Search PubMed⌕ Search

Biomedical subjects

M Shimizu

Publications and source records attributed to M Shimizu.

At least 595 records · Page 33Linked to original sources
PubMed1996

Cloning, characterization and properties of plasmids containing CGG triplet repeats from the FMR-1 gene.

The FMR-1 gene for the human fragile-chi syndrome, a mental retardation disease inherited by non-Mendelian transmission, contains a genetically unstable CGG region in the 5' non-translated region. The severity of the disease is correlated with the length of the CGG tract. The cloning of 28 stable plasmids containing (CGG)n inserts (where n = 6 to 240) with different extents and types of sequence interruptions (polymorphisms), and in different orientations was accomplished by three strategies in Escherichia coli. Some shorter tracts were prepared by the direct cloning of synthetic oligonucleotides, and longer runs were clones of multimers of (CGG)61, (CGG)11AGG(CGG)60CAG(CGG)8, from a cDNA from a fragile-chi patient or from expansions or deletions of these sequences in E. coli. The genetic stability of the inserts, especially for the longer tracts, was dependent on the sequence length, the presence of polymorphisms, the host cell genotypes, the orientation of the inserts in the vector and the position of cloning in a vector. Two-dimensional agarose gel electrophoresis studies on fully methylated and on non-methylated plasmids as well as chemical probe studies revealed the absence of underwound structures or accessible base-pairs. These DNAs enable a range of genetic and biochemical investigations into the molecular basis of the fragile-chi syndrome.

Base Sequence↗
PubMed1996

Triplex-mediated cleavage of DNA by 1,10-phenanthroline-linked 2'-O-methyl RNA.

We have previously reported that 2'-O-methyl RNAs are efficient probes for duplex DNA. Here we describe the design, synthesis, and DNA cleaving activity of 1,10-phenanthroline (OP)-linked 2'-O-methyl RNA (OP-m). Although a local triple helix was formed, both with OP-m and a control OP-linked DNA at the target sequence of the duplex DNA, the promoter region of the human thrombomodulin gene, the cleavage efficiencies on both strands were not proportional when OP-M was used as a cleavage agent. These results may reflect the structural differences of the respective triple helices and the duplex-triplex junction, formed from the two types of triplex-forming oligonucleotides, 2'-O-methyl RNA and DNA. Since the OP-ms were found to work as preferential purine-strand cutters for duplex DNA, they would be useful as unique tools for genome analysis.

Base Sequence↗
PubMed1996

Myocardial scintigraphic study with 123I 15-(p-iodophenyl)-3(R,S)-methylpentadecanoic acid in patients with hypertrophic cardiomyopathy.

123I 15-(p-iodophenyl)-3(R,S)-methylpentadecanoic acid (BMIPP) myocardial scintigraphy and exercise stress thallium (TI)-201 myocardial scintigraphy were performed in 17 patients with hypertrophic cardiomyopathy (HCM) to evaluate the existence of abnormal fatty acid metabolism in the myocardium and the relationship between this abnormality and myocardial ischemia. On the BMIPP scintigraphy, abnormalities were found in 12 of 17 patients (71%). Five patients showing no abnormalities on the BMIPP scintigraphy had well preserved exercise tolerance and had longer exercise duration than the others showing BMIPP scintigraphic abnormalities (P < 0.001). On the evaluation of the segmental abnormalities, TI scintigraphic abnormalities were found in 15 (50%) of 30 segments showing decreased accumulation of BMIPP. On the other hand, BMIPP scintigraphic abnormalities were found in all segments showing decreased accumulation of TI. The sites of decreased accumulation of BMIPP and TI were in good agreement with the sites of wall hypertrophy. Four patients showing BMIPP scintigraphic abnormalities and no T1 scintigraphic abnormalities were in higher New York Heart Association functional classes, had shorter exercise duration (P < 0.05) than the 5 patients showing no abnormalities on either scintigraphy. It is concluded that abnormalities of fatty acid metabolism in the heart are detected at a high rate in patients with HCM, and may be due in part to factors other than myocardial perfusion disturbance.

Adult↗
PubMed1996

Deletion mapping of chromosome region 9p21-p22 surrounding the CDKN2 locus in melanoma.

The cyclin-dependent kinase-4 inhibitor gene CDKN2, localized at chromosome region 9p21, has been shown to be a familial melanoma gene, though we found that mutations of it are rare in uncultured sporadic melanomas. To determine Whether the region of allelic loss at 9p21 frequently observed in sporadic melanomas includes the CDKN2 locus, new polymorphic microsatellite probes were isolated from the genomic segments surrounding the CDKN2 gene and used for the study of loss of heterozygosity (LOH) in melanoma. The LOH study of matched uncultured tumor-constitutional DNA pairs from 66 metastatic cutaneous and 19 primary uveal melanomas showed that 63% and 32% of the respective tumors suffered allelic loss in the 9p21 region. Two regions of common losses which did not include the CDKN2 locus were observed: in a region of common loss near the D9S157 locus, telomeric to the CDKN2 locus, deletions were observed in 51% of informative cases; in the other region of common loss, near the D9S171 locus, centromeric to the CDKN2 locus, deletions were observed in 47% of informative cases. At the D9S974 locus, located within 20 kb of the CDKN2 gene, deletions were observed in 43% of informative cases. Homozygous deletions of the CDKN2 locus were observed in 8 cases of cutaneous melanoma and 2 cases of uveal melanoma; mutations in CDKN2 exon 2 were found in 2 of the 46 cases with allelic deletion in 9p21. Our results support the following conclusions: (i) somatic mutation of the CDKN2 gene is rare in sporadic melanomas with allelic loss at 9p21; (ii) homozygous loss is more frequent than mutation of the CDKN2 gene in sporadic melanomas; (iii) at 9p21-p23 genes other than CDKN2 may be involved in the development of sporadic melanomas.

Alleles↗
PubMed1996

15N labeling method of peptides using a thioredoxin gene fusion expression system: an application to ACTH-(1-24).

For structure analysis of peptides by multinuclear NMR, stable isotope-labeled samples are required. A direct over-expression system by E. coli cells does not work for that purpose because of rapid degradation of the peptides and/or the mRNA in host cells. We here developed an over-expression system by means of thioredoxin gene fusion system. The fused protein composed of thioredoxin and the objective peptide was expressed in E. coli and then the peptide part was released by enterokinase. This system was successfully applied for the production of 15N-labeled human adrenocorticotropic hormone fragment (ACTH-(1-24)) as needed for multinuclear NMR analysis.

Amino Acid Sequence↗
PubMed1996

Novel autoantibodies directed against the common tertiary configuration of transfer RNA in a patient with interstitial lung disease.

OBJECTIVE: To identify and characterize a novel autoantibody, anti-WS, that binds total transfer RNA (tRNA). METHODS: Serum from patient WS, who had polyarthritis, Sjögren's syndrome, Raynaud's phenomenon, and interstitial pulmonary fibrosis, was used in this study. Characteristics of anti-WS and antibody-reactive determinants of tRNA were investigated by 32P immunoprecipitation using HeLa cell RNA and deletion mutants of tRNA transcribed in vitro. RESULTS: WS serum produced nucleolar and cytoplasmic staining on indirect immunofluorescence. 32P immunoprecipitation assays demonstrated that this serum immunoprecipitated total tRNAs and 5.8S and 5S ribosomal RNAs from 32P-labeled HeLa cell extract. When deproteinized RNA was used as antigen source, total tRNAs were still precipitated by WS serum. An immunoprecipitation study, using various deletion mutants of Escherichia coli tRNA, demonstrated that both D and T psi C loops were needed for antibody binding. Substitution of nucleotide 18G with 18A of E coli tRNA(Trp), which is essential in the formation of the tertiary "L" shape of tRNA, inhibited binding by anti-WS antibodies. CONCLUSION: Anti-WS antibodies are novel autoantibodies directed against tRNAs. The antibody binding site is the common L-shaped tertiary structure conformed by the D loop and T psi C loop of tRNA, suggesting that the antibodies are induced by a conserved sequence among all species. Furthermore, these antibodies could be a marker for a newly recognized subset of connective tissue disease.

Autoantibodies↗
PubMed1996

Teratology study of erythritol in rabbits.

The effect of maternal ingestion of the reduced energy sweetener erythritol was investigated in KBL:JW strain pregnant rabbits. Animals received 1.0, 2.24, or 5.0 g/kg intravenously once daily from Days 6 to 18 of gestation. Maternal effects (auricular edema, and bradypragia) were observed in the high-dose group. No deaths or significant abnormalities occurred in animals given 1.0 or 2.24 g/kg. No effect was observed in the reproductive performance of the dams or in fetal development from ingestion at any of the treatment levels.

Abnormalities, Drug-Induced↗
PubMed1996

Protein phylogeny of translation elongation factor EF-1 alpha suggests microsporidians are extremely ancient eukaryotes.

Partial regions of the mRNA encoding a major part of translation elongation factor 1 alpha (EF-1 alpha) from a mitochondrion-lacking protozoan, Glugea plecoglossi, that belongs to microsporidians, were amplified by polymerase chain reaction (PCR) and their primary structures were analyzed. The deduced amino acid sequence was highly divergent from typical EF-1 alpha's of eukaryotes, although it clearly showed a eukaryotic feature when aligned with homologs of the three primary kingdoms. Maximum likelihood (ML) analyses on the basis of six different stochastic models of amino acid substitutions and a maximum parsimony (MP) analysis consistently suggest that among eukaryotic species being analyzed, G. plecoglossi is likely to represent the earliest offshoot of eukaryotes. Microsporidians might be the extremely ancient eukaryotes which have diverged before an occurrence of mitochondrial symbiosis.

Amino Acid Sequence↗
PubMed1996

Highly hydrogenated dietary soybean oil modifies the responses to polychlorinated biphenyls in rats.

The effects of dietary highly hydrogenated soybean oil (HSO) upon the changes caused by dietary polychlorinated biphenyls (PCBs) were examined in rats. Six groups of rats were fed the following diets for 30 d: a 20% soybean oil-containing diet (control diet), a diet in which a half of soybean oil was substituted with HSO (HSO-A diet), a diet in which cellulose powder was replaced with HSO (HSO-B diet) and these diets supplemented with 100 ppm PCBs (control + PCBs, HSO-A + PCBs and HSO-B + PCBs diets). Hepatic concentration of PCBs and relative liver weight were markedly decreased in rats fed with the HSO-A + PCBs diet compared with those fed with the other diets containing PCBs. Liver lipids and liver cholesterol were considerably decreased with a reciprocal increase in fecal sterol excretion by rats fed the HSO-A + PCBs and the HSO-B + PCBs diets compared with those fed with the control + PCBs diet. The fatty acid composition in hepatic phospholipids showed an independent increase of the saturated fatty acid content induced by dietary HSO and PCBs. Dietary PCBs also caused decreases in the amounts of monounsaturated and n-3 polyunsaturated fatty acids. These results suggest that dietary HSO prevents accumulation of PCBs in the liver and promotes the excretion of lipids stimulated by PCBs, accompanied by a change in fatty acid metabolism.

Animals↗
PubMed1996

Extensive soft-tissue involvement of dermatomyositis detected by whole-body scintigraphy with 99mTc-MDP and 201TL-chloride.

The authors present a case of extensive soft-tissue radioactivity visualized on both 99mTc-MDP and 201Tl-chloride scintigrams in a patient with dermatomyositis and colon cancer. Incidentally, diffuse and intense uptake of 99mTc-MDP was observed in the shoulder girdles, anterior chest wall, psoas muscles, both proximal thighs and right lower limb, corresponding to the sites of symptomatic muscles, even though skin lesions were limited and no calcification was detected on radiographs. Moreover, 201Tl-chloride was also intensely accumulated in nearly the same sites as the symptomatic muscles as shown on the 99mTc-MDP bone scintigrams. Whole-body scintigraphy with 99mTc-MDP and 201Tl-chloride is a useful tool to detect occult muscle lesions with dystrophic calcification and hyperemia in dermatomyositis.

Calcinosis↗
PubMed1996

Long-term probucol treatment results in regression of xanthomas, but in progression of coronary atherosclerosis in a heterozygous patient with familial hypercholesterolemia.

A 66-year-old male heterozygous familial hypercholesterolemia (FH) patient with significant coronary atherosclerosis has been treated by us with probucol (1000 mg daily) for eight years. This treatment has produced significant reductions in the cholesterol levels of his serum, low density lipoprotein (LDL), and high density lipoprotein (HDL) from 237 +/- 20 mg/dl (mean +/- S.D.) to 156 +/- 15, from 175 +/- 8 to 111 +/- 16 mg/dl, and from 23 +/- 4 to 19 +/- 2 mg/dl, respectively. These reductions have been maintained for eight years. Serum triglyceride levels also decreased, from 220 +/- 54 to 146 +/- 36 md/dl. During this period, marked regression of xanthomas on the eyelids and finger extensor tendons was observed, while thickness of the Achilles tendons was reduced from 21.0 mm to 13.0 mm. On other hand, effort-induced anginal symptoms requiring additional antianginal medication have been noticed, and angiographically-demonstrated coronary atherosclerosis has progressed significantly during these eight years. These observations lead us to suggest that maintaining low levels of HDL cholesterol with probucol, even though resulting in satisfactory reduction of LDL cholesterol and marked regression of xanthomas, appears to be associated with the progression of atherosclerosis in the coronary arteries.

Aged↗
PubMed1996

Changes of lymphocyte subpopulations in pigs infected with porcine reproductive and respiratory syndrome (PRRS) virus.

Abnormal changes of T-cell subpopulation were observed in the peripheral blood lymphocytes of pigs infected with porcine reproductive and respiratory syndrome (PRRS) virus. Pigs with naturally occurring PRRS revealed increases in CD2+ and CD8+ cells, and decreases in CD4+ cells and the ratios of CD4+/CD8+ cells. Specific-pathogen-free pigs inoculated with PRRS virus showed remarkable decreases in total lymphocytes, CD4+ and CD2+ cells on Postinoculation Day (PID) 3. The decline of CD4+ cells continued for at least 14 days, while CD2+ cells showed a tendency to increase thereafter. On the other hand,CD8+ cells slightly decreased in number on PID 3, and then increased remarkably; their number was significantly larger on PIDs 28 and 35 than on PID 0. The ratios of CD4+/CD8+ cells were significantly low between PIDs 3 and 28 as compared with PID 0. However, there were no differences in thymocyte subpopulations between infected and non-infected pigs, suggesting that the PRRS virus does not modulate intrathymic T-cell differentiation. In an experiment with peripheral blood mononuclear cell cultures, PRRS virus caused neither alteration of T-cell subpopulations nor cell proliferation, suggesting that the virus is not cytotoxic for CD4+ cells and not mitogenic for CD8+ cells.

Animals↗
PubMed1996

Biochemical characterization of a new disintegrin, flavostatin, isolated from Trimeresurus flavoviridis venom.

We biochemically characterized a new disintegrin, flavostatin, isolated from Trimeresurus flavoviridis venom. Flavostatin inhibited ADP-, collagen-, and thrombin receptor agonist peptide-induced platelet aggregation in human platelet-rich plasma (IC50 range: 59 to 111 nM) and blocked the binding of biotinylated human fibrinogen to purified GPIIb/IIIa with an inhibitory potency 31,000-fold higher than that of Arg-Gly-Asp-Ser (RGDS). Flavostatin strongly inhibited high-shear-stress-induced platelet aggregation in platelet-rich plasma (PRP) with an IC50 value of 188 nM. Fluorescein isothiocyanate (FITC)-conjugated flavostatin saturably bound to unstimulated and ADP-stimulated washed platelets with high affinity (Kd values: 38 and 21 nM, respectively); the corresponding number of binding sites was 86460 and 79192 per platelet. In competition experiments with several glycoprotein IIb/IIIa antagonists, the binding of FITC-conjugated flavostatin to platelets was completely inhibited by ReoPro, triflavin, TP9201, MK383 and GR144053, but not by YM207, YM337 and B6A3.

Amino Acid Sequence↗
PubMed1996

Viral antigen and B and T lymphocytes in lymphoid tissues of gnotobiotic piglets infected with hog cholera virus.

Four 4-day-old gnotobiotic piglets infected intranasally with the Kanagawa/74 strain of hog cholera virus (HCV) did not develop severe illness over a period of 3 weeks. Large amounts of HCV were isolated from the lymphoid tissues and serum at necropsy. After the acute phase, hyperplasia of histiocytes and plasmacytopoiesis were observed in two pigs (killed 14 and 21 days after inoculation). The number of CD4+ and CD8+ T lymphocytes increased significantly and their location was consistent with the site of HCV replication. The results suggest that a CD8+ T-lymphocyte reaction is associated with persistent HCV infection.

Acute Disease↗
PubMed1996

Detection of the peptidyltransferase activity of a dipeptide, alanylhistidine, in the absence of ribosomes.

It was shown that a dipeptide, alanylhistidine, can act as a catalyst for the peptidyl transfer reaction in the absence of ribosomes between the amino acid moieties of phenylalanyl, lysyl, prolyl, and glycyl tRNAs depending on their model templates, poly U, poly A, poly C, and poly G, respectively. A template effect was observed: The peptidyl transfer reaction between tRNAGly molecules (anticodon GCC) occurred in the presence of poly G but not in the presence of poly C. The reaction was most efficient for the best stacked poly A (tRNALys) and least efficient for the worst stacked poly U (tRNAPhe).

Catalysis↗
PubMed1996

Complete nucleotide sequences of the genes encoding translation elongation factors 1 alpha and 2 from a microsporidian parasite, Glugea plecoglossi: implications for the deepest branching of eukaryotes.

Complete nucleotide sequences of the genes putatively encoding translation elongation factors 1 alpha (EF-1 alpha) and 2 (EF-2) from a mitochondrion-lacking protozoan, Glugea plecoglossi, that belongs to microsporidians were determined. The deduced amino acid sequences of the putative EF-1 alpha and EF-2 of Gl. plecoglossi showed very unusual features compared with typical eukaryotic sequences. The degree of divergence was especially great in the EF-1 alpha sequence, although it clearly showed a eukaryotic feature when aligned with homologs from the three primary kingdoms. Phylogenetic analyses of EF-1 alpha and EF-2 on the basis of the maximum likelihood method of protein phylogeny clearly and consistently suggested that among eukaryotic species being analyzed, Gl. plecoglossi and another mitochondrion-lacking protozoan, Giardia lamblia, respectively represent the earliest and the second earliest offshoots of eukaryotes. When the EF-1 alpha and EF-2 phylogenies were totally evaluated, the earliest divergence of Gl. plecoglossi in eukaryotes became more clearly confirmed. If the phylogenetic relationship inferred from the present analysis are correct, microsporidians might be extremely ancient eukaryotes that diverged before the occurrence of mitochondrial symbiosis.

Animals↗