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Biomedical subjects

M Nishida

Publications and source records attributed to M Nishida.

At least 235 records · Page 13Linked to original sources
PubMed1997

A simple quantitative measurement of mRNA of human beta-actin by reverse transcription competitive PCR with a compact digital camera.

Reverse transcription (RT) competitive polymerase chain reaction (PCR) is a sensitive and useful technique for the absolute quantitation of mRNA level. We developed a competitive PCR method with a compact digital camera DC 40 (Eastman Kodak Company, NY, U.S.A.) for the measurement of human beta-actin cDNA (mRNA), a reference gene in an analysis for target gene expression. The devised method, compared with currently used methods, was easy, safe, sensitive, and highly reproducible to assay an infinitesimal level of cDNA (mRNA).

Actins↗
PubMed1997

Detection of interleukin 6-producing cells among various organs in normal mice with an improved enzyme-linked immunospot (ELISPOT) assay.

The enzyme-linked immunospot (ELISPOT) assay is an efficient technique for the enumeration of single cells secreting antibodies or cytokines. To assess mice interleukin 6 (IL-6)-secreting cells at a single cell level, a highly sensitive ELISPOT assay with 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt and nitroblue tetrazolium chloride (BCIP/NBT) as a gel-substrate has been developed. The IL-6 specific ELISPOT assay thus developed with an ordinary plastic ELISA plate is as efficient as the previous IL-6 specific ELISPOT assay system which uses a nitrocellulose coated plate, and is much more economic and convenient. We demonstrated with this newly devised ELISPOT assay that the ratio of IL-6 producing cells in the resident peritoneal exuded cells was highest in normal BALB/c mice, and a few IL-6 secreting cells were detected in all organs we tested, namely the spleen, bone marrow, Peyer's patch, axillary lymph node and thymus.

Animals↗
PubMed1997

Effects of swimming training on brown-adipose-tissue activity in obese ob/ob mice: GDP binding and UCP m-RNA expression.

Brown adipose tissue (BAT) of obese animals is generally in a relatively atrophied and thermogenically quiescent state. The aim of the current study was to investigate the effect of swimming training on BAT activity in lean and obese (ob/ob) mice. The trained mice underwent a 6-week endurance swimming training (1 h/day, 5 days/week) in water at 35-36 degrees C. The swimming training significantly increased BAT mass and its protein content in both the lean and obese mice, suggesting hypertrophy. After swimming training, the amounts of protein and guanosine 5'-diphosphate binding in the mitochondria recovered from BAT of both mice increased significantly as compared with the respective sedentary groups, whereas the uncoupling protein (UCP) content increased significantly only in lean mice. After swimming training, the level of UCP mRNA expression did not change substantially in lean mice but appeared to increase in obese mice. The results obtained here suggest that swimming training leads to an increase in the nonshivering thermogenesis of obese mice in addition to lean mice.

Adipose Tissue, Brown↗
PubMed1997

Effects of preconditioning with ebselen on glutathione metabolism and stress protein expression.

Selenium induces several proteins, including glutathione and stress proteins. These proteins have been shown to be cardioprotective against oxidative injury. To determine whether ebselen, a seleno-organic compound, can also induce these proteins and exert cardioprotective action, we examined the effects of preconditioning with ebselen on glutathione metabolism and stress protein expression and on myocyte injury induced by oxidative stress. Treatment of cultured cardiac myocytes with ebselen (0.3-30 microM) for 24 hr increased the reduced glutathione content. Glutathione reductase activity, but not glutathione peroxidase activity, was significantly elevated in a dose-dependent manner. Pretreatment with ebselen increased the expression of such stress proteins as heat shock protein 70 and heme oxygenase-1 (heat shock protein 32) in cardiac myocytes, as assessed by Western blotting. Expression of heat shock protein 70 was increased only at a higher dose of ebselen (30 microM), whereas expression of heme oxygenase-1 was markedly increased at a lower dose of ebselen (3 microM). Under these conditions, the myocyte injury induced by hydrogen peroxide or simulated ischemia/reperfusion, assessed by the release of lactate dehydrogenase into the culture medium, was reduced by ebselen pretreatment in a dose-dependent manner. Results indicated that cardiac myocytes pharmacologically preconditioned with ebselen for 24 hr exhibited resistance to oxidative injury, possibly via the up-regulation of glutathione metabolism and the expression of stress proteins.

Animals↗
PubMed1997

[Angiographical evaluation of extracranial carotid artery: comparison between Japanese and Hungarian].

We investigated the cervical level of carotid bifurcation, diameter of common carotid artery (CCA) and internal carotid artery (ICA) in 147 Japanese patients (150 arteries, range of age 31-79) and in 490 Hungarian patients (517 arteries, range of age 12-77). The cervical level of carotid bifurcation was determined by comparison with cervical vertebra. The carotid bifurcation of Japanese was most frequent at the lower part of 3rd cervical vertebra but in Hungarian the bifurcation was most frequent at the middle part of 4th cervical vertebra. Japanese carotid bifurcation was high-positioned compared with Hungarian. The mean CCA diameter in Japanese was 7.47 mm in male and 7.07 mm in female, in Hungarian 9.24 mm in male and 7.80 mm in female. The mean ICA diameter in Japanese was 4.96 mm in male and 4.83 mm in female, in Hungarian 8.56 mm in male and 7.66 mm in female. CCA and ICA diameter were larger in male than in female. The diameter of CCA had a significantly positive correlation with age (p < 0.002) but ICA had no similar correlation.

Adolescent↗
PubMed1997

[Simplification of on-line connection of analyzers and standardization problems].

On-line connection of automated analyzers to laboratory information system (LIS) reduces mistakes in inputing data for each samples. It also makes reporting faster in clinical laboratory. Moreover, connection of these instruments with sample transporting system enables analyses without touching samples directly. It, however, costs extremely high to construct such a system. It is because every automated analyzer uses different connecting protocol, so that we have to make a different program for each machine. For solving this problem, we have to make a standard for connecting protocol. It is very difficult to make a standard protocol fitting on all of the analyzers, considering its cost and other things. Furthermore, it must take a few decades to spreading the standard through the end users. ICCLS and NCCLS has been taking a central role for this problem since 1996, with a five-year plan to make an international standard. Until the standard will be laid, each clinical laboratories have to pay high costs to construct their systems, connecting different manufacturers' analyzers each other. Thus, we have developed a novel system which enables us to construct a laboratory automation system in a shorter time. For realizing this new system, we have reduced the number of connecting protocols for analyzers. Moreover, we have corrected the flow of laboratory works in order. Each programs are put together into a system as parts, or modules. This software system is now in operation in the clinical laboratory of Kochi Medical School. In this report, we describe the construction of this novel software system, and the effect obtained by using this system. Furthermore, we would show problems and things to be improved for making international standards for communication protocols between host and analyzing instruments.

Automation↗
PubMed1997

[The study of continuous infusion chemotherapy with low-dose cisplatin and 5-fluorouracil for patients with primary liver cancer].

Twelve patients with unresectable primary liver cancer (hepatocellular carcinoma and cholangiocarcinoma) and postoperative recurrence of primary liver cancer received continuous arterial or systemic infusion of low-dose CDDP/5-FU. This infusion chemotherapy was continued for five days, discontinued for two days, and repeated four weeks as one course basally. The partial response rate in patients with HCC or CCC treated with intra-arterial infusion was 20% and 33%, respectively. The rate in patients with HCC or CCC treated with systemic infusion was 0% and 33%, respectively. The response rate included decrease of tumor markers in all patients with HCC or CCC was 33% and 67%, respectively. These results suggest that low-dose CDDP/5-FU therapy may be effective in patients with CCC. Severe side effects such as gastro-duodenal ulcer (4 cases) and pseudomembranous colitis (1 case) were observed. The careful management of side effects should be required during this therapy.

Aged↗
PubMed1997

[A case of rapidly progressive IgA nephropathy with transient hypocomplementemia at onset].

We present a case of IgA nephropathy (IgAGN) which developed rapidly progressive glomerulonephritis and showed marked clinical improvement with treatment. The patient was a 7-year-old boy who initially presented with acute nephritic syndrome with hypocomplementemia. Although the renal function improved with normalization of the serum complement level, it deteriorated again progressively. The first renal biopsy revealed cellular crescents in about 70 percent of 43 glomeruli. Immunofluorescent microscopy demonstrated deposits of IgA, C3 and IgG in the mesangium; they were also deposited along the glomerular capillary walls. He was treated with plasma exchange associated with hemodialysis and methylprednisolone pulse therapy, followed by oral administration of prednisolone, cyclophosphamide and warfarin. Renal function recovered to the normal range about two months after the initiation of treatment. The second biopsy demonstrated a marked decrease in histological activity. In this case, transient hypocomplementemia at onset may indicate that acute glomerulonephritis caused exacerbation of clinically silent IgAGN. Aggressive therapy may be effective in patients with rapidly progressive IgAGN if treated at an early stage.

Child↗
PubMed1996

Inactivation of p16/CDKN2 and p15/MTS2 genes in different histological types and clinical stages of primary ovarian tumors.

To define the involvement of p16/CDKN2 and p15/MTS2 inactivation in ovarian tumorigenesis and the association of these inactivation events with histological types and clinical stages of ovarian tumors, we analyzed homozygous deletion and somatic mutation of p16/CDKN2 and p15/MTS2 genes, as well as hypermethylation of the 5'-CpG island of the p16/CDKN2 gene, in 49 primary ovarian tumors and 6 ovarian carcinoma cell lines. We found homozygous deletions of p16/CDKN2 and p15/MTS2 in 6 (12%) and 5 (10%) primary tumors, respectively. Somatic mutation of p16/CDKN2 was found in only 1 primary tumor, but mutation of p15/MTS2 was not detected in any sample. None of the 28 primary tumors or 6 cell lines was hypermethylated at the 5'-CpG island of p16/CDKN2. The incidence of inactivation of p16/CDKN2 in primary tumors was significantly higher in the advanced stages (7 of 29) than in the early stages (0 of 14). Seven of 9 alterations in p16/CDKN2 and p15/MTS2 were observed in serous (3 of 12), endometrioid (3 of 9) and clear-cell (1 of 4) carcinomas. However, only normal sequences of these genes were detected in mucinous carcinomas. Loss of heterozygosity (LOH) at the IFNA locus was detected in 1 of 19 (5%) tumors, but no change at the D9S171 locus was observed in 17 tumors. These results suggest that: (i) homozygous deletion is the main mechanism of inactivation of p16/CDKN2 and p15/MTS2 in ovarian tumorigenesis; (ii) inactivation of p16/CDKN2 and p15/MTS2 may be the histological type-specific events involved in ovarian tumorigenesis; and (iii) inactivation of p16/CDKN2 is potentially involved in the progression of ovarian tumors in advanced stages.

Carcinoma↗
PubMed1996

Localization of heparin-binding epidermal growth factor-like growth factor in human coronary arteries. Possible roles of HB-EGF in the formation of coronary atherosclerosis.

BACKGROUND: Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a newly identified member of the EGF family. Our previous in vitro studies showed that HB-EGF is a potent mitogen and chemoattractant for vascular smooth muscle cells (SMCs), suggesting the role of HB-EGF in the pathogenesis of atherosclerosis. The purposes of the present study were to investigate the localization of HB-EGF in both normal and atherosclerotic human coronary arteries and to elucidate the possible roles of this growth factor in the formation of atherosclerotic lesions. METHODS AND RESULTS: The immunohistochemical localization of HB-EGF, SMCs, macrophages, and EGF receptors (EGFRs) was examined in human coronary arteries obtained at autopsy. The medial SMCs of coronary arteries in neonates, infants, and children consistently synthesized HB-EGF protein. In normal adults, however, the relative number of HB-EGF-positive medial SMCs decreased gradually with age after about 30 years of age. In nonatherosclerotic coronary arteries with diffuse intimal thickening, SMCs of the intima, especially those located in the area of the medial side of the intima, were strongly positive for HB-EGF protein. In atherosclerotic plaques of coronary arteries with eccentric intimal thickening, both SMCs and macrophages in and around the core lesions, in addition to the intimal and medial SMCs located adjacent to the plaque, produced HB-EGF protein. A strong immunostaining of EGFRs was observed in these SMCs, suggesting a close association of HB-EGF and EGFR expression. CONCLUSIONS: These data suggest that HB-EGF might play important roles in the migration of SMCs from the media to the intima, the proliferation of intimal SMCs, and the interaction between SMCs and macrophages in the process of coronary atherogenesis.

Adult↗
PubMed1996

Anti-erythropoietin receptor monoclonal antibody: epitope mapping, quantification of the soluble receptor, and detection of the solubilized transmembrane receptor and the receptor-expressing cells.

A hybridoma cell line producing the monoclonal antibody against erythropoietin receptor (EpoR) was established using the soluble ectodomain of mouse erythropoietin receptor (sEpoR) as an antigen. The monoclonal antibody termed 1G3 bound to the denatured sEpoR. Epitope mapping with peptide library revealed that 1G3 recognized the amino terminal region including the hexapeptide (positions 6 to 11; LeuProAspProLysPhe). The amino acid sequence in this hexapeptide was identical in mice, rats, and humans, and therefore 1G3 bound to EpoR from all of these sources. Using 1G3, we evaluated sEpoR by a sandwich enzyme-linked immunoassay, and EpoR in the solubilized membrane preparation was detected by Western blotting. The cells expressing EpoR were identified with immunochemical staining. We confirmed the presence of EpoR in a neuronal cell line and PC12 cells, and EpoR was expressed in primary cultured hippocampal neurons.

Amino Acid Sequence↗
PubMed1996

Site-directed mutagenesis of hamster complement C1S: characterization with an active form-specific antibody and possible involvement of C1S in tumorigenicity.

We have previously shown that non-transformed mouse A31 cells became tumorigenic when they were transfected with hamster C1s cDNA expression plasmid BCMGSNeoCS. In the present study, mutations were introduced into the cDNA at the activation cleavage site, Arg423(AGG) and the active center Ser617(AGC). These amino-acids were replaced by His423(CAC) and Thr617(ACC), respectively. The mutated cDNAs were inserted into BCMGSNeo and transfected to A31 and its polyoma-virus-transformed SEA7 cells. C1s produced from these transfectants lost their enzyme activity. Transfectants of these mutated C1s cDNA did not form tumors in nude mice, To distinguish between active and inactive C1s in situ, we have developed novel antibodies, one directed to the NH2-terminal neoepitope of the L chain and the other specific for uncleaved inactive C1s. These antibodies were used to characterize C1s produced by transfectants, so as to determine whether or not it was cleaved at the right position.

Amino Acid Sequence↗
PubMed1996

Amelioration of severity of myocardial injury by a nitric oxide donor in rabbits fed a cholesterol-rich diet.

OBJECTIVES: This study compared the effect of a nitric oxide donor on limiting the size of infarct resulting from myocardial ischemia-reperfusion between atherosclerotic and nonatherosclerotic models. BACKGROUND: Endothelial-derived relaxation in coronary arteries affected by ischemia is substantially impaired after reperfusion, and this impairment may exacerbate the myocardial ischemia-reperfusion injury. In animals with experimental atherosclerosis, release of endothelial-derived relaxing factor is also decreased, and the propagation of myocardial infarction could be exacerbated. METHODS: We examined the extent of myocardial injury induced by ischemia (30 min) and reperfusion (48 hr) in rabbits fed a cholesterol-rich (1%) or normal diet for 10 weeks. We also evaluated the effect of a nitric oxide donor (S-nitroso-N-acetylpenicillamine [SNAP], a nitric oxide precursor (L-arginine) or a degradation product of SNAP (N-acetylpenicillamine) on infarct size in these models. RESULTS: Severity of myocardial injury was significantly exacerbated in cholesterol-fed rabbits (75.2 +/- 4.4% [mean +/- SEM]) compared with that in non-cholesterol-fed rabbits (53.2 +/- 5.2%). This exacerbation was prevented by treatment with SNAP (50.2 +/- 6.4%) but not with L-arginine (70.5 +/- 6.0%) or N-acetylpenicillamine (70.4 +/- 4.8%) in cholesterol-fed-rabbits. However, SNAP did not limit infarct size in non-cholesterol-fed rabbits (60.8 +/- 4.2%). The rate-pressure product was similar during the course of the experiment in all the groups. CONCLUSIONS: Myocardial damage induced by ischemia-reperfusion was significantly exacerbated in rabbits fed a long-term cholesterol-rich diet but was effectively reversed by treatment with a nitric oxide donor. However, this agent did not limit infarct size in normal rabbits. Thus, a nitric oxide donor reduces myocardial infarct size in atherosclerotic but not in nonatherosclerotic rabbits.

Analysis of Variance↗
PubMed1996

Heme oxygenase-1 expression and its relation to oxidative stress during primary culture of cardiomyocytes.

The inducible form of heme oxygenase (heme oxygenase-1) is a heat shock protein 32 (HSP32) whose expression is induced by numerous agents, including heme compounds and heavy metals, and during oxidative stress. The purpose of this study was to examine whether heme oxygenase-1 is induced during primary cell culture of cardiomyocytes and the relation of heme oxygenase-1 expression to oxidative stress levels. Western blot analysis and reverse transcription-polymerase chain reaction analysis showed heme oxygenase-1 expression 12-48 h after isolation of rat neonatal cardiomyocyte for culture. Its expression was barely detected immediately after isolation. Actinomycin D or cycloheximide completely suppressed such expression. Myocardial cells were exposed to oxidative stress during the first 12 h after isolation as assessed by their glutathione redox state; the ratio of reduced glutathione/oxidized glutathione was less than 10. The expression of heme oxygenase-1 was significantly reduced by treatment with reduced glutathione (58% reduction, P < 0.05), but markedly increased by treatment with hydrogen peroxide (65% increase, P < 0.05) 12 h after isolation. Expression of heat shock protein 70 was not significantly changed during primary culture incubation. Results indicate that heme oxygenase-1 is expressed during primary culture of cardiomyocytes. Its expression is closely related to the oxidative stress level of the cultured cells.

Animals↗
PubMed1996

MRI evidence of high signal intensity and temporomandibular arthralgia and relating pain. Does the high signal correlate to the pain?

In order to investigate the relationship between various temporomandibular joint (TMJ) pain levels and the detection of high signal intensity (joint effusion) on T2 weighted magnetic resonance imaging (MRI), 19 consecutive patients who complained of unilateral painful TMJ hypomobility (closed locking) were involved in this study. All patients were clinically examined in a routine manner, and all patients rated their pain levels by a visual analogue scale and eight pain questionnaire prior MRI study. T1 and T2 weighted MRI was taken in sagittal section at unilateral affected joint side. The presence or absence of a high signal intensity spot within the TMJ compartment were judged by three examiners. The high signal intensity was detected in 10 joints, but not in 9 joints. In between these two groups, the pain ratio was calculated and compared. The data showed that there was no significant statistical correlation between pain levels and the presence of high signals. This study disclosed that the MRI detection of high signal intensity in the closed locking TMJ did not directly relate to the presence of TMJ pain nor the increased pain level. These indicate the need of further larger studies.

Adolescent↗
PubMed1996

Dissociation of early and late protective immunity to the nematode Nippostrongylus brasiliensis in Brown Norway and Fischer-344 rats.

Worm expulsion of, and IgE and interferon (IFN)-gamma responses to, Nippostrongylus brasiliensis were studied in 2 rat strains, Brown Norway (BN) and Fischer (F)-344. BN rats expelled the majority of worms by day 14 post-infection (p.i.) with approximately 6% of worms surviving for at least 3 weeks. In F-344 rats, worm expulsion was delayed by 2 days relative to that in BN, while the numbers of residual worms were significantly fewer than in BN, suggesting that different immune mechanisms are involved in early and late phases of immunity. Total serum IgE, as well as in vitro IgE production by mesenteric lymph node (MLN) cells, was increased 2 weeks p.i., the levels being markedly higher in BN than in F-344 rats. Serum rat mast cell protease II was also increased more significantly in BN than in F-344 rats. In contrast, production of IgG2a and IFN-gamma by MLN and spleen cells was found to be higher in F-344 than in BN rats. These results indicate that the early worm expulsion is correlated with the host IgE and mast cell responsiveness, whereas the persistence of infection in the late period may be controlled by different immune mechanisms.

Animals↗