of the fragments suggested that the cleavage occurs at Gln(1337)-Asp(1338) in the spacer region between the G3 and G4 domains. The G4-G5 fragment itself did not show significant activity, but it stimulated cell migration in the presence of a low concentration of the mature laminin-5, suggesting its regulatory role in cell migration.

Amino Acid Sequence↗
PubMed2000

Cold adaptation of a mesophilic subtilisin-like protease by laboratory evolution.

Enzymes isolated from organisms native to cold environments generally exhibit higher catalytic efficiency at low temperatures and greater thermosensitivity than their mesophilic counterparts. In an effort to understand the evolutionary process and the molecular basis of cold adaptation, we have used directed evolution to convert a mesophilic subtilisin-like protease from Bacillus sphaericus, SSII, into its psychrophilic counterpart. A single round of random mutagenesis followed by recombination of improved variants yielded a mutant, P3C9, with a catalytic rate constant (k(cat)) at 10 degrees C 6.6 times and a catalytic efficiency (k(cat)/K(M)) 9.6 times that of wild type. Its half-life at 70 degrees C is 3.3 times less than wild type. Although there is a trend toward decreasing stability during the progression from mesophile to psychrophile, there is not a strict correlation between decreasing stability and increasing low temperature activity. A first generation mutant with a >2-fold increase in k(cat) is actually more stable than wild type. This suggests that the ultimate decrease in stability may be due to random drift rather than a physical incompatibility between low temperature activity and high temperature stability. SSII shares 77. 4% identity with the naturally psychrophilic protease subtilisin S41. Although SSII and S41 differ at 85 positions, four amino acid substitutions were sufficient to generate an SSII whose low temperature activity is greater than that of S41. That none of the four are found in S41 indicates that there are multiple routes to cold adaptation.

Adaptation, Physiological↗
PubMed2000

Isolation and characterization of a novel human gene (NESH) which encodes a putative signaling molecule similar to e3B1 protein.

Using a conventional cloning technique, a novel full-length cDNA was isolated and sequenced from a human placental cDNA library. This cDNA consists of 2129 bp and has a predicted open reading frame encoding 366 amino acids. It possesses a Src homology 3 (SH3) motif, proline-rich region, serine-rich region and no catalytic domain, suggesting that it seems to be a signaling protein most similar to e3B1, an eps8 SH3 binding protein. PCR-based mapping with both a monochromosomal hybrid panel and radiation hybrid cell panels placed the gene to human chromosome 17q21.3 near the marker D17S1795.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Structural requirement of carboxyl-terminal globular domains of laminin alpha 3 chain for promotion of rapid cell adhesion and migration by laminin-5.

The basement membrane protein laminin-5, a heterotrimer of laminin alpha3, beta3, and gamma2 chains, potently promotes cellular adhesion and motility. It has been supposed that the carboxyl-terminal globular region of the alpha3 chain consisting of five distinct domains (G1 to G5) is important for its interaction with integrins. To clarify the function of each G domain, we transfected cDNAs for the full-length (wild type (WT)) and five deletion derivatives (DeltaGs) of the alpha3 chain into human fibrosarcoma cell line HT1080, which expressed and secreted the laminin beta3 and gamma2 chains but not the alpha3 chain. The transfectants with the alpha3 chain cDNAs lacking G5 (DeltaG(5)), G4-5 (DeltaG(4-5)), G3-5 (DeltaG(3-5)), and G2-5 (DeltaG(2-5)) secreted laminin-5 variants at levels comparable to that with WT cDNA. However, the transfectant with the cDNA without any G domains (DeltaG(1-5)) secreted little laminin-5, suggesting that the G domains are essential for the efficient assembly and secretion of the heterotrimer alpha3beta3gamma2. The transfectants with WT, DeltaG(5), and DeltaG(4-5) cDNAs survived in serum-free medium longer than those with DeltaG(3-5), DeltaG(2-5), and DeltaG(1-5) cDNAs. The transfectants with WT, DeltaG(5), and DeltaG(4-5) cDNAs secreted apparently the same size of laminin-5, which lacked G4 and G5 due to proteolytic cleavage between G3 and G4, and these laminin-5 forms potently promoted integrin alpha(3)beta(1)-dependent cell adhesion and migration. However, the laminin-5 forms of DeltaG(3-5) and DeltaG(2-5) hardly promoted the cell adhesion and motility. These findings demonstrate that the G3 domain, but not the G4 and G5 domains, of the alpha3 chain is essential for the potent promotion of cell adhesion and motility by laminin-5.

Cell Adhesion↗
PubMed2000

4-(3-Chloro-4-methoxybenzyl)aminophthalazines: synthesis and inhibitory activity toward phosphodiesterase 5.

We synthesized various 4-(3-chloro-4-methoxybenzyl)aminophthalazines substituted at the 1- and 6-positions and evaluated their inhibitory activity toward phosphodiesterase 5 (PDE5) and their vasorelaxant activity in isolated porcine coronary arteries precontracted with prostaglandin F2alpha (10(-5) M). The preferred substituents at the 1-position of the phthalazine were 4-hydroxypiperidino, 4-hydroxymethylpiperidino, 4-(2-hydroxyethyl)piperidino, and 4-oxopiperidino. Among these compounds, [4-(3-chloro-4-methoxybenzyl)amino-1-(4-hydroxy)piperidino]-6-phthala zinecarbonitrile monohydrochloride (13) exhibited potent PDE5 inhibitory activity (IC(50) = 0.56 nM) with >1700-fold high selectivity over other PDE isozymes (PDE1-4). Compound 13 exhibited the most potent vasorelaxant action (EC(50) = 13 nM) in this series of compounds. Compound 13 reduced mean pulmonary arterial pressure by 29.9 +/- 3.1% when administered intravenously at 30 microg/kg to the chronically hypoxic rats and had an apparent oral bioavailability of about 19.5% in rats and was selected for further biological evaluation.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2000

Interaction with heparin and matrix metalloproteinase 2 cleavage expose a cryptic anti-adhesive site of fibronectin.

We recently found that fibronectin (FN) had a functional site [YTIYVIAL sequence in the heparin-binding domain 2 (Hep 2)] that was capable of suppressing the integrin-mediated cell adhesion to extracellular matrix. However, our results also indicated that this anti-adhesive site seemed to be usually buried within the Hep 2 domain structure because of its hydrophobic nature, raising a question as to the physiological significance of the cryptic anti-adhesive activity of FN. The present study demonstrates that the cryptic anti-adhesive activity can be exposed through the physiological processes. A 30-kDa chymotryptic FN fragment derived from Hep 2 domain (Hep 2 fragment), which had no effect on adhesion of MSV-transformed nonproducer 3T3 cell line (KN(7)8) to FN, expressed the anti-adhesive activity after treatment with 6 M urea. Light scattering and circular dichroism measurements showed that the urea treatment induced the conformational change of the Hep 2 fragment from a more compact form to an unfolded one. Incubation of the Hep 2 fragment with heparin also induced similar conformational changes and expression of anti-adhesive activity. Additionally, both the urea and heparin treatments made the Hep 2 fragment and intact FN much more accessible to the polyclonal antibody (alphaIII14A), with a recognition site near the anti-adhesive site of FN. Specific cleavage of either the Hep 2 fragment or intact FN by matrix metalloproteinase 2 (MMP-2) released a 10-kDa fragment with the anti-adhesive activity, which was shown to have the exposed anti-adhesive site on the amino-terminal region. Thus, the cryptic anti-adhesive activity of FN can be expressed upon conformational change and proteolytic cleavage of Hep 2 domain.

3T3 Cells↗
PubMed2000

Structural requirements of heparan sulfate for the binding to the tumor-derived adhesion factor/angiomodulin that induces cord-like structures to ECV-304 human carcinoma cells.

Tumor-derived adhesion factor/angiomodulin (AGM) is accumulated in tumor blood vessels and on the endothelial cell surface (Akaogi, K., Okabe, Y., Sato, J., Nagashima, Y., Yasumitsu, H., Sugahara, K., and Miyazaki, K. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8384-8389). In cell culture, it promotes cell adhesion and morphological changes to form cord-like structures of the human bladder carcinoma cell line ECV-304. The cord formation is prevented by heparin, which inhibits the binding of AGM to ECV-304 cells. This observation suggests that AGM interacts with cell surface heparan sulfate (HS) proteoglycans. In this study, HS glycosaminoglycans and core proteins of integral transmembrane proteoglycans, syndecan-1 and -4, were identified by immunocytochemistry on ECV-304 cells, and the structural requirements for the interaction of HS with AGM were characterized. Inhibition experiments with sulfated polysaccharides and chemically modified heparin derivatives indicated that sulfate groups were essential for both AGM-HS binding and cord-like structure formation and that the rank order of the different sulfate groups in terms of their contribution was N-sulfate > 6-O-sulfate > 2-O-sulfate. The minimum size of heparin, a chemical analog of HS, required for the binding to AGM was a dodecasaccharide as determined by competition experiments using size-defined heparin oligosaccharides. Thus, a specific sulfation pattern in the HS of cell surface syndecans of ECV-304 cells is required for AGM binding and the morphological changes.

Animals↗
PubMed2000

Molecular cloning and characterization of a novel human gene (NESCA) which encodes a putative adapter protein containing SH3.

A full-length cDNA encoding a novel protein was isolated and sequenced from a human placental cDNA library. This cDNA consists of 1990 bp and has a predicted open reading frame encoding 433 amino acids. It possesses an Src homology 3 (SH3) motif, a leucine zipper motif and no catalytic domain, suggesting that it seems to be an adapter protein. PCR-based mapping with both a monochromosomal hybrid panel and radiation hybrid cell panels placed the gene to human chromosome 1q21-22.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Directed evolution study of temperature adaptation in a psychrophilic enzyme.

We have used laboratory evolution methods to enhance the thermostability and activity of the psychrophilic protease subtilisin S41, with the goal of investigating the mechanisms by which this enzyme can adapt to different selection pressures. A combined strategy of random mutagenesis, saturation mutagenesis and in vitro recombination (DNA shuffling) was used to generate mutant libraries, which were screened to identify enzymes that acquired greater thermostability without sacrificing low-temperature activity. The half-life of seven-amino acid substitution variant 3-2G7 at 60 degrees C is approximately 500 times that of wild-type and far surpasses those of homologous mesophilic subtilisins. The dependence of half-life on calcium concentration indicates that enhanced calcium binding is largely responsible for the increased stability. The temperature optimum of the activity of 3-2G7 is shifted upward by approximately 10 degrees C. Unlike natural thermophilic enzymes, however, the activity of 3-2G7 at low temperatures was not compromised. The catalytic efficiency, k(cat)/K(M), was enhanced approximately threefold over a wide temperature range (10 to 60 degrees C). The activation energy for catalysis, determined by the temperature dependence of k(cat)/K(M) in the range 15 to 35 degrees C, is nearly identical to wild-type and close to half that of its highly similar mesophilic homolog, subtilisin SSII, indicating that the evolved S41 enzyme retained its psychrophilic character in spite of its dramatically increased thermostability. These results demonstrate that it is possible to increase activity at low temperatures and stability at high temperatures simultaneously. The fact that enzymes displaying both properties are not found in nature most likely reflects the effects of evolution, rather than any intrinsic physical-chemical limitations on proteins.

Adaptation, Physiological↗
PubMed2000

v-Src suppresses SHPS-1 expression via the Ras-MAP kinase pathway to promote the oncogenic growth of cells.

We investigated the effect of cell transformation by v-src on the expression and tyrosine phosphorylation of SHPS-1, a putative docking protein for SHP-1 and SHP-2. We found that transformation by v-src virtually inhibited the SHPS-1 expression at mRNA level. While nontransforming Src kinases including c-Src, nonmyristoylated forms of v-Src had no inhibitory effect on SHPS-1 expression, transforming Src kinases including wild-type v-Src and chimeric mutant of c-Src bearing v-Src SH3 substantially suppressed the SHPS-1 expression. In cells expressing temperature sensitive mutant of v-Src, suppression of the SHPS-1 expression was temperature-dependent. In contrast, tyrosine phosphorylation of SHPS-1 was rather activated in cells expressing c-Src or nonmyristoylated forms of v-Src. SHPS-1 expression in SR3Y1 was restored by treatment with herbimycin A, a potent inhibitor of tyrosine kinase, or by the expression of dominant negative form of Ras. Contrary, active form of Mekl markedly suppressed SHPS-1 expression. Finally, overexpression of SHPS-1 in SR3Y1 led to the drastic reduction of anchorage independent growth of the cells. Taken together, our results suggest that the suppression of SHPS-1 expression is a pivotal event for cell transformation by v-src, and the Ras-MAP kinase cascade plays a critical role in the suppression.

3T3 Cells↗