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Biomedical subjects

M Nishimura

Publications and source records attributed to M Nishimura.

At least 451 records · Page 25Linked to original sources
PubMed1996

Structure and expression of two highly related genes encoding SCM-1/human lymphotactin.

SCM-1/lymphotactin is a chemokine-like molecule produced selectively, if not exclusively, by activated CD8+ T cells. Here we report that there are two highly homologous SCM-1 genes, which we designate as SCM-1alpha and SCM-1beta. Both genes have three exons and two introns. The 1st intron of SCM-1alpha contains a pseudogene of the ribosomal large subunit L7a. In SCM-1beta, a 1.5-kb region including about a quarter of the L7a pseudogene is deleted from the 1st intron. Otherwise, the two genes are highly homologous including the 5' and 3' flanking regions. Both genes were mapped to human chromosome 1q23. The two genes were similarly induced in peripheral blood mononuclear cells by mitogenic stimulation. Primer extension and RNase protection revealed several transcription initiation sites. The biological activities of SCM-1alpha and SCM-1beta, which have two amino acid differences at positions 7 and 8 in the mature proteins, remain to be compared.

Amino Acid Sequence↗
PubMed1996

Formation of crossline as a fluorescent advanced glycation end product in vitro and in vivo.

Crossline is one of the major advanced glycation end products resulting the reaction mixture of free amino group(s) such as epsilon-one in lysine with D-glucose in vitro. To study crossline formation on proteins in vitro and in vivo, polyclonal antiserum to the crossline hapten was prepared. This antiserum reacted with bovine and human serum albumin that had been modified by prolonged incubation with glucose as well as with crossline itself. Antisera did not react with unmodified serum albumin or the other Maillard-related compounds. Crossline was formed in a time-dependent manner when a mixture of six different proteins was incubated with glucose at pH 7.2 or 9.0. Crossline levels could be measured in rat lens proteins and the levels increased with age. The crossline content of lens proteins in diabetic rats was more than two-fold higher than that of age-matched controls. Results of this study suggest that most proteins containing advanced glycation end products have crossline-like structures. Measurement of crossline-like structures in biological specimens may provide an index of aging and of the development of diabetic complications.

Animals↗
PubMed1996

Post-infectious encephalitis with anti-galactocerebroside antibody subsequent to Mycoplasma pneumoniae infection.

Galactocerebroside (Gc) is a major component of myelin in both the peripheral and central nervous systems. Although it is regarded as an important glycolipid hapten of myelin in rabbit experimental allergic neuritis (EAN), its role in human demyelinating diseases is not known. We studied three post-infectious encephalitis (PIE) patients related to Mycoplasma pneumoniae infection. All three of three patients with encephalitis and M. pneumoniae infection were positive for Gc antibodies (100%), while 25% of 32 M. pneumoniae-infected patients without neurological disease were positive, and 3.8% of 52 healthy controls. This indicates anti-Gc antibody is induced by M. pneumoniae infection. One of the PIE patients, who had extraordinary high titer antibody to Gc, showed an extensive, diffuse white matter demyelination and poor recovery. Since circulating anti-Gc antibody induces central nervous system demyelination in animals with elevated antibody titers and disruption of the blood-brain barrier, anti-Gc antibody may have an important function in the increased demyelination in PIE patients after M. pneumoniae infection.

Antibodies↗
PubMed1996

PCR-based restriction fragment length polymorphism (RFLP) analysis and serotyping of Campylobacter jejuni isolates from diarrheic patients in China and Japan.

A molecular typing approach for Campylobacter jejuni was applied with restriction fragment length polymorphism (RFLP) analysis of a 702-bp PCR-amplified portion of the flagellin-A (flaA) gene. We analyzed a total of 179 strains, including 69 independent clinical isolates from diarrheic patients in Japan, 85 isolates in China, and 25 heat-stable (HS) serotype strains by Penner and Hennessy (1980) J. Clin. Microbiol. 12, 732-737). Six AfaI, seven MboI, and five HaeIII RFLPs were found in the 702-bp flaA segment from the 179 strains. Using a combination of these three enzymes, 25 separate RFLP groups were recognized. While 59 of 154 (38.3%) strains obtained in Japan and China were nontypeable by the HS antigenic scheme, all but two of 154 (98.7%) could be typed by RFLP typing. All 11 isolates of HS-19 strains, which are frequently isolated from Guillain-Barré syndrome (GBS) patients, showed an identical RFLP pattern (Cj-1), and Cj-1 consisted only of HS-19 strains. This suggests that the HS-19:Cj-1 strain is distinct among C. jejuni strains. This molecular typing method provides a rapid and reliable typing scheme for epidemiological studies of C. jejuni, and may also be useful for the analysis of C. jejuni subtypes from GBS patients.

Bacteriological Techniques↗
PubMed1996

Five candidate genes for hamster cardiomyopathy did not map to the cardiomyopathy locus by FISH analysis.

The Syrian cardiomyopathic hamster (BIO14.6), that develops both muscular dystrophy and progressive cardiomyopathy, is widely used as an animal model of autosomal recessive cardiomyopathy mimicking human hypertrophic cardiomyopathy, and five genes have been proposed as strong candidates for the cause of cardiomyopathy. We recently mapped the cardiomyopathy locus of the hamster to the centromeric region of chromosome 9qa2.1-b1 by construction of a genetic linkage map of the Syrian hamster. Thus, we analyzed the loci of the five candidate genes, alpha tropomyosin, cardiac troponin T, adhalin, calpain 3 and cardiac myosin binding protein-C, by the FISH method, and found that these genes were mapped on the distal portion of chromosome 12qa5 and 4pa2 and the proximal portion of chromosomes 9qb7, 1qc1.1 and 1qb3, respectively. These results provide strong evidence that the five candidate genes previously proposed are not related to the hamster cardiomyopathy.

Animals↗
PubMed1996

Molecular cloning of a novel T cell-directed CC chemokine expressed in thymus by signal sequence trap using Epstein-Barr virus vector.

Precursors of most secreted and cell surface molecules carry signal sequences at their amino termini. Here we describe an efficient signal sequence trap method and isolation of a novel CC chemokine. An expression library was constructed by inserting 5' portion-enriched cDNAs from phytohemagglutinin-stimulated peripheral blood mononuclear cells into upstream of signal sequence-deleted CD4 cDNA in an Epstein-Barr virus shuttle vector. After electroporation into Raji cells, CD4 antigen-positive cells were enriched by repeated cell sorting and plasmids were recovered in Escherichia coli. Out of 100 plasmid clones examined, 42 clones directed expression of CD4 antigen on the cell surface. Among them were signal sequences of CD6, beta2-microglobulin, MGC-24, and T cell receptor epsilon-chain, and at least four novel potential signal sequences. A cDNA clone encoding a novel CC chemokine was isolated by using one of the trapped fragments. The gene designated as TARC from Thymus and Activation-Regulated Chemokine was expressed transiently in phytohemagglutinin-stimulated peripheral blood mononuclear cells and constitutively in thymus. Radiolabeled recombinant TARC specifically bound to T cell lines and peripheral T cells but not to monocytes or granulocytes. The binding of radiolabeled TARC to the high-affinity receptor (Kd, 2.1 nM) on Jurkat was displaced by TARC but not by interleukin-8, MIP-1alpha, RANTES, or MCP-1. TARC also bound to the promiscuous chemokine receptor on erythrocytes (Kd, 17 nM). TARC induced chemotaxis in T cell lines Hut78 and Hut102. Pretreatment of Hut78 with pertussis toxin abolished the TARC-induced cell migration. Collectively, T cells express a highly selective receptor for TARC that is coupled to pertussis toxin-sensitive G-protein. TARC may a factor playing important roles in T cell development in thymus as well as in trafficking and activation of mature T cells.

Amino Acid Sequence↗
PubMed1996

Synchronous onset of NGF and TrkA survival dependence in developing dorsal root ganglia.

Determinations of dorsal root ganglion (DRG) neuron loss in nerve growth factor (NGF) and neurotrophin-3 (NT-3) null mutant mice have supported the concept that neurons can switch neurotrophin dependence by revealing that many neurons must require both of these factors acting either sequentially or simultaneously during development. The situation is complex, however, in that NT-3(-/-) mutant mice show far greater neuron loss than mice deficient in the NT-3 receptor TrkC, suggesting that NT-3 may support many DRG neurons via actions on the NGF receptor TrkA. To assess the possibility of ligand-receptor cross-talk as a developmental mechanism, we have compared the onset of survival dependence of lumbar DRG neurons on NT-3, TrkC, NGF, and TrkA signaling in mice deficient in these molecules as a result of gene targeting. At embryonic day 11.5 (E11.5), virtually all lumbar DRG cells express TrkC mRNA and many require NT-3 and TrkC signaling for survival. In contrast, although many lumbar DRG cells also express TrkA at E11.5, there is little survival dependence on TrkA signaling. By E13.5, most lumbar DRG cells have downregulated TrkC mRNA. The onset of survival dependence on NGF and TrkA-signaling is concurrent and of equal magnitude at E13.5, demonstrating that NT-3 alone does not support DRG neurons via TrkA, nor can NT-3 compensate for the loss of NGF. We conclude that many murine DRG cells require NT-3 activation of TrkA is unimportant to these early NT-3 survival-promoting actions. We suggest that the discrepancy in cell loss between NT-3(-/-) and trkC(-/-) mutants is attributable to the ability of NT-3 to support DRG neurons via TrkA in the artificial situation where TrkC is absent.

Animals↗
PubMed1996

Immunological characterization of antibodies against synthetic peptides of glutamic acid decarboxylase.

We characterized antibodies against synthetic N-terminal peptides (glutamic acid decarboxylase; GAD65N and GAD67N) and a C-terminal peptide (GAD67C) of human GAD isoforms. On Western blots, the GAD65N antibody specifically stained the 65 kDa isoform and the GAD67N antibody the 67 kDa one in various mammalian brain tissues, whereas the GAD67C antibody stained both. The immunotrapped GAD enzyme activity increased in a dose-dependent manner with increasing concentration of the N-terminal peptide antibodies, but the activity was completely inhibited by the C-terminal peptide antibody. By an enzyme-linked immunosorbent assay using rat brain GAD purified on a GAD67C antibody-affinity column, we detected GAD antibodies in 40% (24/60) of the patients with long-standing insulin-dependent diabetes mellitus (IDDM). These antipeptide antibodies are a useful tool not only for identifying the GAD isoforms, but also for purifying GAD.

Animals↗
PubMed1996

Costimulation enhances the active immunotherapy effect of recombinant anticancer vaccines.

Activation of T lymphocytes in the absence of a costimulatory signal can result in anergy or apoptotic cell death. Two molecules capable of providing a costimulatory signal, B7-1 (CD80) and B7-2 (CD86), have been shown to augment the immunogenicity of whole-tumor cell vaccines. To explore a potential role for costimulation in the design of recombinant anticancer vaccines, we used lacZ-transduced CT26 as an experimental tumor and beta-galactosidase (beta-gal) as the model tumor antigen. Attempts to augment the function of a recombinant vaccinia virus (rVV) expressing beta-gal by admixture with rVV expressing murine B7-1 were unsuccessful. However, a double recombinant vaccinia virus engineered to express both B7-1 and the model antigen beta-gal was capable of significantly reducing the number of pulmonary metastases when administered to mice bearing tumors established for 3 or 6 days. Most important, the double recombinant vaccinia virus prolonged the survival of tumor-bearing mice. These effects were antigen specific. The related costimulatory molecule B7-2 was found to have a similar, although less impressive enhancing effect on the function of a rVV expressing beta-gal. Thus, the addition of B7-1 and, to a lesser extent, B7-2 to a rVV encoding a model antigen significantly enhanced the therapeutic antitumor effects of these poxvirus-based, therapeutic anticancer vaccines.

Animals↗
PubMed1996

Alterations in basal and glucose-stimulated voltage-dependent Ca2+ channel activities in pancreatic beta cells of non-insulin-dependent diabetes mellitus GK rats.

In genetically occurring non-insulin-dependent diabetes mellitus (NIDDM) model rats (GK rats), the activities of L- and T-type Ca2+ channels in pancreatic beta cells are found to be augmented, by measuring the Ba2+ currents via these channels using whole-cell patch-clamp technique, while the patterns of the current-voltage curves are indistinguishable. The hyper-responsiveness of insulin secretion to nonglucose depolarizing stimuli observed in NIDDM beta cells could be the result, therefore, of increased voltage-dependent Ca2+ channel activity. Perforated patch-clamp recordings reveal that the augmentation of L-type Ca2+ channel activity by glucose is markedly less pronounced in GK beta cells than in control beta cells, while glucose-induced augmentation of T-type Ca2+ channel activity is observed neither in the control nor in the GK beta cells. This lack of glucose-induced augmentation of L-type Ca2+ channel activity in GK beta cells might be causatively related to the selective impairment of glucose-induced insulin secretion in NIDDM beta cells, in conjunction with an insufficient plasma membrane depolarization due to impaired closure of the ATP-sensitive K+ channels caused by the disturbed intracellular glucose metabolism in NIDDM beta cells.

Animals↗
PubMed1996

Cerebral adenosine triphosphate-sensitive K+ channels may be impaired during acute cerebral ischemia in spontaneously hypertensive rats.

To elucidate the role of cerebral adenosine triphosphate (ATP)-sensitive K+ channels (KATP) on arterial pressure regulation during acute cerebral ischemia in spontaneously hypertensive rats (SHR), intracerebroventricular (i.c.v.) injections of either glibenclamide, a specific blocker of KATP, or pinacidil, a KATP opener, were performed in SHR and Wistar-Kyoto rats (WKY). Intracerebroventricular injections of glibenclamide elicited a vasopressor response in WKY with bilateral ligation of the carotid arteries, whereas the response was smaller in SHR. It increased plasma AVP, but decreased pituitary AVP in WKY with ligation, but not in SHR. Systemic administration of an AVP V1 receptor antagonist, OPC-21268, abolished the vasopressor responses to i.c.v. injections of glibenclamide in WKY. Bilateral ligation of the carotid arteries augmented the vasodepressor responses to i.c.v. injections of pinacidil in WKY, but not in SHR. Cerebral KATP may play a role in buffering a rise in arterial pressure by inhibiting the release of AVP from the pituitary glands during acute cerebral ischemia in WKY, but this mechanism might be deranged in SHR, probably due to impaired responsiveness of cerebral KATP to ischemia.

Adenosine Triphosphate↗
PubMed1996

Leukocyte depletion attenuates reperfusion injury in patients with left ventricular hypertrophy.

BACKGROUND: Reperfusion injury can occur after a long period of aortic cross-clamping in patients with left ventricular hypertrophy during open-heart surgery, even with the most up-to-date techniques of myocardial protection. In the present study, we examined whether leukocyte depletion as an adjunct to terminal blood cardioplegia (LDTC) attenuates reperfusion injury in patients with left ventricular hypertrophy (LV mass, >300 g; left ventricular end-systolic volume index, >100 mL/m2) in a group of 30 patients undergoing aortic valve replacement. METHODS AND RESULTS: We used basic cold potassium crystalloid cardioplegic solution. Terminal blood cardioplegic solution (TC) or LDTC was accomplished by mixing a cold potassium crystalloid cardioplegic solution with warm arterial blood obtained through cardiopulmonary bypass and administered to the aortic root for the first 10 minutes of reperfusion. During delivery of LDTC, warm arterial blood was passed through a leukocyte-removal filter. Patients were randomized into one of three groups for reperfusion: whole blood (WB) (n=10), TC (n=10), and LDTC (n=10). Left ventricular biopsies were obtained before ischemia, at the end of ischemia, and 15 minutes after reperfusion. Semiquantitative scoring for ultrastructural alterations indicated that the LDTC group achieved significantly better recoveries of both scores at reperfusion for myocyte damage and for endothelial cell damage of capillaries than did the WB and TC groups. The LDTC group had significantly fewer neutrophils adhering to endothelial cells at reperfusion and a lower level of malondialdehyde derived from myocardium than did the WB and TC groups. Regarding the clinical data, the LDTC group had a lower maximum creatine kinase-MB, a higher percentage of spontaneous defibrillation, a lower pulmonary capillary wedge pressure, and a lower requirement for dopamine that did the WB group, whereas the TC group failed to do better than the WB group. CONCLUSIONS: These results demonstrate that leukocyte-depleted reperfusion is potentially beneficial as an adjunct to terminal cardioplegia during cardiac surgery to attenuate reperfusion injury in patients with left ventricular hypertrophy.

Adult↗
PubMed1996

Electron microscopic study of optic nerves of macular mice.

The optic nerve of the macular mouse as a model of Menkes' disease was examined by electron microscopy. Since hemizygote macular mice die at 14 or 15 days of age, they were treated with 50 micrograms CuCl2 per 0.1 ml distilled water at 7 days of age. The optic nerves of 1-month-old hemizygote macular mice treated with copper showed hypomyelination and unmyelinated axons, while 1-month-old heterozygote macular mice had focal demyelination of axons. The number of myelinated axons in treated hemizygotes and heterozygotes was statistically significantly lower than that in the control littermates. Oligodendrocytes form myelin sheaths. Since oligodendrocytes of the hemizygote macular mice may have lower activities of cuproenzymes, such as cytochrome oxidase and superoxide dismutase, hypomyelination is assumed to be caused by the dysfunction of oligodendrocyte.

Animals↗
PubMed1996

Pumpkin hydroxypyruvate reductases with and without a putative C-terminal signal for targeting to microbodies may be produced by alternative splicing.

Two full-length cDNAs encoding hydroxypyruvate reductase were isolated from a cDNA library constructed with poly(A)+ RNA from pumpkin green cotyledons. One of the cDNAs, designated HPR1, encodes a polypeptide of 386 amino acids, while the other cDNA, HPR2 encodes a polypeptide of 381 amino acids. Although the nucleotide and deduced amino acid sequences of these cDNAs are almost identical, the deduced HPR1 protein contains Ser-Lys-Leu at its carboxy-terminal end, which is known as a microbody-targeting signal, while the deduced HPR2 protein does not. Analysis of genomic DNA strongly suggests that HPR1 and HPR2 are produced by alternative splicing.

Alcohol Oxidoreductases↗
PubMed1996

cDNA cloning and expression of a gene for 3-ketoacyl-CoA thiolase in pumpkin cotyledons.

A cDNA clone for 3-ketoacyl-CoA thiolase (EC 2.3.1.16) was isolated from a lambda gt11 cDNA library constructed from the poly(A)+ RNA of etiolated pumpkin cotyledons. The cDNA insert contained 1682 nucleotides and encoded 461 amino acid residues. A study of the expression in vitro of the cDNA and analysis of the amino-terminal sequence of the protein indicated that pumpkin thiolase is synthesized as a precursor which has a cleavable amino-terminal presequence of 33 amino acids. The amino-terminal presequence was highly homologous to typical amino-terminal signals that target proteins to microbodies. Immunoblot analysis showed that the amount of thiolase increased markedly during germination but decreased dramatically during the light-inducible transition of microbodies from glyoxysomes to leaf peroxisomes. By contrast, the amount of mRNA increased temporarily during the early stage of germination. In senescing cotyledons, the levels of the thiolase mRNA and protein increased again with the reverse transition of microbodies from leaf peroxisomes to glyoxysomes, but the pattern of accumulation of the protein was slightly different from that of malate synthase. These results indicate that expression of the thiolase is regulated in a similar manner to that of other glyoxysomal enzymes, such as malate synthase and citrate synthase, during seed germination and post-germination growth. By contrast, during senescence, expression of the thiolase is regulated in a different manner from that of other glyoxysomal enzymes.

Acetyl-CoA C-Acyltransferase↗
PubMed1996

HLA-A null allele with a stop codon, HLA-A*0215N, identified in a homozygous state in a healthy adult.

A healthy adult having no serologically detectable HLA class I A locus antigens was identified. The parents of the individual are consanguineous. Results of a family study indicated that the individual is homozygous for the B46-Cw1-DR8.1 haplotype, which was shown to be positively associated with A*0207 in our previous study. The HLA-A null individual is healthy and exhibits no apparent immunological abnormality. Total RNAs extracted from peripheral blood were converted to cDNAs. The reverse transcriptase-polymerase chain reaction (PCR) product, which is of the same size as the normally expressed gene, was easily obtained from the cDNAs with HLA-A locus-specific primers. The nucleotide sequence of this null allele (A*0215N) was the same as that of A*0207 except for a single nucleotide substitution which resulted in a stop codon in exon 4. From its nucleotide sequence, a truncated molecule was expected to be produced; however, the immunoprecipitation study failed to detect the predicted product. Genomic DNAs from 29 unrelated individuals who expressed only one HLA-A antigen with HLA-B46, were analyzed by a PCR-sequence-specific oligonucleotide method. None of the samples possessed this stop codon. Therefore, A*0215N is likely to be a rare allele generated by a single point mutation from A*0207.

Adult↗
PubMed1996

The role of Ca2+ release from sarcoplasmic reticulum in the regulation of sinoatrial node automaticity.

The role of Ca2+ release channels in the sarcoplasmic reticulum in modulating physiological automaticity of the sinoatrial (SA) node was studied by recording transmembrane action potentials and membrane ionic currents in small preparations of the rabbit SA node. Ryanodine, which modifies the conductance and gating behavior of the Ca2+ release channels, was used to block Ca2+ release from the sarcoplasmic reticulum. Superfusion of 1-mM ryanodine decreased the spontaneous firing frequency as well as the maximal rate of depolarization of the SA, and these reductions reached a steady state within approximately 5 min. The action potential recordings revealed that the latter part of diastolic depolarization was depressed and that the take-off potential became less negative. This suggested that the negative chronotropic effect of ryanodine resulted from the blockade of physiological Ca2+ release from the sarcoplasmic reticulum. In voltage clamp experiments, using double-microelectrode techniques, ryanodine did not markedly reduce the Ca2+ current (ICa) but decreased the delayed rectifying K+ current (IK), the steady-state inward current (Iss), and the hyperpolarization-activated inward current (Ih). These observations suggest that, even when the function of C2+ channels in the cell membrane is normally maintained, depression of Ca2+ release channels in the sarcoplasmic reticulum would prevent sufficient elevation of the Ca2+ concentration in SA node cells for the activation of various ionic currents, and, thus adversely affect the physiological automaticity of this primary cardiac pacemaker.

Action Potentials↗