Biomedical subjects

M Kinoshita

Publications and source records attributed to M Kinoshita.

At least 55 records · Page 3Linked to original sources

PubMed2001

VIPP1, a nuclear gene of Arabidopsis thaliana essential for thylakoid membrane formation.

The conversion of light to chemical energy by the process of photosynthesis is localized to the thylakoid membrane network in plant chloroplasts. Although several pathways have been described that target proteins into and across the thylakoids, little is known about the origin of this membrane system or how the lipid backbone of the thylakoids is transported and fused with the target membrane. Thylakoid biogenesis and maintenance seem to involve the flow of membrane elements via vesicular transport. Here we show by mutational analysis that deletion of a single gene called VIPP1 (vesicle-inducing protein in plastids 1) is deleterious to thylakoid membrane formation. Although VIPP1 is a hydrophilic protein it is found in both the inner envelope and the thylakoid membranes. In VIPP1 deletion mutants vesicle formation is abolished. We propose that VIPP1 is essential for the maintenance of thylakoids by a transport pathway not previously recognized.

Amino Acid Sequence↗

PubMed2001

Establishment of medaka (Oryzias latipes) transgenic lines with the expression of green fluorescent protein fluorescence exclusively in germ cells: a useful model to monitor germ cells in a live vertebrate.

We have generated transgenic medaka (teleost, Oryzias latipes), which allow us to monitor germ cells by green fluorescent protein (GFP) fluorescence in live specimens. Two medaka strains, himedaka (orange-red variety) and inbred QurtE, were used. The transgenic lines were achieved by microinjection of a construct containing the putative promoter region and 3' region of the medaka vasa gene (olvas). The intensity of GFP fluorescence increases dramatically in primordial germ cells (PGCs) located in the ventrolateral region of the posterior intestine around stage 25 (the onset of blood circulation). Whole-mount in situ hybridization and monitoring of ectopically located cells by GFP fluorescence suggested that (i) the increase in zygotic olvas expression occurs after PGC specification and (ii) PGCs can maintain their cell characteristics ectopically after stages 20-25. Around the day of hatching, the QurtE strain clearly exhibits sexual dimorphisms in the number of GFP fluorescent germ cells, a finding consistent with the appearance of leucophores, a sex-specific marker of QurtE. The GFP expression persists throughout the later stages in the mature ovary and testis. Thus, these transgenic medaka represent a live vertebrate model to investigate how germ cells migrate to form sexually dimorphic gonads, as well as a potential assay system for environmental substances that may affect gonad development. The use of a transgenic construct as a selective marker to efficiently isolate germ-line-transmitting founders during embryogenesis is also discussed.

Animals↗

PubMed2001

Structural organization, complete genomic sequences and mutational analyses of the Fukuyama-type congenital muscular dystrophy gene, fukutin.

Fukuyama-type congenital muscular dystrophy (FCMD) is an autosomal recessive severe muscular dystrophy in combination with cerebral cortical dysplasia. Previously, we identified the gene responsible for FCMD, termed fukutin, through positional cloning. In this study, we have sequenced 131892 bp of genomic DNA in the region of the fukutin gene on chromosome 9q31 and obtained its complete genomic structure. The fukutin genomic sequence spans approximately 100 kb and is organized into 10 exons (41-6067 bp) and nine introns (1841-21460 bp). Using these sequence data, we have identified three novel fukutin mutations in FCMD patients. We have also located a putative TATA box in the flanking 5' region and identified numerous alternatively spliced fukutin mRNA transcripts. Analysis of expressed sequence tag clusters within the region revealed two novel genes upstream of the fukutin gene. These data provide fundamental information to support detailed genetic and functional analyses of the fukutin gene.

Alternative Splicing↗

PubMed2001

Cancer cells surviving hypoxia obtain hypoxia resistance and maintain anti-apoptotic potential under reoxygenation.

In solid tumors hypoxia and reoxygenation may be important factors in secondary expansion after anti-cancer therapy. Our study examined the effect of hypoxia and reoxygenation on the apoptotic potential of cancer cells. Four experimental groups were studied using a human colorectal cancer cell line (HCT116) that is apoptosis-competent in conventional culture: (1) sham, cells grown under conventional conditions; (2) hypoxic, cells cultured in 95% N2 and 5% CO2 for 24 hr; (3) continued hypoxic, cells cultured for 48 hr; and (4) reoxygenation, cells grown in hypoxic conditions for 24 hr followed by another 24 hr under conventional conditions. Protein expression of p53, bcl-2 and PCNA were determined by immunohistochemistry and immunoblotting (p53), and viable cell growth rate was determined. Hypoxia for 24 hr induced significant up-regulation of p53 and bcl-2 expression, accompanied by significant decreases of cell growth rate and PCNA expression. Up-regulation of p53 and bcl-2 expression persisted with both continued hypoxia and reoxygenation, despite increased cell growth rate and PCNA expression. Cells escaping hypoxia acquired sustained resistance to apoptosis and proliferate despite an elevated p53 level, suggesting that p53 transfer to hypoxic solid tumor should be reevaluated as a cancer gene therapy approach.

Apoptosis↗

PubMed2001

Fertile and diploid nuclear transplants derived from embryonic cells of a small laboratory fish, medaka (Oryzias latipes).

Fertile and diploid nuclear transplants were successfully generated by using embryonic cells as donors in a small laboratory fish, medaka (Oryzias latipes). Embryonic cell nuclei from transgenic fish carrying the green fluorescent protein (GFP) gene were transplanted into unfertilized eggs enucleated by x-ray irradiation. In this study, 1 out of 588 eggs transplanted in the first experiment and 5 out of 298 eggs transplanted in the second experiment reached the adult stage. All of these nuclear transplants were fertile and diploid, and the natural and GFP markers of the donor nuclei were transmitted to the F(1) and F(2) offspring in a Mendelian fashion. This systematic study proves the feasibility of generating nuclear transplants by using embryonic cells from fish as donors, and it is supported by convincing evidence.

Animals↗

PubMed2001

Frequency of BRCA1 and BRCA2 germline mutations in Japanese breast cancer families.

The purpose of this investigation is to study the frequency of BRCA1 and BRCA2 germline mutations in Japanese breast cancer families. Mutation analysis of BRCA1 and BRCA2 by SSCP was conducted on the 113 breast cancer patients (probands) with at least 1 breast cancer (site-specific breast cancer families, n = 101) or 1 ovarian cancer (breast/ovarian cancer families, n = 12) patient in their first-degree relatives. Fifteen deleterious mutations (13.3%), including 8 nonsense and 7 frameshift mutations, were identified in BRCA1, and 21 deleterious mutations (18.6%), including 8 nonsense, 12 frameshift and 1 splice-site mutations, were identified in BRCA2. In site-specific breast cancer families, mutation frequency of BRCA1 or BRCA2 was high in families with 3 or more breast cancer patients (36%, 9/25), early onset (40 < or = years old) breast cancer patients (38%, 19/50) or bilateral breast cancer patients (40%, 6/15). In breast/ovarian cancer families, mutations of BRCA1 (58.3%, n = 7), but not BRCA2 (0%, n = 0), were observed. BRCA1 codon 63 (TTA to TAA) nonsense mutation and BRCA2 frameshift mutation (5802 del AATT) were observed in 4 and 7 independent families, respectively. Haplotype analysis has suggested that carriers of each of these mutations have common ancestors. These results demonstrate that family profiles are important determinants of risk for carrying a BRCA1 or BRCA2 mutation and that cumulative frequency of BRCA1 and BRCA2 mutations in Japanese breast cancer families (31.9%) is within the range observed in Caucasian breast cancer families. Presence of Japanese founder mutations has also been suggested.

Adult↗

PubMed2001

Anomalous migration of hyaluronic acid oligomers in capillary electrophoresis: correlation to susceptibility to hyaluronidase.

During high-resolution capillary electrophoresis analysis in an electrolyte solution containing a neutral polymer, small oligomers of regularly arranged acidic polysaccharides such as hyaluronic acid and N-acetylneuraminic acid polymers showed reversal of the migration order. This anomalous migration was well correlated with their reported biological activity. In the present study, we analyzed hyaluronidase action on the purified hyaluronic acid oligomers using capillary electrophoresis and found that hydrolytic and transglycosylation actions by hyaluronidase were dependent on the molecular sizes of hyaluronic acid oligomers, and well correlated to their migration profiles. Furthermore, fluorescent polarization technique was employed for understanding the relationship between molecular size of hyaluronic acid oligomers and their electromigrations.

Animals↗

PubMed2001

Lactone formation of N-acetylneuraminic acid oligomers and polymers as examined by capillary electrophoresis.

We examined lactone-formation reaction of oligomers and polymers of N-acetylneuraminic acid in diluted hydrochloric acid solution and found that the time course of the lactonization reaction was easily traced by capillary electrophoresis. The reaction proceeded more rapidly with increasing the molecular mass of oligomers because the conformation of inner units became rigid and more favorable for the formation of lactone linkage. Present results obtained using capillary electrophoresis will be useful in understanding of physical and chemical properties of oligo/polysialic acids.

Carbohydrate Sequence↗

PubMed2001

Detection of denervated but viable myocardium in cardiac sarcoidosis with I-123 MIBG and Tl-201 SPECT imaging.

A 58-year-old man, who had biopsy-proven cardiac sarcoidosis, underwent TI-201 and I-123 MIBG cardiac scintigraphy. Although no perfusion defect was identified by Tl-201, mild heterogeneity of I-123 MIBG uptake was present in the myocardium. The denervated but viable myocardium was demonstrated in the heart with sarcoidosis. Cardiac sympathetic nerve function was impaired in cardiac sarcoidosis, slightly improved with steroid therapy. I-123 MIBG scintigraphy may be useful to assess extent of myocardial involvement and response to therapy.

3-Iodobenzylguanidine↗

PubMed2001

Retinal regionalization and heterogeneity of butterfly eyes.

The regional characteristics of the eyes of butterflies from different families have been surveyed using epi-illumination microscopy, utilizing the eyeshine visible due to the tapetum situated proximally to the rhabdom. All butterflies studied have a high spatial acuity in the frontal region. The facet diameter varies slightly across the eye, and the interommatidial angle and the eye parameter p are especially large dorsally. Whereas the ommatidial lattice is generally highly regular, the eyeshine colours distinctly depend on the species. Sometimes the eyeshine is locally uniform, but often it is heterogeneous. It is hypothesized that the regional characteristics as well as the local heterogeneity are adaptations that optimize spectral discrimination.

Animals↗

PubMed2001

Stapedial reflex in myotonic dystrophy type 1 and CTG repeat expansion.

The severity of clinical symptoms of myotonic dystrophy type 1 (DM1) has been shown to be closely related to the size of a CTG triplet repeat in the gene encoding myotonin protein kinase. Neuro-otological examinations that include eye movement and stapedial reflex (SR) tests can contribute to the quantitative evaluation of muscular involvement in DM1. We previously found that saccadic eye movement velocity in DM1 patients was significantly lower than that in control subjects and that the saccadic velocity and size of the CTG triplet repeat in DM1 patients had a strong inverse correlation. We now report a case-control study that compared the SR wave form (latency: L, contraction time: C50, and relaxation time: D50) measured by the acoustic impedance method in 13 patients with DM1 and in 14 control subjects matched for age and sex. The correlation between the SR wave form and CTG repeat length in DM1 patients obtained by Southern blot analysis with EcoRI was also examined. We found (1) no significant difference between the pure tone audiometric threshold at 500 Hz in the DM1 patients and that in the control subjects; (2) or between the SR thresholds in the patients and controls (500 Hz stimuli); (3) C50 and D50 in DM1 patients to be significantly prolonged, whereas L was not; (4) C50 and D50 in DM1 patients to be significantly correlated with CTG repeat length, whereas L was not. Measurement of SR by the acoustic impedance method is completely non-invasive, causes no discomfort to the subject, and does not depend on the person's effort or cooperation. Our findings show that SR measurement can be used for a quantitative evaluation of muscle involvement in DM1. We believe that the prolongation of D50 in DM1 is caused by myotonia, which has to be confirmed by further clinical and pathological studies.

Adult↗

PubMed2001

Transgenic medaka overexpressing a melanin-concentrating hormone exhibit lightened body color but no remarkable abnormality.

Melanin-concentrating hormone (MCH) is a cyclic heptadecapeptide that concentrates melanin granules in the melanophores and lightens the body color of a fish. To investigate the utility of MCH as a reporter gene, a transgenic medaka strain overexpressing the MCH gene was established and its phenotypic features were examined. The salmon MCH gene driven by cytomegalovirus promoter was injected into 100 fertilized eggs of the HNI-1 medaka strain, which exhibits black body color. One F(0) female transmitted the transgene and a lightened body color phenotype to the F(1) generation. A homozygous transgenic strain was established by crossing F(2) fish homozygous for the transgene. Expression of the transgene was detected in several organs by Northern blotting. The melanin granules of transgenics were highly shrunk. Bioassay using scales confirmed the secretion of MCH into blood, and the MCH concentration was estimated between 0.5 and 5 microM. Development, growth, feeding behavior, and reproduction of transgenics did not differ significantly among transgenic and nontransgenic siblings. The result whereby enhanced MCH expression induced a change in body color, but no remarkable abnormality, suggests the usefulness of MCH as a novel reporter gene with unique features.

Journal Article↗

PubMed2001

Chromosomal instability detected by fluorescence in situ hybridization in Japanese breast cancer patients.

The relationship between chromosomal instability (CIN) and prognostic factors was investigated in 31 breast cancers and 5 benign breast lesions (three fibroadenomas and two papillomas). Using fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes of chromosomes 1, 2, 6, 7, 10, 11, 17 and 18, CIN for each case was determined. CIN varied from 8.1% to 59.3% among the breast cancer patients tested, and was significantly higher than that observed in the benign breast lesions (p<0.01). Moreover, CIN showed a significant correlation with lymph node metastases (p<0.05) and estrogen receptor negativity (p<0.01). These findings suggest that CIN might be useful in the prediction of the biological aggressiveness of breast cancers.

Asian People↗

PubMed2001

Effect of spironolactone on plasma brain natriuretic peptide and left ventricular remodeling in patients with congestive heart failure.

OBJECTIVES: We sought to evaluate the effects of spironolactone on neurohumoral factors and left ventricular remodeling in patients with congestive heart failure (CHF). BACKGROUND: Aldosterone (ALD) promotes collagen synthesis and structural remodeling of the heart. Spironolactone, an ALD receptor antagonist, is reported to reduce mortality in patients with CHF, but its influence on left ventricular remodeling has not been clarified. METHODS: Thirty-seven patients with mild-to-moderate nonischemic CHF were randomly divided into two groups that received treatment with spironolactone (n = 20) or placebo (n = 17). We measured left ventricular volume and mass before treatment and after four months of treatment. We also measured the plasma levels of neurohumoral factors, such as atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), as well as plasma procollagen type III aminoterminal peptide (PIIINP), a marker of myocardial fibrosis. RESULTS: Left ventricular volume and mass were significantly decreased and ejection fraction was significantly increased in the spironolactone group, while there were no changes in the placebo group. Plasma levels of ANP, BNP and PIIINP were significantly decreased after spironolactone treatment, but were unchanged in the placebo group. There was a significant positive correlation between the changes of PIIINP and changes of the left ventricular volume index (r = 0.45, p = 0.045) as well as the left ventricular mass index (r = 0.65, p = 0.0019) with spironolactone treatment. CONCLUSIONS: These findings indicate that four months of treatment with spironolactone improved the left ventricular volume and mass, as well as decreased plasma level of BNP, a biochemical marker of prognosis and/or ventricular hypertrophy, suggesting that endogenous aldosterone has an important role in the process of left ventricular remodeling in nonischemic patients with CHF.

Aged↗

PubMed2001

Bradykinin stimulates the release of tissue plasminogen activator in human coronary circulation: effects of angiotensin-converting enzyme inhibitors.

OBJECTIVES: The goal of this study was to determine: 1) whether bradykinin (BK) directly stimulates tissue plasminogen activator (tPA) secretion in human coronary circulation, and 2) whether angiotensin-converting enzyme (ACE) inhibition favorably alters the fibrinolytic balance regulated by BK. BACKGROUND: Bradykinin is a potent stimulator of tPA secretion in endothelial cells; however, the effect of BK on tPA release in the human coronary circulation has not been studied. METHODS: Fifty-six patients with atypical chest pain were randomly assigned to two groups: 25 patients were treated with the ACE inhibitor enalapril (ACE inhibitor group), and 31 were not treated with ACE inhibitors (non-ACE inhibitor group). Graded doses of BK (0.2, 0.6, 2.0 microg/min), acetylcholine (ACh) (30 microg/min) and papaverine (PA) (12 mg) were administered into the left coronary artery. Coronary blood flow (CBF) was evaluated by Doppler flow velocity measurement. Blood samples were taken from the aorta (Ao) and the coronary sinus (CS). RESULTS: Bradykinin induced similar increases in CBF in both groups. The net tPA release induced by BK was dose-dependently increased in both groups, and the extent of that increase in the ACE inhibitor group was greater than that in the non-ACE inhibitor group. Bradykinin did not alter plasminogen activator inhibitor-1 (PAI-1) levels in the Ao or CS in either group. Neither ACh nor PA altered tPA levels or PAI-1 levels in either group. CONCLUSIONS: Intracoronary infusion of BK stimulates tPA release without causing any change in PAI-1 levels in the human coronary circulation. In addition, this effect of BK is augmented by an ACE inhibitor.

Acetylcholine↗

PubMed2001

Doxazosin, an alpha1-adrenergic antihypertensive agent, decreases serum oxidized LDL.

Oxidized low-density lipoprotein (ox-LDL) is well known to play an important role in atherogenesis through the recruitment of monocytes into vessel walls and the deposition of cholesterol ester in the macrophages, which leads to the formation of lipid-rich plaque. It was assumed that only trace amounts of ox-LDL were present in plasma because the half-life of ox-LDL was only a few minutes. Recently, through the use of a monoclonal antibody against ox-LDL, a quantitative method to measure serum ox-LDL concentration has been developed. Metabolites of doxazosin, an alpha1-adrenergic antihypertensive agent, have been reported to inhibit oxidation of LDL in vitro. In this study, we investigated the in vivo effect of doxazosin on LDL oxidation using this new method to measure serum ox-LDL concentration. After the administration of doxazosin for 1 to 2 months, serum concentration of ox-LDL decreased significantly (P < .05). Although the reduction of ox-LDL concentration does not strictly indicate doxazosin's antiatherosclerotic effect, it may constitute one of doxazosin's additional weapons beside lowering blood pressure and serum lipid values in the prevention of atherosclerosis.

Adrenergic alpha-Antagonists↗

PubMed2001

Bupivacaine hydrochloride induces muscle fiber necrosis and hydroxyl radical formation-dimethyl sulphoxide reduces hydroxyl radical formation.

We induced acute skeletal muscle necrosis in rats using bupivacaine hydrochloride and found that both 2,5- and 2,3-dihydroxybenzoic acid significantly increased in skeletal muscle. A single administration of dimethyl sulphoxide, a free radical scavenger, significantly lowered concentrations of 2,5- and 2,3-dihydroxybenzoic acid. These results suggest that dimethyl sulphoxide is an effective hydroxyl radical scavenger and may be useful in the treatment of myopathy.

Anesthetics, Local↗

PubMed2001

Borg proteins control septin organization and are negatively regulated by Cdc42.

The Cdc42 GTPase binds to numerous effector proteins that control cell polarity, cytoskeletal remodelling and vesicle transport. In many cases the signalling pathways downstream of these effectors are not known. Here we show that the Cdc42 effectors Borg1 to Borg3 bind to septin GTPases. Endogenous septin Cdc10 and Borg3 proteins can be immunoprecipitated together by an anti-Borg3 antibody. The ectopic expression of Borgs disrupts normal septin organization. Cdc42 negatively regulates this effect and inhibits the binding of Borg3 to septins. Borgs are therefore the first known regulators of mammalian septin organization and provide an unexpected link between the septin and Cdc42 GTPases.

Amino Acid Sequence↗