[Genome science--aiming for new life science (discussion)].
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Animals↗
Biomedical subjects
K Matsubara
Publications and source records attributed to K Matsubara.
c-axis resistivity of MoCl5 graphite intercalation compounds.
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Journal Article↗
Differences in dopamine efflux induced by MPP+ and beta-carbolinium in the striatum of conscious rats.
The effects of N-methyl-4-phenylpyridinium cation (MPP+) and of an endogenously formed analog, 2,9-di-methyl-norharmanium cation (2,9-Me2NH+), on extracellular dopamine were studied in the striatum of freely moving rats. Perfusion of either 2,9-Me2NH+ or MPP+ through a microdialysis probe evoked a marked and dose-dependent increase of dopamine levels. Tetrodotoxin and Ca(2+)-free medium prevented the increase in dopamine levels induced by 2,9-Me2NH+, but not that induced by MPP+. Cocaine, 3 microM, intensified the 2,9-Me2 NH(+)-induced increase in extracellular dopamine and slightly attenuated the MPP(+)-induced efflux. S(-)-3-(3-Hydroxy-phenyl)-N-propylpiperidine, that acts as an antagonist of dopamine autoreceptors in the presence of a dopamine reuptake inhibitor, markedly enhanced the increase in extracellular dopamine elicited by 2,9-Me2NH+, but not that by MPP+. These results suggested that 2,9-Me2NH+ was a potent dopamine reuptake blocker, whereas MPP+ acts as an amphetamine-like dopamine releaser rather than a reuptake inhibitor on the membrane transporter.
1-Methyl-4-phenylpyridinium↗
A cDNA cloning of human AEBP1 from primary cultured osteoblasts and its expression in a differentiating osteoblastic cell line.
By comparing lists of 3'-directed partial cDNA sequences (gene signatures) randomly collected from various tissues, genes uniquely expressed in individual tissue can be identified. A full length cDNA clone, corresponding to one such gene signature, unique to human osteoblast and adipose tissue, was isolated. This cDNA clone encodes a 845-amino acid protein that is almost identical to the mouse adipocyte transcription factor AEBP1 except that it has additional 105 amino acids in the N terminus. A northern hybridization showed that in the differentiating murine osteoblastic cell line, AEBP1 is also expressed but that it is shut off in the final calcification phase, suggesting a transcriptional repressive effect on genes for bone formation.
3T3 Cells↗
Identification of the subunit loci in the extracellular multisubunit hemoglobin from annelid Perinereis aibuhitensis.
Giant hemoglobin (Hb) from Perinereis aibuhitensis is made of several types of protein components including single-chain globin (a), disulfide-bridged globin trimer (A-b-B), disulfide-bridged dimers of nonglobin chain (or linkers; L1-L1, L2-L2, and L1-L2), and oligomers of L1-L2 [(L1-L2)n]. The intact form of this giant Hb is a two-tiered hexagonal structure composed of 12 identical units, or so-called submultiples (six submultiples to a tier). To obtain a view of the three-dimensional architectural arrangement of these components in the intact form, we identified the subunit loci by using two mutually complementary chemical modifications and a colloidal gold labeling technique. Using the chemical modifications, we discovered that (i) linkers L1-L2 and L2-L2 were located at the exterior of the Hb, (ii) linker L1-L1 and globin a were buried in the interior, and (iii) linker (L1-L2)n and globin trimer A-b-B were located at both exterior and interior loci. The labeling with an L2-specific colloidal gold revealed the predominant loci of L2 at the outer and inner boundaries between neighboring submultiples in a hexagonal form. By combining these results with those from our previous reports [S. Ebina, K. Matsubara, K. Nagayama, M. Yamaki, and T. Gotoh (1995) Proc. Natl. Acad. Sci. USA 92, 7367-7371; K. Matsubara, M. Yamaki, Nagayama, H. Ishii, K. Imai, T. Gotoh, and S. Ebina (1996), in press], we deduced the following conclusions concerning the Hb architecture. The L1-L1 chains perhaps together with (L1-L2)n chains form a scaffold on which submultiples assemble into a two-tiered hexagonal arrangement, probably by connecting the carbohydrates in globin a. The L1-L2 and L2-L2 chains reinforce the connections of the submultiples by binding carbohydrates, perhaps those carbohydrates in globin A. We proposed to call this type of non-protein-dependent structural level as seen in such a carbohydrate-glued protein aggregate "protein-plus structure."
Animals↗
Identification and cloning of neuroblastoma-specific and nerve tissue-specific genes through compiled expression profiles.
An expression profile of active genes in a human neuroblastoma cell line CHP134 was obtained by collecting 1222 partial sequences from a 3'-directed cDNA library representing a non-biased mRNA population. By comparing this expression profile with the compiled profiles of multiple tissues, several novel gene transcripts that appeared only in the profile of the neuroblastoma cell line were identified. Further analyses by Northern blotting revealed two specific cDNA clones that are expressed in most of the human neuroblastomas examined, and three that are in some of the human neuroblastoma cell lines as well as in the adult human brain. Full-size cDNAs were cloned using these five partial cDNA sequences as probes and sequenced. A database search revealed that they are all novel and unique sequences: one sharing some amino acid sequence similarities with a cytoskeletal protein, two clones likely to be transcriptional factors, a clone that has characteristic potassium channel properties, and a clone that is non-homologous to any one of the known proteins. Thus, we argue that the collection of 3'-directed cDNA sequences in combination with the compiled expression profiles of active genes in multiple tissues is a powerful tool for discovering novel genes that are specifically expressed in a given cell or tissue, in this case neuroblastomas and/or nerve tissue.
Amino Acid Sequence↗
Expression profiles of active genes in human and mouse livers.
An expression profile of active genes in the human liver was obtained by collecting sequences with a 3'-directed cDNA library that faithfully represents composition of the mRNA population. The results show the relative activity of ca. 600 genes in maintaining the hepatocytes and sustaining their liver-specific phenotypes. The most active group of genes are those for the production of plasma proteins, followed by the genes for the synthesis of lipoproteins, protease inhibitors, coagulation factors, and complements. This balance of gene activity was maintained for four independently obtained expression profiles from human livers, including those of adult and fetus. The expression profiling was extended to the liver of adult mouse, used as a model for the molecular etiology of hepatocytes and for examining the effects of drugs. Subtle biological differences between the human and mouse livers are reflected in the global expression profiles of active genes, especially with regard to the synthesis of plasma proteins, lipoproteins and complements. This comparative analysis using expression profiling should find a wide application in comparative biology.
Adult↗
Effect of a pyridinium metabolite derived from haloperidol on the activities of striatal tyrosine hydroxylase in freely moving rats.
The effects of a pyridinium metabolite (HPP+) derived from haloperidol (HP) on in vivo tyrosine hydroxylation was evaluated in freely moving rats. As an index of the in vivo activity of tyrosine hydroxylase (TH), the rat striatum was perfused with NSD-1015, and extracellular 3,4-dihydroxyphenylalanine (DOPA) levels were measured. HPP+ (1 mM) gradually reduced tyrosine hydroxylation to 30% of the basal level, although the effect was less potent than 1-methyl-4-phenylpyridinium ion (MPP+). On the contrary, HPP+ at a 0.1 mM dose decreased in 5-hydroxyindoleacetic acid (5-HIAA) level, but did not affect dopamine metabolites. The present study revealed that HPP+ irreversible inhibited in vivo tyrosine hydroxylation by the same manner of MPP+. However, the neurotoxic effects of HPP+ in vivo would be selective for serotonergic over dopaminergic neurons, which distinguishes the toxic profile of this compound compared to that of MPP+.
1-Methyl-4-phenylpyridinium↗
Wheat germ agglutinin-reactive chains of giant hemoglobin from the polychaete Perinereis aibuhitensis.
Wheat germ agglutinin-reactive chains of multisubunit extracellular hemoglobin from the polychaete Perinereis aibuhitensis were identified to clarify the carbohydrate gluing which is the carbohydrate-dependent supramolecular architecture of the hemoglobin (Ebina S. et al. (1995) Proc. Natl. Acad. Sci. USA 92, 7367-7371). Electron microscope micrographs of Perinereis hemoglobin showed a characteristic shape of two-tiered hexagonal rings whose diameter and height were determined to be 29.4 +/- 1.7 nm and 20.0 +/- 1.8 nm, respectively. Four types of globins and two types of linkers were isolated from the giant hemoglobin by reverse-phase chromatography and SDS-PAGE. These constituents showed similar NH2-terminal sequences as those previously reported for corresponding chains of Tylorrhynchus hemoglobin (Suzuki T. and Gotoh T. (1986) J. Biol. Chem. 261, 9257-9267; Suzuki T. et al. (1990) J. Biol. Chem. 265, 12168-12177). Thus, each globin of Perinereis hemoglobin was identified in terms of amino acid sequence homology and designated using names common to Tylorrhynchus hemoglobin, namely, a, A, b, and B. The linkers were stained by horseradish peroxidase (HRP)-lectins and PAS staining kits, indicating the presence of carbohydrate oligomers. Lectin staining was also significantly positive to globins a and A, which belong to strain A, but negative to globins b and B, which belong to strain B. Results showed that linkers and globins of strain A had a site in a carbohydrate oligomer to which wheat germ agglutinin (WGA) could bind. On the other hand, an alignment between known amino acid sequences of annelid globins and linkers and the sequences of lectins revealed that only the domain of the cysteine-rich motif in linkers has a homology with WGA-type lectins. The results of this study clarify the structuring mechanism of a supramolecule by lectin-like binding, called carbohydrate gluing.
Amino Acid Sequence↗
Platelet aggregation is inhibited by phycolectins.
Lectins from four marine algal species were examined for interaction with human platelets. The lectin designated hypnin A, from the red alga Hypnea japonica, inhibited adenosine diphosphate (ADP)- or collagen-induced human platelet aggregation in a dose-dependent manner. Complete inhibition was observed at concentrations of 100 and 5 micrograms/ml of the lectin with ADP (2 microM) and collagen (0.2 microgram/ml)-induced platelet aggregation, respectively. At the inhibitory concentration of 0.5 to 100 micrograms/ml, the lectin did not induce aggregation of resting platelets. Lectins from the other three algal species also inhibited ADP-induced human platelet aggregation. These results indicate that the algal lectins are a new group of inhibitors and may be useful to study glycoconjugates on platelet membranes and to design novel platelet aggregation inhibitors.
Adenosine Diphosphate↗
Species identification by the positional analysis of fatty acid composition in triacylglyceride of adipose and bone tissues.
In a new attempt at species identification, the total composition and positional distribution of fatty acid in triacylglyceride (TG) of adipose and bone tissues were analyzed in human, bovine, pig, dog, cat and chicken tissues. Although the total fatty acid compositions of bovine and pig tissues were significantly different from those of human (different in more than half the fatty acids tested), dog, cat and chicken tissues showed a comparatively similar composition to human fatty acids composed of TG in both tissues. The TG in these tissues was also subjected to stereospecific analysis using pancreatic lipase, that is, the fatty acid distribution in positions 1,3 (not distinguished between 1 and 3) and 2 of the TG were determined. The distribution of fatty acids among the positions 1,3 and 2 in the TG of animal adipose and bone tissues was non-random. The distribution between position 2 and positions 1,3 seems to be governed by chain length and unsaturation in each animal. The shorter and more unsaturated fatty acids showed a greater tendency to occupy position 2 of TG. Although this rule appeared in all animals except the pig, the distribution of each fatty acid into position 2 was species-specific. The positional distribution of fatty acid in TG was identical among the same species and in different regions of the same body. Thus, even when species identification is difficult using the pattern of total fatty acid composition, the analysis of the positional distribution of fatty acid makes it possible to determine the species. From the present results, the evaluation of positional distribution of fatty acid in the TG is a useful tool for the identification of human tissues.
Adipose Tissue↗
cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1 (AdiPose Most abundant Gene transcript 1).
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Adiponectin↗
An expression profile of active genes in cultured human keratinocytes.
An expression profile of genes active in cultured human keratinocytes was obtained by collecting 770 partial sequences from a 3'-directed cDNA library that faithfully represents the mRNA population in the source cells. Ninety-four species composed of 258 clones occurred recurrently, and 512 clones appeared only once. The gene which showed the most abundant expression codes for type I keratin 14, the major keratin that is known to be strongly expressed in the basal layer of the epidermis. Altogether 242 gene species were identified in GenBank, 9.5% of which encode cytoskeletal proteins and 14.5% the components of protein synthesis. Keratin synthesis, the unique property of the keratinocyte, has been measured by elevating the level of transcription of several keratin genes. However, there was no expression of the differentiation markers, type II keratin 1 (K1) or type I keratin 10 (K10), indicating that the cells used for our cDNA library construction were in the pre-differentiation stage. By comparing this expression profile with seven expression profiles from other tissues/cells, four clones, including a novel clone were tentatively identified as specific to keratinocytes.
Base Sequence↗
Isolation and chromosomal localization of the human cone cGMP phosphodiesterase gamma cDNA (PDE6H).
By comparing lists of 3'-directed partial cDNA sequences (gene signatures) randomly collected from various tissues, genes unique to each tissue can be identified. A full-size cDNA clone, corresponding to one such tissue-specific gene signature that recurred only in a retina library, was isolated and analyzed. This clone encoded 83 amino acids highly homologous to the bovine blue cone cGMP phosphodiesterase gamma subunit. The retina-specific expression of this gene was confirmed by multiple tissue Northern blotting, and its cone specificity was confirmed by in situ hybridization to a human retina specimen. From these results, we concluded that this clone (HGMW-approved symbol PDE6H) encodes for the gamma subunit of human cone-specific cGMP phosphodiesterase, and it was assigned to chromosome 12p13 by fluorescence in situ hybridization.
3',5'-Cyclic-GMP Phosphodiesterases↗
Identification of 3-nitro-L-tyrosine, a product of nitric oxide and superoxide, as an indicator of oxidative stress in the human brain.
3-Nitro-L-tyrosine synthesized from L-tyrosine by peroxynitrite, a product of superoxide and nitric oxide, was identified for the first time in human brains. By quantitative analysis using high-performance liquid chromatography with multi-electrochemical detectors, 3-nitro-L-tyrosine concentration in the gray matter was higher in the cerebrum than in the cerebellum; 0.96 and 0.29 nmol/g wet weight, respectively. On the other hand, L-tyrosine concentration was not different. 3-Nitro-L-tyrosine in the brain may be used as an indicator of oxidative stress induced by reactive oxygen species and nitric oxide.
Aged↗
Chromosomal assignment of human genomic NotI restriction fragments in a two-dimensional electrophoresis profile.
Using DNA from sorted human chromosomes and two-dimensional gel electrophoresis, we assigned 2295 NotI sites, 43% of the total, to specific chromosomes and designated the procedure CA-RLGS (chromosome-assigned restriction landmark genomic scanning). Although the NotI enzyme is sensitive to DNA methylation, our results suggested that the majority of the spots did not seem to be affected by this modification. The NotI sites were distributed at higher levels in chromosomes 17, 19, and 22, suggesting higher gene content in these chromosomes. Most spots were assigned to unique chromosomes, but some spots were found on two or more chromosomes. Quantitative analysis revealed the intensity of the DNA spots on the sex chromosomes to be haploid and that of the chromosome 21 spots in DNA from a male with Down syndrome to be trisomic, although there were exceptions. We report here the first-generation CA-RLGS map of the human genome.
Chromosome Aberrations↗
Increased serotonin release across the coronary bed during a nonischemic interval in patients with vasospastic angina.
BACKGROUND: Platelet activation and coagulation abnormality have been observed during coronary spasm. It is crucial whether platelet activation occurs even during a nonischemic period. HYPOTHESIS: This study was designed to determine whether platelets might be activated across the coronary bed during a nonischemic interval in patients with vasospastic angina. METHODS: Plasma levels of serotonin, 6-keto-prostaglandin F1 alpha, and catecholamines in the aorta and the coronary sinus were simultaneously measured in 16 patients with vasospastic angina and 13 control patients with nonischemic heart disease. RESULTS: None of these patients showed myocardial ischemia during sampling. The difference in transcardiac plasma levels of serotonin in patients with vasospastic angina was significantly higher than that in controls (1.48 +/- 1.08 ng/ml vs. 0.07 +/- 0.12 ng/ml, respectively, p < 0.001). Coronary sinus plasma norepinephrine levels in these two groups were almost the same (204.8 +/- 110.8 pg/ml vs. 190.4 +/- 131.6 pg/ml, respectively). The ratio of 6-keto-prostaglandin F1 alpha in the coronary sinus and the aorta was not different between the two groups (1.17 +/- 0.96 in patients with vasospastic angina vs. 1.15 +/- 0.68 in controls). CONCLUSIONS: These data suggest that platelet activation across the coronary bed should be ascribed to endothelial dysfunction. Lack of compensatory enhancement of prostacyclin production might be concerned with dysfunction of coronary endothelial cells in these patients.
6-Ketoprostaglandin F1 alpha↗
HLA class II is associated with the frequency of glutamic acid decarboxylase M(r) 65,000 autoantibodies in Japanese patients with insulin-dependent diabetes mellitus.
Autoantibodies to glutamic acid decarboxylase (GAD65Ab) are common in both caucasian and Japanese patients with insulin-dependent diabetes mellitus (type 1), while the type 1-associated HLA haplotypes differ. In the present study, we analyzed GAD65Ab in relation to HLA-DQ and -DR alleles in Japanese type 1 patients. GAD65Ab were found in 58% short-duration (less than 5 years) type 1, 23% long-duration type 1, 56% slowly progressive type 1, 3% type 2 patients, and 1.7% healthy individuals. In 75 HLA-typed type 1 patients, the GAD65Ab frequency was higher in short-duration patients with DRB1*08 allele (100%, Pc < 0.05). GAD65Ab frequencies in DQB1*0302, DQB1*0303, and DRB1*09-positive, long-duration type 1 patients were lower than those in short-duration type 1 patients (14%, 19%, and 20%, Pc < 0.02 compared with short-duration type 1, 90%, 75%, and 71%, respectively), while the frequency varied less in DQB1*04 individuals (44% and 30% in short- and long-duration type 1 patients, respectively). These findings were also observed among patients with DRB1*04, i.e., the haplotype DRB1*0405-DQB1*0401 showed less variation in frequency of GAD65Ab (44% and 35% in short- and long-duration type 1 patients, respectively), while DRB1*04xx-DQB1*0302 showed lower frequency in long-duration type 1 than short-duration (13% and 100%, respectively). Thus, HLA class II is associated with frequency GAD65Ab, and this association might be affected by disease duration in Japanese type 1 patients.
Adolescent↗