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Biomedical subjects

H Nishio

Publications and source records attributed to H Nishio.

At least 19 recordsLinked to original sources
PubMed1996

Ca(2+)-independent fusion of synaptic vesicles with phospholipase A2-treated presynaptic membranes in vitro.

To clarify the mechanism of exocytosis in neurotransmitter release, the fusion of synaptic vesicles with presynaptic membranes prepared from rat brain synaptosomes and concomitant acetylcholine (ACh) release induced by fusion of them were studied in vitro. Fusion of the synaptic vesicles with presynaptic membranes was measured by a fluorescence-dequenching assay with octadecyl rhodamine B. Synaptic vesicles fused with presynaptic membranes which had been pretreated with porcine phospholipase A2 (PLA2) in the presence of 20 microM Ca2+ and released ACh, whereas synaptic vesicles did not interact with non-pretreated membranes. The fusion followed by ACh release depended (i) on the activity of PLA2 during the membrane pretreatment, (ii) on the amount of pretreated membrane and (iii) on the duration of the pretreatment. The presence of Ca2+ ions during the pretreatment was essential for inducing a fusogenic activity of the membranes, but Ca2+ ions were not required for the fusion itself because the fusion experiment was carried out in the presence of 5mM EGTA without added Ca2+. The presence of quinacrine, an antagonist of PLA2, during the membrane pretreatment inhibited their fusogenic activity, suggesting the importance of activation of PLA2. Presence of albumin during the pretreatment, which is an adsorbent of free fatty acids, also inhibited the fusogenic activity. Arachidonic acid, when added during the pretreatment, potentiated the fusogenic activity of the membrane. These findings suggest that the conformational change in the presynaptic membrane phospholipids induced by PLA2 and the presence of arachidonic acid produced by PLA2 are important in the process of fusion of synaptic vesicles with the presynaptic membranes of rat brain, and that the fusion process itself is independent of Ca2+.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1996

Complete structure of the human alpha-albumin gene, a new member of the serum albumin multigene family.

The nucleotide sequence of the human alpha-albumin gene, including 887 bp of the 5'-flanking region and 1311 bp of the 3-flanking region (24,454 in total), was determined from three overlapping lambda phage clones. The sequence spans 22,256 bp from the cap site to the polyadenylylation site, revealing a gene structure of 15 exons separated by 14 introns. The methionine initiation codon ATG is within exon 1; the termination codon TGA is within exon 14. Exon 15 is entirely untranslated and contains the polyadenylylation signal AATAAA. The deduced polypeptide chain is composed of a 21-amino-acid leader peptide, followed by 578 amino acids of the mature protein. There are seven repetitive DNA elements (Alu and Kpn) in the introns and 3-flanking region. The sizes of the 15 alpha-albumin exons match closely those of the albumin, alpha-fetoprotein, and vitamin D-binding protein genes. The exons are symmetrically placed within the three domains of the individual proteins, and they share a characteristic codon splitting pattern that is conserved among members of the gene family. The results provide strong evidence that alpha-albumin belongs to, and most likely completes with, the serum albumin gene family. Based on structural similarity, alpha-albumin appears to be most closely related to alpha-fetoprotein. The complete structure of this family of four tandemly linked genes provides a well-characterized approximately 200 kb locus in the 4q subcentromeric region of the human genome.

Albumins↗
PubMed1996

Role of cytokines in leukemic type growth of myelodysplastic CD34+ cells.

The clonal growth of progenitor cells from myelodysplastic syndromes (MDS) can be subdivided into four growth patterns: (1) normal, (2) no growth or low plating efficiency, (3) low colony and high cluster number, and (4) normal or high colony number with a large number of clusters. The former two (1 and 2) can be referred to as nonleukemic patterns and latter two (3 and 4) as leukemic. In a search for a role for cytokines in leukemic-type growth of MDS progenitor cells, marrow CD34+ cells were purified up to 94% for 8 normal individuals and 88% for 12 MDS patients, using monoclonal antibodies and immunomagnetic microspheres (MDS CD34+ cells). The purified CD34+ cells were cultured for 14 days with various combinations of cytokines, including recombinant human macrophage colony-stimulating factor (rM-CSF), granulocyte-CSF (rG-CSF), granulocyte-macrophage-CSF (rGM-CSF), interleukin-3 (rIL-3), and stem cell factor (SCF; a ligand for c-kit) in serum-free medium. The clonal growth of MDS CD34+ cells supported by a combination of all of the above cytokines was subdivided into the two patterns of leukemic or nonleukemic, and then the role of individual or combined cytokines in proliferation and differentiation of MDS CD34+ cells was analyzed in each group. Evidence we obtained showed that SCF plays a central role in the leukemic-type growth of MDS CD34+ cells and that G-CSF, GM-CSF; and/or IL-3 synergize with SCF to increase undifferentiated blast cell colonies and clusters over that seen in normal CD34+ cells. SCF is present in either normal or MDS plasma at a level of nanograms per milliliter, and this physiologic concentration of SCF can stimulate progenitor cells. This means that progenitor cells are continuously exposed to stimulation by SCF in vivo and that MDS leukemic cells have a growth advantage over normal blast cells. This depends, at least in part, on cytokines such as G-CSF, GM-CSF, IL-3, and SCF.

Acute Disease↗
PubMed1996

Tandem arrangement of the human serum albumin multigene family in the sub-centromeric region of 4q: evolution and chromosomal direction of transcription.

The albumin gene family is comprised of four genes encoding: serum albumin (ALB), alpha-fetoprotein (AFP), alpha-albumin (ALF), and vitamin D-binding protein (DBP; also known as GC). The genes are regulated developmentally, expressed in the liver, and the proteins are secreted into the bloodstream. The GC gene, and the tandemly linked ALB and AFP genes, have been previously localized to human chromosome 4q11-13. Using techniques of fluorescence in situ hybridization to chromatin fibres, chromosome walking and DNA sequencing of genomic clones, we now report on the chromosomal location of the ALF gene and the organization of the entire gene family. The four genes are tandemly linked in the 4q sub-centromeric region: 5'ALB-5'AFP-5'ALF-5'GC3'-centromere, and hence are transcribed in the same, centromere-bound, direction. The linear arrangement of the four genes along the chromosome is not correlated with their temporal expression in the human ontogeny. It appears that GC is very close (and may be the gene proximal) to the centromere. The linear chromosomal arrangement of the four genes and the structural differences between them are congruent with the following evolutionary divergence of the gene family. Starting with the first duplication of an ancestral progenitor gene, a single evolutionary line led to the contemporary GC, leaving ALB/AFP/ALF on the other line of descent. The second duplication occurred in this ALB lineage, giving rise to ALB and the AFP/ALF progenitor, and the third, most recent one, gave rise to the AFP-ALF pair.

Albumins↗
PubMed1996

A common mutation in methylenetetrahydrofolate reductase gene among the Japanese population.

Hyperhomocysteinemia has been reported as an independent risk factor for atherosclerotic cerebrovascular and coronary heart diseases. 5, 10-Methylenetetrahydrofolate reductase (MTHFR) is one of the enzymes responsible for hyperhomocysteinemia. The C to T transition of the MTHFR gene at nucleotide position 677 results in decreasing the enzymatic activity and increasing the plasma homocysteine level. We studied the distribution of the MTHFR gene mutation among the Japanese population. The subjects were 129 Japanese males (aged 40-59 years). The allele frequency of the mutation was 0.38. The frequencies of the three genotypes were as follows: +/+, 11%; +/-, 54%; -/-, 35% (+ and-indicate the presence and absence of the mutation, respectively). We also studied the frequency of the MTHFR gene mutation in the middle-aged Japanese males with hypertension to investigate the possibility that this mutation is related to essential hypertension. The normotensive and hypertensive subjects were identical in the distribution of the mutated allele and the frequencies of the three genotypes. Furthermore, the prevalence of hypertension in each genotype group was same, although the mean diastolic pressure of the group with homozygous mutation was significantly higher than that of other groups (p < 0.05). Therefore, we concluded that there was no significant relationship between the MTHFR gene mutation and hypertensive subjects studied in this study.

Adult↗
PubMed1996

Re-examination for pharmacological properties of serotonin-induced tachycardia in isolated guinea-pig atrium.

In the presence of 10(-6) M atropine, 5-HT induced a positive chronotropic and inotropic effect in isolated guinea-pig atrium preparation. Both propranolol, a beta-adrenoceptor blocker, and imipramine, a 5-HT uptake inhibitor, did not affect the response induced by 5-HT, indicating that a tyramine-like mechanism is not involved. The positive chronotropic effect of 5-HT was mimicked by several 5-HT3 receptor agonists. N omega-Methyl-5-HT, reported to have a high affinity for 5-HT1B recognition sites, was found to act as a 5-HT3 receptor agonist with higher efficacy. The other 5-HT receptor agonists tested did not produced any responses. The positive chronotropic effect of 5-HT was inhibited by various 5-HT3 receptor antagonists, such as tropisetron, granisetron, ondansetron, cisapride and zacopride, but it was unaffected by various 5-HT receptor antagonists which are not selective for 5-HT3 receptor. Thus, the positive chronotropic response to 5-HT is a direct effect, and it was suggested to be mediated by 5-HT3 receptor subtype with rather an atypical profile.

Adrenergic Uptake Inhibitors↗
PubMed1996

5-HT3 receptor blocking properties of the antiparkinsonian agent, talipexole.

1. Talipexole showed moderate displacement activity of 3H-GR 65630 binding to 5-HT3 receptors in both rat cortical and intestinal membrane fractions with Ki values of 0.35 microM and 0.22 microM, respectively. 2. Bromocriptine failed to displace the binding activity in either experimental system even at a concentration of 10 microM. 3. Both talipexole and tropisetron were found to significantly inhibit 5-HT3 receptor-mediated effects of 5-HT in isolated guinea-pig ileum or atrium; however, the effect of talipexole was weaker than that of tropisetron. 4. Bromocriptine, in contrast, had no antagonistic effects on 5-HT3 receptor-mediated activity in guinea-pig ileum or atrium. 5. It was concluded that talipexole might act as an antagonist on 5-HT3 receptors in both brain and intestinal tissues.

Animals↗
PubMed1996

A Japanese boy with myalgia and cramps has a novel in-frame deletion of the dystrophin gene.

We report a Japanese Becker muscular dystrophy (BMD) patient with occasional myalgia and cramps during normal activity that developed at the age of 28 months. His family history was negative for neuromuscular diseases. Muscle biopsy analyses, including dystrophin immunostaining, disclosed no clinically relevant findings. The diagnosis of BMD was initially made at the age of 10 years, when indications of persistent high serum levels of CK prompted us to screen deletions in the dystrophin gene by amplification of 19 deletion-prone exons from the genomic DNA by the polymerase chain reaction (PCR). Among the exons examined, exons 13 and 17 were deleted. To clarify the size of the deletion, the dystrophin transcript was analyzed by reverse transcription PCR. The determined nucleotide sequence of the amplified product encompassing exons 10 to 20 disclosed that the entire segment corresponding to exons 13 to 18 (810 bp) was absent, a deletion that would be expected to cause the production of a dystrophin protein lacking 270 amino acids from the rod domain. This result indicates that occasional myalgia and cramps could be early clinical manifestations of mild BMD, especially in patients who have a deletion in the rod domain, and that deletion screening of the dystrophin gene might be the only reliable method to diagnose such cases.

Base Sequence↗
PubMed1996

[Benign infantile mitochondrial myopathy caused by reversible cytochrome c oxidase deficiency].

A 2-month-old girl had generalized weakness, profound muscular hypotonia, hepatomegaly and severe lactic acidosis. She needed ventilatory support. Muscle specimen taken at 2 months showed ragged-red fibers, abnormal mitochondria, and reduced cytochrome c oxidase (CCO) staining Biochemical analysis showed CCO activity to be reduced to about 16% of the normal mean. She received carnitine and coenzyme Q10 supplementation from the age of 3 months and abnormal blood lactate values declined to near normal values during the first three weeks. Gradually her condition started to improved: she held her head at 9 months, and walked alone at 15 months. The second biopsy specimen at 3 years and 8 months showed almost normal CCO staining and she was free of clinical signs. This case is an example of a rare benign infantile mitochondrial myopathy caused by CCO deficiency. Early diagnosis is crucial to provide intensive treatment until spontaneous clinical improvement appears. We concluded that carnitine and coenzyme Q10 supplementation was a useful treatment for clinical improvement in patients with a benign infantile mitochondrial myopathy caused by CCO deficiency.

Carnitine↗
PubMed1995

The chimpanzee alpha-fetoprotein-encoding gene shows structural similarity to that of gorilla but distinct differences from that of human.

The chimpanzee (Pan troglodytes) alpha-fetoprotein (AFP)-encoding gene (AFP) spans 18,867 bp from the transcription start point to the polyadenylation site, and the nucleotide (nt) sequence reveals that the gene is composed of 15 exons, which are symmetrically placed within three domains of AFP. In addition, we report 3121 bp of 5'-flanking sequence and 4886 bp of 3'-flanking sequence. The entire 26,874 bp of contiguous DNA reported here was determined from three overlapping lambda phage clones. The deduced polypeptide chain is composed of a 19-amino-acid (aa) putative leader peptide, followed by 590 aa of the mature protein. The sequence of chimpanzee AFP was compared with those of the previously published human AFP [Gibbs et al., Biochemistry 26 (1987) 1332-1343] and gorilla AFP [Ryan et al., Genomics 9 (1991) 60-72]. At the aa level, the human AFP differs from the chimpanzee at 6 aa positions and from the gorilla at 4 aa positions; the chimpanzee and gorilla differ at 8 aa positions. There are four types of repetitive sequence elements (X, Alu, Xba and Kpn) in the introns and flanking regions of chimpanzee AFP, and they are located in orthologous positions in the human and gorilla AFP. However, one specific Alu and one Xba repeat in introns 4 and 7, respectively, found in human AFP, are absent from orthologous positions in chimpanzee and gorilla AFP. These two repeats represent human-specific novelties that arose from recent DNA transpositions in primate phylogeny.

Amino Acid Sequence↗
PubMed1995

Ca(2+)-independent fusion of secretory granules with phospholipase A2-treated plasma membranes in vitro.

The fusion of secretory granules with plasma membranes prepared from rat parotid gland was studied in vitro to clarify the mechanism of exocytosis. Fusion of the granules with plasma membranes was measured by a fluorescence-dequenching assay with octadecyl rhodamine B, and release of amylase was also measured to confirm the fusion as a final step of the secretory process. Plasma membranes that had been pretreated with porcine phospholipase A2 (PLA2) in the presence of 20 microM Ca2+ fused with the granules within 30 s, and induced amylase release by reacting with the membranes of granules, whereas without this pretreatment they had no significant effect. The fusion process accompanied by amylase release was induced in the presence of 10 mM EGTA, and therefore was apparently Ca(2+)-independent. On the other hand, the presence of EGTA or 100 microM quinacrine, an inhibitor of PLA2, during treatment of plasma membranes with PLA2 inhibited their fusogenic activity, suggesting the importance of activation of PLA2. Arachidonic acid and linoleic acid were released from the plasma membranes during the PLA2 treatment. The presence of albumin, an adsorbent of fatty acids, during the treatment also inhibited the activity. Pretreatment of the membranes with arachidonic acid or linoleic acid did not have any effect, but the presence of exogenously added arachidonic acid during PLA2 treatment enhanced the membrane-fusion-inducing effect of PLA2. Pretreatment of the membranes with lysophosphatidylcholine induced fusogenic activity. These findings suggest that the conformational change in the plasma-membrane phospholipids induced by PLA2 and the presence of arachidonic acid or linoleic acid produced by PLA2 are important in the process of fusion of secretory granules with the plasma membranes of rat parotid acinar cells and that the fusion process itself is independent of Ca2+.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1995

pp60c-src expression in rat spermatogenesis.

A fractionation of the membrane extract of rat testes revealed the existence of pp60c-src kinase activity. The expression of pp60c-src was examined in the developing rat testes. The immunecomplex kinase assay using a monoclonal antibody specific to pp60c-src (mAb327) showed that the expression of pp60c-src kinase activity increased during the development of rat testes and declined in the adult. The increase in pp60c-src kinase activity observed during the development of rat testes was accompanied with an increase in the amount of pp60c-src protein. The peak period in the increase of pp60c-src kinase activity well coincided with the timing, when the spermatogenesis by meiosis just began. The immunohistochemical staining of pp60c-src in rat testes demonstrated that pp60c-src is most abundantly expressed in the spermatids which are the spermatogenic cells in the post-meiotic phase of the spermatogenesis. These findings strongly suggest that pp60c-src is a developmentally regulated gene product which is involved in rat spermatogenesis.

Amino Acid Sequence↗
PubMed1995

Role of calcium ion in platelet serotonin uptake regulation.

It is generally accepted that intracellular Ca2+ is a key substance in the intracellular signal transducing mechanism of platelets. We investigated the possibility that extracellular and/or intracellular Ca2+ might regulate the transport activity of serotonin (5-HT) into platelets. We found that extracellular Ca2+ chelation with EGTA caused inhibition of 5HT uptake activity, which was recovered by extracellulary applied excess Ca2+. Intracellular Ca2+ chelation with acetoxymethyl bis(O-aminophenoxy)ethane-N,N,N'-tetraacetate (BAPTA-AM) did not, however, have any inhibitory effect on 5HT uptake activity in the presence of extracellular Ca2+. In the absence of extracellular Ca2+, BAPTA-AM significantly inhibited 5-HT uptake. The restorative effect of Ca2+ on 5-HT transport into EGTA-treated platelets was mimicked by Ba2+, but not by Sr2+. It was antagonised by inorganic Ca2+ channel antagonist including Ni2+, La3+ and Gd3+, but not by organic Ca2+ channel blockers including verapamil, nifedipine, diltiazem, omega-conotoxin GVIA and omega-agatoxin IVA. Furthermore, 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester hydrochloride (TMB-8), an intracellular Ca2+ antagonist, was found to inhibit the restorative effect of Ca2+. These results have led to the suggestion that depletion of intracellular Ca2+ pool(s) by EGTA might result in a reduction of 5-HT uptake activity. Thus, the intracellular Ca2+ pool(s) susceptible to EGTA might have a regulatory role in maintaining 5-HT transport into blood platelets.

Animals↗
PubMed1995

Relation of angiographically defined coronary artery disease and plasma concentrations of insulin, lipid, and apolipoprotein in normolipidemic subjects with varying degrees of glucose tolerance.

We investigated the association between hyperinsulinemia and changes in lipid, lipoprotein, and apolipoprotein that would increase the risk of coronary artery disease (CAD) independent of glucose tolerance. A coronary angiogram was recorded in 127 male subjects, including 41 with normal glucose tolerance, 41 with impaired glucose tolerance, and 45 with non-insulin-dependent diabetes mellitus (NIDDM). Subjects were divided into 2 groups according to results: the group with CAD (n = 94) and the group with normal coronary arteries (n = 33). All subjects were normolipidemic (total cholesterol < 230 mg/dl and triglycerides < 150 mg/dl). The CAD group had a significantly lower plasma level of high-density lipoprotein (HDL) cholesterol and apolipoprotein A-I (apo A-I) and a higher level of apolipoprotein B (apo B) than the normal group with normal glucose tolerance. In considering subjects with impaired glucose tolerance or NIDDM, the CAD group had a significantly lower plasma level of HDL cholesterol and apo A-I and a significantly higher plasma level of total cholesterol, triglycerides, and apo B than the normal group. In each of the subjects with normal and impaired glucose tolerance, and NIDDM, the elevation of plasma insulin concentration during both the complete test period and the early phase of an oral glucose challenge was significantly higher in the CAD than in the normal group. In all subjects, graded reductions in HDL cholesterol and apo A-I and graded increases in plasma total cholesterol, triglycerides, and apo B were observed with increasing tertiles of the postglucose challenge measurements of insulinemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein A-I↗
PubMed1995

Modulation of in vitro splicing of the upstream intron by modifying an intra-exon sequence which is deleted from the dystrophin gene in dystrophin Kobe.

Molecular analysis of dystrophin Kobe showed that exon 19 of the dystrophin gene bearing 52-bp deletion was skipped during splicing, although the known consensus sequences at the 5' and 3' splice sites of exon 19 were maintained (Matsuo, M., T. Masumura, H. Nishio, T. Nakajima, Y. Kitoh, T. Takumi, J. Koga, and H. Nakamura. 1991. J. Clin. Invest. 87:2127-2131). These data suggest that the deleted sequence of exon 19 may function as a cis-acting element for exact splicing for the upstream and downstream introns. To investigate this potential role of exon 19, an in vitro splicing system using artificial dystrophin mRNA precursors (pre-mRNAs) was established. Pre-mRNA containing exon 18, truncated intron 18, and exon 19 was spliced precisely in vitro, whereas splicing of intron 18 was almost completely abolished when the wild-type exon 19 was replaced by the dystrophin Kobe exon 19. Splicing of intron 18 was not fully reactivated when dystrophin Kobe exon 19 was restored to nearly normal length by inserting other sequences into the deleted site. These results suggest that the presence of the exon 19 sequence which is lost in dystrophin Kobe is more critical for splicing of intron 18 than the length of the exon 19 sequence. Characteristically, the efficiency of splicing of this intron seemed to correlate with the presence of polypurine tracks within the downstream exon 19. Moreover, an antisense 31-mer 2'-O-methyl ribonucleotide complementary to the 5' half of the deleted sequence in dystrophin Kobe exon 19 inhibited splicing of wild-type pre-mRNA in a dose- and time-dependent manner. This first in vitro evidence that dystrophin pre-mRNA splicing can be modulated by an antisense oligonucleotide raises the possibility of a new therapeutic approach for Duchenne muscular dystrophy.

Base Sequence↗
PubMed1995

Synthesis of the esterified cerebroside in the epidermis of guinea pigs.

(2S,3R,4E,23'Z)-1-O-(beta-D-Glucopyranosyl)-N-(32'-linoleoyloxy -23'-dotriacontenoyl)-4-sphingenine (1), a component of the esterified cerebrosides in the epidermis of guinea pigs, was synthesized by selective N-acylation of (2S,3R,4E)-1-O-(beta-D-glucopyranosyl)-4-sphingenine (18) with activated omega-linoleoyloxy fatty acid ester 17.

Acylation↗