[Epidemiologic study of muscular dystrophy at school age in Gifu city].
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Adolescent↗
Biomedical subjects
S Nishimura
Publications and source records attributed to S Nishimura.
[Mechanism of action of azatyrosine: recent process].
Azatyrosine is known to convert ras, raf or c-erbB-2-transformed NIH3T3 cells to a normal phenotype. We attempted to identify the signal-transduction process triggered by oncogenic c-ErbB-2 that was inhibited by azatyrosine. Azatyrosine did not suppress activation of Ras induced by introduction of c-ErbB-2. However, it inhibited increases in phosphorylation of c-Raf-1 induced by oncogenic c-ErbB-2. Furthermore, azatyrosine inhibited activation of the 12-O-tetradecanoylphorbol-13-acetate (TPA) response element in response to stimulation by oncogenic c-ErbB-2. These results suggest that this agent acts downstream of Ras in signal transduction from oncogenic c-ErbB-2 to nuclear factors. Moreover, we found that azatyrosine was incorporated into proteins instead of tyrosine. The simultaneous presence of a high concentration of tyrosine inhibited the conversion to a normal phenotype of transformed cells by azatyrosine. These results strongly suggest that incorporation of azatyrosine into proteins might convert the transformed cells in to cells with a normal phenotype.
3T3 Cells↗
Burkitt's type acute lymphoblastic transformation associated with t(8;14) in a case of B cell chronic lymphocytic leukemia.
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Aged↗
Expression of alpha vbeta3 and alpha vbeta8 integrins during oligodendrocyte precursor differentiation in the presence and absence of axons.
We have shown previously that switching of the alpha v-associated beta1 and beta5 integrin subunits during differentiation of myelin-forming oligodendrocytes may regulate important aspects of cell behaviour such as migration (Milner et al., 1996: J Neurosci 16:7240-7252). In this study we have examined the developmental regulation of other alpha v-associated beta subunits in oligodendroglial cell cultures and also the control of their expression by neurons, using xenocultures to distinguish glial and neuronal integrins. We have found that oligodendroglia express alpha vbeta8 in addition to the previously-described alpha vbeta1, alpha vbeta3, and alpha vbeta5. Beta8 and beta3 together comprise the 80kD band seen in alpha v immunoprecipitations that represents the most abundant alpha v-associated beta subunit and show reciprocal patterns of expression during development. Alpha vbeta8 is expressed at high levels on oligodendrocyte precursors and differentiated oligodendrocytes but diminishes during the intermediate stages of differentiation. Alpha vbeta3, in contrast, shows an opposite pattern of expression, with the highest levels seen at the intermediate stages of differentiation and little expression on either oligodendrocyte precursors and differentiated oligodendrocytes. The expression of alpha vbeta3 is not altered by coculture with neurons, unlike that of alpha vbeta8, in which the decrease seen at the intermediate stages of differentiation is less marked in the presence of neurones. Our results confirm that switching of alpha v-associated beta subunits is an important feature of oligodendrocyte differentiation and suggest that alpha vbeta8 and alpha vbeta3 have distinct functions during myelination.
Animals↗
[Pharmacokinetic, bacteriological and clinical studies on cefozopran in neonates].
Cefozopran (CZOP) was administered to nine newborn patients with infections at a dose of 20 mg/kg twice or three times daily for 5 to 6 days to evaluate the efficacy, safety and pharmacokinetics of cefozopran. 1. Blood concentrations CZOP was intravenously given to 6 newborn patients by drip infusion at a dose of 20 mg/kg over 30 minutes. The maximum blood concentrations (Cmax) were 38.4 micrograms/ml in a patient aged 0 day, 37.7 and 54.3 micrograms/ml in two patients aged 1 day, 51.3 and 64.1 micrograms/ml in two patients aged 3 days and 51.0 micrograms/ml in a patient aged 5 days. Cmax was lower in the patient aged 0 day. The elimination half life (T 1/2) was 9.2 hours in the patient aged 0 day, 4.9 and 3.7 hours in the patients aged 1 day, 3.1 and 2.4 hours in the patients aged 3 days and 2.9 in the patient aged 5 days, showing a prolongation of T 1/2 in patients of lower age. 2. Urinary excretion Of the 6 patients given CZOP at a dose of 20 mg/kg by intravenous drip infusion over 30 minutes, urine was collected in 5 patients. The cumulative excretion rate within 6 hours after infusion was as low as 19.8% of dose in the patient aged 0 day. The rates were elevated as high as 46.3 and 57.0% of dose in the patients aged 1 day. In the patient aged 3 days, the recovery within 4 hours after infusion was 47.3%. It was 70.6% of dose within 6 hours after dosing in the patient aged 5 days. The urinary recovery within 6 hours after dosing increased with the advance of age. 3. Clinical results Efficacy was evaluable in 7 patients. Of them, 3 had suspected septicemia, 2 pneumonia, 1 intrauterine infection and 1 urinary tract infection. The clinical efficacy was judged "excellent" in all the evaluable patients. Neither adverse drug reactions of signs and symptoms nor abnormal alterations of the laboratory test values were recognized in the 9 patients evaluable for safety. These results suggest that CZOP is an effective and safe drug for treatment of infections in the newborns. As for the dosage and method of administration from the view of the pharmacokinetic data obtained, intravenous drip infusion of 20 mg/kg once or twice daily was considered to be sufficient for patients aged 0 day. For patients aged 1 to 7 days and those aged 8 days or elder, the administration of twice to 3 times daily and 3 to 4 times daily were considered to be sufficient, respectively.
Bacterial Infections↗
The consensus motif for phosphorylation by cyclin D1-Cdk4 is different from that for phosphorylation by cyclin A/E-Cdk2.
Cyclin D-Cdk4/6 and cyclin A/E-Cdk2 are suggested to be involved in phosphorylation of the retinoblastoma protein (pRB) during the G1/S transition of the cell cycle. However, it is unclear why several Cdks are needed and how they are different from one another. We found that the consensus amino acid sequence for phosphorylation by cyclin D1-Cdk4 is different from S/T-P-X-K/R, which is the consensus sequence for phosphorylation by cyclin A/E-Cdk2 using various synthetic peptides as substrates. Cyclin D1-Cdk4 efficiently phosphorylated the G1 peptide, RPPTLS780PIPHIPR that contained a part of the sequence of pRB, while cyclins E-Cdk2 and A-Cdk2 did not. To determine the phosphorylation state of pRB in vitro and in vivo, we raised the specific antibody against phospho-Ser780 in pRB. We confirmed that cyclin D1-Cdk4, but not cyclin E-Cdk2, phosphorylated Ser780 in recombinant pRB. The Ser780 in pRB was phosphorylated in the G1 phase in a cell cycle-dependent manner. Furthermore, we found that pRB phosphorylated at Ser780 cannot bind to E2F-1 in vivo. Our data show that cyclin D1-Cdk4 and cyclin A/E Cdk2 phosphorylate different sites of pRB in vivo.
Amino Acid Sequence↗
Generation of cell transfectants expressing cardiac calcium ion channel and calcium indicator protein aequorin.
Chinese hamster ovary (CHO) cells stably coexpressing cardiac calcium ion channel [L-type calcium channel or ryanodine receptor (RyR)] and the calcium-sensitive bioluminescent protein aequorin were generated by transfecting aequorin cDNA. In a selected clone, C1-17, carrying the L-type calcium channel, depolarization induced by high concentration of K+ produces aequorin luminescence. In another clone, R3-7, carrying RyR, caffeine produces aequorin luminescence. In the presence of selective calcium ion channel blockers, the aequorin luminescence was inhibited in a dose-dependent manner. These results indicate that functionally expressed calcium ion channels in these transformants can be monitored through the activation of endogenous aequorin luminescence following a physiological signal similar to that of native calcium channel. Moreover, the aequorin system compared very well with Fura-2 measurements. Thus, the recombinant cell models, which expressed cloned calcium channel and aequorin, will contribute to the elucidation of Ca2+ movement through the cell surface and intracellular calcium ion channels.
Aequorin↗
Structural changes in the peptide backbone in complex formation between activated rhodopsin and transducin studied by FTIR spectroscopy.
Structural changes in the complex formation between transducin and metarhodopsin II, the activated form of photolyzed rhodopsin, in visual transduction processes were analyzed by Fourier transform infrared spectroscopy. The spectrum of the complex was obtained by subtracting the contribution of metarhodopsin I and uncomplexed metarhodopsin II. The averaged spectrum upon the complex formation was then compared with that in the conversion of rhodopsin-to-metarhodopsin II. Frequency shifts of the peptide carbonyl vibrations at 1686, 1674, and 1661 cm-1 to 1640 cm-1 were observed upon complex formation from metarhodopsin II plus transducin. These changes must have resulted from the strengthening of H-bonding of one or a few peptide groups but is not ascribable to global conformation change. Changes in the frequencies of the peptide amides were also detected. With regard to intramembrane carboxylic acid residues, no further changes were noticed in the carboxyl vibrations of Asp83, Glu122, and Glu113. Only a small change possibly due to Glu134 was detected.
Animals↗
Cloning of the RhoB gene from the mouse genome and characterization of its promoter region.
Rho proteins have been implicated in a variety of cytoskeletal functions, but it is unclear how Rho proteins regulate these cellular functions and how Rho proteins are regulated. In this study, we cloned the rhoB gene from the mouse genome and characterized its promoter region. The predicted amino acid sequence was identical to that encoded by the human rhoB gene. A site for initiation of transcription was found at position -376 relative to the site for initiation of translation. Deletion analysis of the 5'-flanking region of the rhoB gene revealed that the minimum region of the promoter was located between positions -507 and -376. Northern blotting analysis showed that the expression of the mouse rhoB gene was induced by serum, suggesting that expression of the rhoB gene might be controlled by some signal-responsive element(s).
3T3 Cells↗
Hepatocyte growth factor (HGF) produced by peritoneal fibroblasts may affect mesothelial cell morphology and promote peritoneal dissemination.
Mesothelial cell monolayers have been reported to prevent infiltration of cancer cells into the peritoneum. We have previously reported that peritoneal fibrosis induced by gastric cancer cells prior to metastatization may provide a congenial environment for peritoneal metastases. In this study, we investigated the effects of peritoneal fibroblasts on peritoneal mesothelial cell morphology. Human gastric cancer (OCUM-2MD3), peritoneal fibroblast (NF-2P) and mesothelial (MS-1) cell lines were established in our laboratory. Histology of the peritoneum was investigated following intraperitoneal inoculation of serum-free conditioned media (SF-CM) from OCUM-2MD3 cells into nude mice. SF-CM from peritoneal fibroblasts was added to monolayer-cultured mesothelial cells, and their morphology was examined by phase-contrast microscopy. This experiment was conducted in the presence and absence of neutralizing antibodies against various factors. Mesothelial cells exposed to fibroblasts proliferation became hemispherical and separated from each other, while unexposed mesothelium remained as a flat monolayer. Cultured-mesothelial cells rounded up or exhibited a fibroblast-like shape following the addition of peritoneal fibroblast SF-CM. Anti-hepatocyte growth factor (HGF) neutralizing antibody partly inhibited this effect. We suggest that soluble factors, such as HGF, produced by peritoneal fibroblasts affect the morphology of mesothelial cells in monolayers so that the resulting environment may become prone to the peritoneal dissemination of cancer cells.
Animals↗
Induction of E. coli oh8Gua endonuclease by oxidative stress: its significance in aerobic life.
The induction of 8-hydroxyguanine (oh8Gua) endonuclease, a DNA repair enzyme for an oxidatively modified guanine, oh8Gua was studied in various growth conditions in Escherichia coli (AB1157). Anaerobically grown E. coli were found to have a very low activity of this enzyme while aerobically grown cells showed activity about 20 times that of the anaerobic level. Under the same condition, superoxide dismutase (SOD) showed about 6-fold increase in activity. A shift in growth conditions from anaerobic to aerobic resulted in rapid induction of this enzyme, and this induction was blocked (but not completely) by chloramphenicol. It is indicated that molecular oxygen is an effective stimulator to the induction of this enzyme and its induction depends partly on protein synthesis. Superoxide-producing compounds such as paraquat and menadione also increased the activity of endonuclease as well as SOD, but H2O2 showed no effect. Thus, superoxides are also implied as a stimulator. In contrast, hyperoxia induced only SOD not the endonuclease. This induction of the endonuclease by hyperoxia was only observed in a SOD-deficient strain (QC774). The aerobic activity of the endonuclease in QC774 was the same as that of wild types (AB1157, GC4468). It is implied that the responsiveness of oh8Gua endonuclease to superoxides is less sensitive than that of SOD. The endonuclease was also induced by a temperature shift from 30 to 43 degrees C and treatment with nalidixic acid. Among the stimuli used, molecular oxygen seems to be most effective for its induction. The inducible nature of this enzyme will serve as an important mechanism for the protection of oxidative DNA damage in the aerobic environment.
Aerobiosis↗
Suppression of translation frameshift by upstream termination codon.
Retroviruses employ translation frameshift for gene expression. Translation frameshift takes place at the slippery heptanucleotide sequence on mRNA. A downstream stem-loop structure (and a pseudoknot formed by the stem-loop) enhances the level of ribosomal frameshifting. Here, we describe that an upstream termination codon suppresses the ribosomal frameshifting, leading to reduction in the expression of downstream genes.
Amino Acid Sequence↗
DNA-dependent protein kinase inhibitor (OK-1035) suppresses p21 expression in HCT116 cells containing wild-type p53 induced by adriamycin.
The tumor growth suppressor p21 has been shown to be induced by wild-type p53 (wt-p53) and to be a potent inhibitor of cyclin-dependent kinases and PCNA/DNA polymerase delta. Although wt-p53 is reported to be phosphorylated by several protein kinases, the function and significance of the phosphorylation of wt-p53 are not yet fully understood. Using OK-1035, a selective inhibitor of DNA-dependent protein kinase (DNA-PK), we demonstrated the importance of the phosphorylation of wt-p53 by DNA-PK in the DNA damage-mediated expression of the p21 gene. Treatment of HCT116, a human colon carcinoma cell line, with adriamycin induced the expression of wt-p53 and p21. By addition of OK-1035 to this culture, the induction of p21 protein was significantly decreased in a dose-dependent manner, whereas wt-p53 induction was not affected. Northern blot analysis revealed that suppression of p21 protein expression by OK-1035 resulted from reduction in the level of p21 mRNA. OK-1035 did not directly affect the binding ability of wt-p53 to its consensus DNA sequence. Our observations support the idea that wt-p53 induces the transcriptional activation of the p21 gene only after it is phosphorylated by DNA-PK.
Amino Acid Sequence↗
Fibrosis in the peritoneum induced by scirrhous gastric cancer cells may act as "soil" for peritoneal dissemination.
BACKGROUND: Peritoneal metastases are one reason for the poor prognosis of scirrhous gastric cancer. However, the mechanism of peritoneal metastasis remains unclear. We therefore investigated the interaction between gastric cancer cells and peritoneal fibroblasts, in vivo and in vitro. METHODS: A human scirrhous gastric cancer cell line, OCUM-2MD3, and a human peritoneal fibroblast cell line, NF-2P, were established in our laboratory. We then investigated the effect of OCUM-2MD3 cells on the peritoneum of nude mice and on the proliferation of NF-2P cells, the effect of NF-2P cells on the migratory capability of OCUM-2MD3 cells, and the peritoneal fibrosis on the development of peritoneal metastasis. RESULTS: The proliferation of peritoneal fibroblasts was recognized in areas with and without cancer cell infiltration. Serum-free conditioned medium from the OCUM-2MD3 cells had induced peritoneal fibrosis in vivo and significantly stimulated the proliferation of NF-2P cells in vitro. The molecular weight (molecular weight ratio 25,000-43,000) of the growth factors produced by the OCUM-2MD3 cells was estimated by gel filtration high performance liquid chromatography. The migratory capability of the OCUM-2MD3 cells was significantly increased by the presence of NF-2P cells. Tumorigenicity in mice with peritoneal fibrosis was greater than in mice with normal peritoneum. CONCLUSIONS: Peritoneal fibrosis, formed by a factor secreted by scirrhous gastric cancer cells, may be congenial environment ("soil") for peritoneal metastases of scirrhous gastric carcinoma.
Adenocarcinoma, Scirrhous↗
Cyclin-dependent kinase-2 (Cdk2) forms an inactive complex with cyclin D1 since Cdk2 associated with cyclin D1 is not phosphorylated by Cdk7-cyclin-H.
Cyclin-dependent kinases (Cdks) form complexes with cyclins, and as a consequence they generally express kinase activities. One of these Cdks, Cdk2, is known to bind with cyclins A and E, and plays an important role in the progression of the cell cycle via phosphorylation of target proteins such as the product of the retinoblastoma tumor-suppressor gene (pRB). It has been suggested that Cdk2 bound with cyclin D1 and Cdk2-cyclin-D1 complex show neither H1 histone nor pRB kinase activity. However, it is not clear whether Cdk2-cyclin-D1 has unknown targets and why Cdk2 is not activated by binding with cyclin D1. We investigated these questions using Cdk, cyclin and Cdk-cyclin complexes produced in a baculovirus expression system. Cdk2 formed a complex with cyclin D1 in this system. After extensive purification, Cdk2 was still bound to cyclin D1. The Cdk2-cyclin-D1 complex did not phosphorylate any tested substrates, such as H1 histone, pRB, SV40 large T antigen, p53, E2F-1 or a preparation of nuclear proteins from HeLa cells; in contrast, Cdk2-cyclin-E and Cdk2-cyclin-A phosphorylated these proteins. Moreover, the Cdk2-cyclin-D1 complex was not activated by incubation with Cdk4 or cyclin E. Thus, Cdk2 and cyclin D1 formed a stable complex that was not activated. In order to determine why Cdk2-cyclin-D1 lacks kinase activity, we investigated the phosphorylation of Cdk2. Under-shifted Cdk2, the active form of Cdk2, was not detected in the Cdk2-cyclin-D1 complex in the baculovirus system. In human WI-38 cells, cyclin D1 began to form a complex with Cdk2 as well as with Cdk4 from the mid-G1 phase of the cell cycle. The Cdk2 bound to cyclin D1 in human cells was also the inactive form that was slowly migrated. Moreover, we found that Cdk2 bound to cyclin D1 was not phosphorylated by Cdk7-cyclin-H, while Cdk2 bound to cyclin E, as well as free Cdk2, was was phosphorylated by Cdk7-cyclin-H. Additionally, Cdk2 phosphorylated by Cdk7-cyclin-H did not bind to cyclin D1. These results strongly suggest that Cdk2 forms a stable complex with cyclin D1 but is not activated because the Cdk2 molecule in the complex is not phosphorylated by Cdk7-cyclin-H and the phosphorylated Cdk2, an active form, does not bind to cyclin D1.
Animals↗
Effects of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine on intestinal polyp development in Apc delta 716 knockout mice.
Damage to the human adenomatous polyposis coli (APC) gene is responsible for not only familial adenomatous polyposis but also many sporadic cancers of the entire digestive tract. Using homologous recombination in embryonic stem cells, we recently constructed gene knockout mice with a truncation mutation in the Apc gene. These heterozygous mice developed intestinal polyps. We found that all microadenomas dissected from the earliest polyps had already lost the wild-type allele, indicating loss of heterozygosity (LOH) (Oshima et al., Proc. Natl. Acad. Sci. USA 92:4482-4486, 1995). Using these knockout mice, we investigated the effects of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhlP), one of the most abundant heterocyclic amines found in cooked meat and fish. When PhIP was fed to these mice at 400 ppm for 8 wk, the polyp distribution shifted to a larger size range, although the total polyp number did not change significantly. Similar, but weaker, effects were observed with the other heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline. On the other hand, intraperitoneal injections of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhlP) at a higher dose (50 mg/kg) for five consecutive days increased the polyp number significantly. This increment was not associated with mutations in the Apc gene; however, most polyps showed loss of the full-length Apc allele (LOH). These results suggest that PhIP affects intestinal polyp development by accelerating the growth rate of microadenomas. It is also possible that high doses of N-OH-PhIP increase the frequency of Apc gene LOH.
Age Factors↗
Peritoneal metastatic model for human scirrhous gastric carcinoma in nude mice.
We established a peritoneal-metastatic model for scirrhous gastric carcinoma. Peritoneal metastasis had developed after intraperitoneal inoculation of OCUM-2MD3 cells in nude mice. This cell line was derived from a peritoneal-metastatic nodule at the mesenterium after orthotopic implantation of OCUM-2M cells which developed no peritoneal metastasis after intraperitoneal inoculation. The histologic findings of orthotopic-implanted tumor in the stomach show scirrhous type while those of subcutaneous-implanted tumor show medullary type. There might be factors, in OCUM-2MD3 cells, which are responsible for peritoneal metastasis. We next investigated the differences in the biological behavior of the original OCUM-2M and the derived variant OCUM-2MD3. Morphology and growth activity of the two cell lines were similar to each other. The specific chromosomes, add(6)(q13), del(7)(q21.2) and inv(11)(p13q21), were found in OCUM-2MD3 cells but not in OCUM-2M cells. While the oncogenes amplification by OCUM-2M cells was found in K-sam and c-myc, that by OCUM-2MD3 cells was found only in c-myc. The expression of E-cadherin by OCUM-2MD3 cells was decreased compared with that of OCUM-2M cells. Expression level of beta 1-integrin of OCUM-2MD3 cells were higher than that of OCUM-2M cells. The binding and invasion activity of OCUM-2MD3 cells were higher than those of OCUM-2M cells, and were decreased by anti-beta 1-integrin antibody. The invasion activity of OCUM-2MD3 cells was increased in the presence of peritoneal fibroblast. In this study, it was suggested that orthotopic implantation of cancer cells might have an effect on the acquisition of metastatic ability. beta 1-integrin and peritoneal fibroblasts might be correlated with peritoneal metastasis. This peritoneal-metastatic model should be useful for analysing the mechanism of peritoneal metastasis of human scirrhous gastric cancer.
Adenocarcinoma, Scirrhous↗
Biological evaluation of 5-methyl-branched-chain omega-[18F]fluorofatty acid: a potential myocardial imaging tracer for positron emission tomography.
5-Methyl-17-[18F]fluoroheptadecanoic acid (5-MFHA) has been proposed as a new myocardial imaging tracer for positron emission tomography (PET), containing methyl-branching at the odd-numbered position, except the 3-position. To compare the site of methyl-branching of fatty acids on the contribution to myocardial imaging, the biological evaluation of 5-MFHA-using accumulation studies, metabolic studies, and PET studies of the heart muscle-was investigated. In the comparative biodistribution studies for 16-[18F]fluoropalmitic acid (FPA), 3-methyl-17-[18F]fluoroheptadecanoic acid (3-MFHA) and 5-MFHA, the initial myocardial uptake of 5-MFHA (2.64 [%dose/g tissue]) was relatively high between those of FPA and 3-MFHA (3.45 and 1.58, respectively), and the washout from myocardium of 5-MFHA was midway between those of FPA and 3-MFHA. In the lipid analysis studies, 5-MFHA was mainly metabolized to triglycerides in the myocardium, and its metabolic pattern was similar to that of straight-chain fatty acids (FPA). In the PET studies using 5-MFHA in canines, good myocardial images were obtained for up to 30 min after injection.
Animals↗