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Biomedical subjects

Y Murakami

Publications and source records attributed to Y Murakami.

At least 433 records · Page 24Linked to original sources
PubMed1996

Life with 6000 genes.

The genome of the yeast Saccharomyces cerevisiae has been completely sequenced through a worldwide collaboration. The sequence of 12,068 kilobases defines 5885 potential protein-encoding genes, approximately 140 genes specifying ribosomal RNA, 40 genes for small nuclear RNA molecules, and 275 transfer RNA genes. In addition, the complete sequence provides information about the higher order organization of yeast's 16 chromosomes and allows some insight into their evolutionary history. The genome shows a considerable amount of apparent genetic redundancy, and one of the major problems to be tackled during the next stage of the yeast genome project is to elucidate the biological functions of all of these genes.

Amino Acid Sequence↗
PubMed1996

c-Jun stimulates origin-dependent DNA unwinding by polyomavirus large Tantigen.

The AP1 protein c-Jun has previously been shown to stimulate polyomavirus (Py) DNA replication in vivo. In order to define the mechanism, we added purified c-Jun protein to the origin-dependent and large T antigen (LT)-dependent in vitro DNA unwinding assay. c-Jun protein was found to stimulate by approximately 5-fold the unwinding of a 290 bp linear DNA fragment containing both the Py origin and the AP1 recognition sequence to which c-Jun binds. Efficient levels of stimulation were specifically observed at limiting concentrations of LT for unwinding. Under similar conditions, Py DNA replication was stimulated to a comparable extent by AP1 in a purified in vitro replication assay. Mobility shift and DNase I footprinting assays showed that c-Jun stimulates the ATP-dependent binding of LT to the origin core by approximately 7-fold. Furthermore, c-Jun was found to interact directly with LT, but not with replication protein A. The activities of c-Jun to stimulate unwinding and origin binding of LT were found to be harbored within the N-terminal region of c-Jun, which is distinct from the DNA binding domain. We speculate that certain transcription factors may possess specific DNA replication domains that function to stimulate the loading of replication factors at the origin during the initiation of DNA synthesis.

Adaptor Protein Complex 1↗
PubMed1996

Construction of high-resolution physical maps from yeast artificial chromosomes using restriction landmark genomic scanning (RLGS).

We have established a new system for chromosome-specific yeast artificial chromosome (YAC) contig construction using restriction landmark genomic scanning (RLGS-based YAC contig mapper). RLGS is a powerful tool for detecting more than 1000 restriction landmarks distributed on an entire genome in one procedure. In this system, RLGS is applied to sorted chromosomes to cover the target chromosome. Using these landmarks as guideposts, chromosome-specific YAC clones are then ordered. In this paper, we report the construction of a map for a human chromosome 21 YAC contig spanning q22.1 using this new approach. Applying RLGS to sorted chromosomes 21 enables detection of approximately 1400 spots (equivalent of 1050 PacI landmarks), covering the entire region of this chromosome. We constructed the 2.5-Mb YAC contig encompassing 21q22.1 with 66 spots (equivalent of 50 PacI landmarks). With this contig map, we could detect two deleted regions and chimerism in the YAC insert DNA. Our results demonstrated the usefulness of this approach for finding DNA alterations of YACs, such as deletions and chimerism.

Chimera↗
PubMed1996

Identification of plexin family molecules in mice.

By screening of E17.5 mouse brain cDNA libraries, we isolated two cDNAs encoding new plexin-like proteins. Sequencing revealed that these two proteins were type 1 membrane proteins which showed over 60% identity at the amino acid level to mouse plexin 1. Moreover, putative extracellular segments of these two proteins had three repeats of a cysteine-rich domain which is a common motif for plexin proteins. Thus, we named these two proteins mouse plexin 2 and mouse plexin 3. We obtained mouse plexin 3 cDNA clones in which a part of protein-coding region was deleted. Also, Northern blot analysis showed molecular heterogeneity in mouse plexin 2 mRNAs. These findings indicate that in the mouse, plexins comprise a molecular family (the plexin family).

Alternative Splicing↗
PubMed1996

Identification of a neuronal cell surface molecule, plexin, in mice.

We searched for mouse homologues of the cell adhesion protein plexin which was originally found in Xenopus, and obtained a cDNA encoding a plexin-like protein. We referred to this protein as mouse plexin 1. The overall amino acid identity between mouse plexin 1 and Xenopus plexin was 84%. As in the Xenopus plexin, the extracellular segment of mouse plexin 1 protein possessed three cysteine-rich domains which showed significant homology with the cysteine-rich domain of the c-Met proto-oncogene protein product (c-Met protein) and Met-like receptor protein tyrosine kinases. Northern blot analysis indicated that mouse plexin 1 was predominantly expressed in the brain.

Amino Acid Sequence↗
PubMed1996

Potent inhibitors of secretory phospholipase A2: synthesis and inhibitory activities of indolizine and indene derivatives.

Phospholipase A2 is an enzyme which hydrolyzes the sn-2 position of certain cellular phospholipids. The liberated lysophospholipid and arachidonic acid are precursors in the biosynthesis of various biologically active products. As human nonpancreatic sPLA2 is present in high levels in the blood of patients in several pathological conditions, the potent sPLA2 inhibitors have been suggested to be useful drugs. Here we describe the synthesis, structure-activity relationship, and inhibitory activities of indolizine and indene derivatives. 1-(Carbamoylmethyl)indolizine derivatives and 1-oxamoylindolizine derivatives exhibited very potent inhibitory activity. The former was unstable to air oxidation, but the latter exhibited an improvement both in stability and in potency. Some compounds approached the stoichiometric limit of the chromogenic assay.

Alkylation↗
PubMed1996

Functional and bioenergetic consequences of postinfarction left ventricular remodeling in a new porcine model. MRI and 31 P-MRS study.

BACKGROUND: The underlying mechanisms by which left ventricular remodeling (LVR) leads to congestive heart failure (CHF) are unclear. This study examined the functional and bioenergetic abnormalities associated with postinfarction ventricular remodeling in a new, large animal model. METHODS AND RESULTS: Remodeling was induced by circumflex coronary artery ligation in young pigs. LV mass, volume, ejection fraction (EF), the ratio of scar surface area to LV surface area, and LV wall stresses were calculated from magnetic resonance imaging anatomic data and simultaneously measured LV pressure. Hemodynamics, transmural blood flow, and high-energy phosphates (spatially localized 31P-nuclear magnetic resonance) were measured under basal conditions, during hyperperfusion induced by pharmacological vasodilation with adenosine, and during pyruvate infusion (11 mg/kg per minute IV). Six of 18 animals with coronary ligation developed clinical CHF while the remaining 12 animals had LV dilation (LVR) without CHF. The results were compared with 16 normal animals. EF decreased from 55.9 +/- 5.6% in normals to 34.6 +/- 2.3% in the LVR group (P < .05) and 24.2 +/- 2.8% in the CHF group (P < .05 versus LVR). The infarct scar was larger in CHF hearts than in LVR hearts (P < .05). In normals, LV myocardial creatine phosphate (CP)/ATP ratios were 2.10 +/- 0.10, 2.06 +/- 0.16, and 1.92 +/- 0.12 in subepicardium (EPI), mid myocardium (MID), and subendocardium (ENDO), respectively. In LVR hearts, the corresponding ratios were decreased to 1.99 +/- 0.13, 1.80 +/- 0.14, and 1.57 +/- 0.15 (ENDO P < .05 versus normal). In CHF hearts, CP/ATP ratios were 1.41 +/- 0.14, 1.33 +/- 0.15, and 1.25 +/- 0.15; (P < .05 versus LVR in EPI and MID). The calculated myocardial free ADP levels were significantly increased only in CHF hearts. CONCLUSIONS: Bioenergetic abnormalities in remodeled myocardium are related to the severity of LV dysfunction, which, in turn, is dependent on the severity of the initiating myocardial infarction.

Adenosine Triphosphate↗
PubMed1996

Isolation of DNA sequences amplified at chromosome 19q13.1-q13.2 including the AKT2 locus in human pancreatic cancer.

In the human pancreatic cancer cell line PANC1, we detected several DNA fragments with abnormally intensified signals by restriction landmark genomic scanning. Major five of these fragments were cloned. All of the cloned fragments were mapped at the 19q13.1-13.2 region where the AKT2 oncogene was located. Southern blotting using the cloned DNA fragments and a fragment of AKT2 cDNA as probes revealed that the AKT2 gene was amplified in 3 of 12 pancreatic cancer cell lines analyzed including PANC1 and in 3 of 20 primary pancreatic cancers. The AKT2 gene was overexpressed in the 3 cell lines with the amplified gene. The results suggest that the AKT2 gene is a candidate oncogene activated by amplification in some human pancreatic cancers.

Base Sequence↗
PubMed1996

Purification, gene cloning, and reconstitution of the heterotrimeric single-stranded DNA-binding protein from Schizosaccharomyces pombe.

We have purified a single-stranded DNA-binding protein (SSB) from Schizosaccharomyces pombe (Sp) and have shown that it is composed of three subunits of 68, 30, and 12 kDa. The SpSSB supports T antigen-dependent unwinding of SV40 ori containing DNA, but is not functional in the SV40 in vitro replication reaction. All three genes that encode the SpSSB subunit have been isolated. The cloned cDNA of the ssb1(+), encoding the p68 subunit, contains 609 amino acids (68.3 kDa), while that of the ssb2(+), encoding the p30 subunit, contains a 279 amino acids (30.3 kDa). The genomic DNA clone of the p12 subunit gene (ssb3(+)) has 2 introns and an open reading frame of 104 amino acids (11.8 kDa). Significant homology is observed among the largest and middle subunits of eukaryotic SSBs, but there is poor homology among the smallest subunits. In addition, we have reconstituted the SpSSB complex by coexpression of all three subunits in Escherichia coli. The reconstituted complex is active in single-stranded DNA binding and the T antigen-dependent unwinding of SV40 ori DNA. Finally, we observed a cell cycle-dependent phosphorylation pattern of the p30 subunit of SpSSB, which is similar to that observed for the human and Saccharomyces cerevisiae SSB.

Amino Acid Sequence↗
PubMed1996

Inactive allele-specific methylation and chromatin structure of the imprinted gene U2af1-rs1 on mouse chromosome 11.

The imprinted U2af1-rs1 gene that maps to mouse chromosome 11 is predominately expressed from the paternal allele. We examined the methylation of genomic sequences in and around the U2af1-rs1 locus to establish the extent of sequence modifications that accompanied the silencing of the maternal allele. The analysis of HapII or HhaI sites showed that the silent maternal allele was hypermethylated in a block of CpG sequences that covered more than 10 kb. By comparison, the expressed paternal allele was unmethylated from a CpG island upstream of the transcribed region through 2 kb. An analysis of DNaseI hypersensitivity of a putative promoter of U2af1-rs1 showed an open chromatin conformation only on the unmethylated, expressed paternal allele. These results suggest that allele-specific hypermethylation covering the gene and its upstream CpG island plays a role in maternal allele repression of U2af1-rs1, which is reflected in altered chromatin conformation of DNaseI hypersensitive sites.

Alleles↗
PubMed1996

Proteasome pathway operates for the degradation of ornithine decarboxylase in intact cells.

Ornithine decarboxylase (ODC) is degraded in an ATP-dependent manner in vitro by the 26 S proteasome in the presence of antizyme, an ODC destabilizing protein induced by polyamines. In the present study we examined whether the proteasome catalyses ODC degradation in living mammalian cells. Lactacystin, the most selective proteasome inhibitor, strongly inhibited the degradation of ODC that had been induced in hepatoma tissue-culture (HTC) cells by refeeding with fresh medium. Furthermore the inhibitor inhibited the rapid degradation of ODC that had been induced by hypotonic shock. Interestingly, hypertonic shock was found to increase the proportion of OD present as a complex with antizyme (the ratio of ODC-antizyme complex to total ODC). Cycloheximide, which partly inhibited rapid ODC degradation caused by hypertonic shock, also part inhibited the increase in the ratio of ODC-antizyme complex total ODC. These results suggest that a common ODC degradation pathway, namely the antizyme-dependent and 26 proteasome-catalysed ODC degradation pathway, is also operating in intact cells for osmoregulated ODC degradation.

Acetylcysteine↗
PubMed1996

Stimulating effect of 6R-tetrahydrobiopterin on Ca2+ channels in neurons of rat dorsal motor nucleus of the vagus.

We have recently found that 6R-tetrahydrobiopterin (6R-BH4), a natural cofactor for aromatic L-amino acid hydroxylases and nitric oxide synthase, enhances dopamine release. Here, using a slice patch method, we examined the effect of 6R-BH4 on Ca2+ channels in neurons of rat dorsal motor nucleus of the vagus, where dopaminergic neurons are densely located. 6R-BH4 enhanced N-type Ca2+ channel currents, whereas 6S-BH4, a diastereoisomer of 6R-BH4, had little effect. Neither sodium nitroprusside, a nitric oxide generator, nor L-DOPA, a product of tyrosine hydroxylation, mimicked the effect of 6R-BH4. These findings suggest that 6R-BH4 enhances N-type Ca2+ channel currents in stereospecifically and independently of its cofactor activities as observed in its dopamine releasing action, and raise possibility that 6R-BH4 enhances dopamine release by activating Ca2+ channels.

Animals↗
PubMed1996

Ursodeoxycholic acid inhibits the induction of nitric oxide synthase.

Ursodeoxycholic acid was recently recognized as an effective agent in the treatment of primary biliary cirrhosis. Since the beneficial effect of ursodeoxycholic acid therapy appears to be mediated in part by an immune mechanism, we evaluated the effects of ursodeoxycholic acid on the synthesis of nitric oxide (NO), elevated production of which could be important in the pathogenesis of autoimmunity. Ursodeoxycholic acid (0.1-1000 microM) inhibited NO production by bacterial lipopolysaccharide-activated J774 macrophages in a concentration-dependent fashion, but the cytotoxicity was also evident at higher concentrations (250 and 1000 microM). Ursodeoxycholic acid did not have any effect on the activity of NO synthase that had already been induced. Treatment with lipopolysaccharide led to a significant expression of NO synthase mRNA that was significantly reduced by ursodeoxycholic acid. Findings indicated that ursodeoxycholic acid inhibited NO synthesis by inhibiting the induction of NO synthase, rather than its catalytic activity. Ursodeoxycholic acid therapy may exert a beneficial effect, in part, by attenuating the production of NO.

Animals↗
PubMed1996

Cloning of antizyme inhibitor, a highly homologous protein to ornithine decarboxylase.

The degradation of ornithine decarboxylase (ODC) catalyzed by the 26 S proteasome is accelerated by antizyme, an ODC inhibitory protein induced by polyamines. Previously, we have found another possible regulatory protein of ODC degradation, antizyme inhibitor. Antizyme inhibitor binds to the antizyme with a higher affinity than that of ODC, releasing ODC from ODC-antizyme complex. We report here the cDNA sequence of rat heart antizyme inhibitor. The deduced sequence of the protein is highly similar to, but distinct from, sequences of ODCs from various species. Antizyme inhibitor contains amino acid residues required for formation of active sites of ODC, but it completely lacks ODC activity. Antizyme inhibitor has no homology with peptide sequence in the mammalian ODC carboxyl terminus, which is needed for rapid turnover of ODC. It inhibits antizyme-dependent ODC degradation, but, unlike ODC, its degradation is not accelerated by antizyme.

Amino Acid Sequence↗