Biomedical subjects

T Nishimoto

Publications and source records attributed to T Nishimoto.

At least 19 recordsLinked to original sources

PubMed2003

A novel nuclear protein, Twa1, and Muskelin comprise a complex with RanBPM.

A truncated human RanBPM has been isolated as a protein binding to Ran, Ras-like nuclear small GTPase. Full-sized human RanBPM cDNA which was recently isolated, was found to encode a protein of 90 kDa which comprises a large protein complex. Consistent with this finding, several proteins were found to be co-precipitated with RanBPM by immunoprecipitation analysis. Accordingly, in the present study, we screened the human cDNA library by the two-hybrid method using RanBPM cDNA as bait. One novel protein designated as Twa1 (Two hybrid associated protein No. 1 with RanBPM), and two known proteins, a human homologue (hMuskelin) of mouse Muskelin and HSMpp8 were isolated repeatedly. Twa1 was well conserved through evolution and was localized within the nucleus. Interestingly, in addition to Muskelin and RanBPM, Twa1 was found to possess the LisH-CTLH motif which is detected in proteins involved in microtubule dynamics, cell migration, nucleokinesis and chromosome segregation. These functions overlap with functions suggested for the RanGTPase cycle. Immunoprecipitation and gel-filtration analyses indicated that both Twa1 and hMuskelin did indeed comprise a protein complex with RanBPM. Taken together with the fact that RanBPM interacts with Ran, our present findings suggested that there is an as yet uncovered function of the RanGTPase cycle.

Adaptor Proteins, Signal Transducing↗

PubMed2003

The pilot trial of the prevention of the increase in electrical taste thresholds by zinc containing fluid infusion during chemotherapy to treat primary lung cancer.

It is well known that there are various adverse effects during chemotherapy for cancer treatment. A taste disorder is also seen in 35-70% of patients. It has been reported that a zinc deficiency is associated with the development of these alterations in taste sensation. The purpose of this pilot study was to determine whether the zinc including infusion had the effect on taste disorder in patients with lung cancer. Taste disorder was evaluated as the increase in electrical taste thresholds using an electrogustometer. The plasma zinc concentration was also measured. Although there was no significant correlation, the increase in taste thresholds was detected in many patients who had a low zinc concentration even before receiving chemotherapy. Moreover, after 2 weeks of chemotherapy, almost all patients who did not have a zinc containing infusion showed development of taste disorder (5/5, 100% at chorda tympani area; 4/5, 80% at glossopharyngeal area), whereas no development of taste disorder was observed in those patients receiving a zinc containing infusion. These results suggest the possibility that the administration of zinc during chemotherapy could be a useful supportive therapy for preventing taste disorder and to help maintain a better quality of life.

Aged↗

PubMed2001

The human licensing factor for DNA replication Cdt1 accumulates in G1 and is destabilized after initiation of S-phase.

S-phase onset is controlled, so that it occurs only once every cell cycle. DNA is licensed for replication after mitosis in G(1), and passage through S-phase removes the license to replicate. In fission yeast, Cdc6/18 and Cdt1, two factors required for licensing, are central to ensuring that replication occurs once per cell cycle. We show that the human Cdt1 homologue (hCdt1), a nuclear protein, is present only during G(1). After S-phase onset, hCdt1 levels decrease, and it is hardly detected in cells in early S-phase or G(2). hCdt1 can associate with the DNA replication inhibitor Geminin, however these two proteins are mostly expressed at different cell cycle stages. hCdt1 mRNA, in contrast to hCdt1 protein, is expressed in S-phase-arrested cells, and its levels do not change dramatically during a cell cycle, suggesting that proteolytic rather than transcriptional controls ensure the timely accumulation of hCdt1. Consistent with this view, proteasome inhibitors stabilize hCdt1 in S-phase. In contrast, hCdc6/18 levels are constant through most of the cell cycle and are only low for a brief period at the end of mitosis. These results suggest that the presence of active hCdt1 may be crucial for determining when licensing is legitimate in human cells.

Animals↗

PubMed2001

Full-sized RanBPM cDNA encodes a protein possessing a long stretch of proline and glutamine within the N-terminal region, comprising a large protein complex.

Previously isolated RanBPM, a Ran-binding protein in the microtubule-organizing center, which had been thought to play a role in Ran-stimulated microtubule assembly, turned out to be a truncated protein. To clarify the function of RanBPM, we cloned the full-sized RanBPM cDNA that encodes a 90 kDa protein, compared to the previously isolated cDNA that encoded a 55 kDa protein. The newly cloned 5' coding region contains a great number of cytidine and guanidine nucleotides, like the CpG island. Thus, full-sized RanBPM cDNA encodes a long stretch of proline and glutamine residues in the N-terminal region. It comprises a protein complex of more than 670 kDa. Ran was detected in this complex when RanBPM and Ran were both ectopically expressed. New antibodies to RanBPM were prepared against three different regions of RanBPM. All of them detected a 90 kDa protein that is predominantly localized both in the nucleus and in the cytoplasmic region surrounding the centrosome, but none of them stained the centrosome. In this context, our previous notion that RanBPM is a centrosomal protein should be discarded. RanBPM is well conserved in the animal kingdom. It may play an important role in uncovering Ran-dependent nuclear events.

Adaptor Proteins, Signal Transducing↗

PubMed2001

The N-terminal helix of Xenopus cyclins A and B contributes to binding specificity of the cyclin-CDK complex.

Mitotic cyclins A and B contain a conserved N-terminal helix upstream of the cyclin box fold that contributes to a significant interface between cyclin and cyclin-dependent kinase (CDK). To address its contribution on cyclin-CDK interaction, we have constructed mutants in conserved residues of the N-terminal helix of Xenopus cyclins B2 and A1. The mutants showed altered binding affinities to Cdc2 and/or Cdk2. We also screened for mutations in the C-terminal lobe of CDK that exhibited different binding affinities for the cyclin-CDK complex. These mutations were at residues that interact with the cyclin N-terminal helix motif. The cyclin N-terminal helix mutations have a significant effect on the interaction between the cyclin-CDK complex and specific substrates, Xenopus Cdc6 and Cdc25C. These results suggest that the N-terminal helix of mitotic cyclins is required for specific interactions with CDKs and that to interact with CDK, specific substrates Cdc6 and Cdc25C require the CDK to be associated with a cyclin. The interaction between the cyclin N-terminal helix and the CDK C-terminal lobe may contribute to binding specificity of the cyclin-CDK complex.

Amino Acid Sequence↗

PubMed2001

Fabrication of quartz microchips with optical slit and development of a linear imaging UV detector for microchip electrophoresis systems.

We have developed quartz microchips for electrophoresis and a linear imaging UV detector along with the microchip. The microchips have an optical slit, which cut off the stray light in order to improve the sensitivity of UV absorption detection on the chip, at the bonding interface. They have been successfully fabricated on synthesized quartz glass substrates using the hydrofluoric acid (HF) solution bonding method. The signal level of UV absorption detection was effectively improved by applying microchips with the "on-chip" optical slit. It is also possible to improve the signal-to-noise ratio by repetitive scanning of linear photodiode array located along the separation channel, and signal averaging during elimination of the potential. Furthermore, the analysis may be performed until the separation of the target component is complete, because the real-time migration pattern of each component in the sample can be seen just as in a slab-gel electrophoresis, thus enabling a shorter analysis time.

Electrophoresis↗

PubMed2001

The Saccharomyces cerevisiae small GTPase, Gsp1p/Ran, is involved in 3' processing of 7S-to-5.8S rRNA and in degradation of the excised 5'-A0 fragment of 35S pre-rRNA, both of which are carried out by the exosome.

Dis3p, a subunit of the exosome, interacts directly with Ran. To clarify the relationship between the exosome and the RanGTPase cycle, a series of temperature-sensitive Saccharomyces cerevisiae dis3 mutants were isolated and their 5.8S rRNA processing was compared with processing in strains with mutations in a S. cerevisiae Ran homologue, Gsp1p. In both dis3 and gsp1 mutants, 3' processing of 7S-to-5.8S rRNA was blocked at three identical sites in an allele-specific manner. In contrast, the 5' end of 5.8S rRNA was terminated normally in gsp1 and in dis3. Inhibition of 5.8S rRNA maturation in gsp1 was rescued by overexpression of nuclear exosome components Dis3p, Rrp4p, and Mtr4p, but not by a cytoplasmic exosome component, Ski2p. Furthermore, gsp1 and dis3 accumulated the 5'-A0 fragment of 35S pre-rRNA, which is also degraded by the exosome, and the level of 27S rRNA was reduced. Neither 5.8S rRNA intermediates nor 5'-A0 fragments were observed in mutants defective in the nucleocytoplasmic transport, indicating that Gsp1p regulates rRNA processing through Dis3p, independent of nucleocytoplasmic transport.

Active Transport, Cell Nucleus↗

PubMed2001

Enzymatic synthesis of novel oligosaccharides from L-sorbose, maltose, and sucrose using kojibiose phosphorylase.

Glucosyl-L-sorbose, -maltose, and -sucrose were synthesized using kojibiose phosphorylase (KPase) from Thermoanaerobacter brockii ATCC35047 with beta-D-glucose-1-phosphate (beta-G1P) as a glucosyl donor. One disaccharide and two trisaccharides thus synthesized were isolated by Toyopearl HW-40S column chromatography. The results of KPase digestion, methylation analysis, and 13C-NMR studies indicated that these oligosaccharides were alpha-D-glucopyranosyl-(1-->5)-alpha-L-sorbopyranose, alpha-D-glucopyranosyl-(1-->2)-alpha-D-glucopyranosyl-(1-->4)-D-glucopyranose (4-alpha-D-kojibiosyl-glucose), and alpha-D-glucopyranosyl-(1-->2)-alpha-D-glucopyranosyl-(1-->2)-beta-D-fructofuranoside, which are all novel oligosaccharides. Glucosyl-L-sorbose was partially hydrolyzed to glucose and L-sorbose by alpha-glucosidases, while glucosyl-sucrose and glucosyl-maltose were not hydrolyzed by glucoamylase, alpha-glucosidases, or CGTase.

Journal Article↗

PubMed2001

Enzymatic synthesis of kojioligosaccharides using kojibiose phosphorylase.

We have attempted to synthesize kojioligosaccharides (oligosaccharides having the alpha-1,2 glycosidic linkage at the nonreducing end) using two methods. In the first, mixtures of various proportions of glucose and beta-D-glucose-1-phosphate (beta-G1P) were allowed to react in the presence of kojibiose phosphorylase (KPase). In the second, maltose was allowed to react with KPase and maltose phosphorylase (MPase) simultaneously. In the former method, kojioligosaccharides having only the alpha-1,2 glucosidic linkage were synthesized and the average degree of polymerization (D.P.) of oligosaccharides increased with decreasing proportions of glucose. In the second method, kojioligosaccharides were obtained at approximately 70% yields under optimum conditions. 4-alpha-D-Kojibiosyl-glucose, kojitriose and kojitetraose, the principal kojioligosaccharides synthesized, were not hydrolyzed by salivary amylase, artificial gastric juice, pancreatic amylase, or small intestinal enzymes.

Journal Article↗

PubMed2001

Change in cytokeratin 19 fragment level according to the severity of pulmonary alveolar proteinosis.

A 48-year-old man was diagnosed with primary alveolar proteinosis (PAP). Whole lung lavage was performed for treatment, and the opacity on his chest X-ray completely disappeared. The value of cytokeratin 19 fragment (CYFRA) in the serum was high at the beginning, decreased after lung lavage, and became elevated again when the disease recurred 7 months later. As PAP is thought to be a problem of surfactant secreted from type II pneumocytes, and a cytokeratin is present in the alveolar epithelial tissue, the value of serum CYFRA might be related to the severity of PAP.

Biomarkers↗

PubMed2000

Novel G proteins, Rag C and Rag D, interact with GTP-binding proteins, Rag A and Rag B.

Rag A/Gtr1p are G proteins and are known to be involved in the RCC1-Ran pathway. We employed the two-hybrid method using Rag A as the bait to identify proteins binding to Rag A, and we isolated two novel human G proteins, Rag C and Rag D. Rag C demonstrates homology with Rag D (81.1% identity) and with Gtr2p of Saccharomyces cerevisiae (46.1% identity), and it belongs to the Rag A subfamily of the Ras family. Rag C and Rag D contain conserved GTP-binding motifs (PM-1, -2, and -3) in their N-terminal regions. Recombinant glutathione S-transferase fusion protein of Rag C efficiently bound to both [(3)H]GTP and [(3)H]GDP. Rag A was associated with both Rag C and Rag D in their C-terminal regions where a potential leucine zipper motif and a coiled-coil structure were found. Rag C and D were associated with both the GDP and GTP forms of Rag A. Both Rag C and Rag D changed their subcellular localization, depending on the nucleotide-bound state of Rag A. In a similar way, the disruption of S. cerevisiae GTR1 resulted in a change in the localization of Gtr2p.

Amino Acid Motifs↗

PubMed2000

Yrb1p interaction with the gsp1p C terminus blocks Mog1p stimulation of GTP release from Gsp1p.

Mog1p, a multicopy suppressor of gsp1, the temperature-sensitive mutant of the Saccharomyces cerevisiae Ran homologue, binds to GTP-Gsp1p but not to GDP-Gsp1p. The function of Mog1p in the Ran cycle is as yet unknown. This study found that Mog1p releases a nucleotide from GTP-Gsp1p but not from GDP-Gsp1p. Yrb1p, the S. cerevisiae homologue of RanBP1, which is a strong inhibitor of RCC1-stimulated nucleotide release, also inhibited the Mog1p-stimulated nucleotide release from GTP-Gsp1p. At a concentration corresponding to the molar concentration of GTP-Gsp1p, Yrb1p completely inhibited the Mog1p-stimulated nucleotide release. Consistently, the Yrb1p.GTP-Gsp1p complex was more stable than the Mog1p.GTP-Gsp1p complex. Yrb1p did not inhibit the Mog1p-stimulated nucleotide release from GTP-Gsp1DeltaC. The Gsp1DeltaC protein lacks the final eight amino acids of the C terminus, and for this reason, the interaction between GTP-Gsp1DeltaC and Yrb1p was strongly reduced. On the other hand, Mog1p binds to GTP-Gsp1DeltaC more efficiently than to GTP-Gsp1p.

Carrier Proteins↗

PubMed2000

Inhibition of N-linked glycosylation causes apoptosis in hamster BHK21 cells.

The tsBN7 cell line is one of the temperature-sensitive mutants for cell proliferation derived from hamster BHK21 cell line. It has a mutation in the DAD1 gene and enters apoptosis at the restrictive temperature of 39 degrees C. The defect of Dad1p causes a loss of N-linked glycosylation; therefore, it was thought that an inhibition of N-linked glycosylation induced apoptosis.However, tunicamycin, a potent inhibitor of N-linked glycosylation, had not caused apoptosis in wild-type BHK21 cells. In order to clarify this discrepancy, wild-type BHK21 cells treated with tunicamycin and tsBN7 cells incubated at 39.5 degrees C were examined by the annexin V staining and TUNEL methods. Both methods showed that tunicamycin induces apoptosis in wild-type BHK21 cells, similar to the defect of Dad1p. Thus, we concluded that loss of N-linked glycosylation causes apoptosis.

Animals↗

PubMed2000

The Cdt1 protein is required to license DNA for replication in fission yeast.

To maintain genome stability in eukaryotic cells, DNA is licensed for replication only after the cell has completed mitosis, ensuring that DNA synthesis (S phase) occurs once every cell cycle. This licensing control is thought to require the protein Cdc6 (Cdc18 in fission yeast) as a mediator for association of minichromosome maintenance (MCM) proteins with chromatin. The control is overridden in fission yeast by overexpressing Cdc18 (ref. 11) which leads to continued DNA synthesis in the absence of mitosis. Other factors acting in this control have been postulated and we have used a re-replication assay to identify Cdt1 (ref. 14) as one such factor. Cdt1 cooperates with Cdc18 to promote DNA replication, interacts with Cdc18, is located in the nucleus, and its concentration peaks as cells finish mitosis and proceed to S phase. Both Cdc18 and Cdt1 are required to load the MCM protein Cdc21 onto chromatin at the end of mitosis and this is necessary to initiate DNA replication. Genes related to Cdt1 have been found in Metazoa and plants (A. Whitaker, I. Roysman and T. Orr-Weaver, personal communication), suggesting that the cooperation of Cdc6/Cdc18 with Cdt1 to load MCM proteins onto chromatin may be a generally conserved feature of DNA licensing in eukaryotes.

Cell Cycle↗

PubMed2000

Normal values and age-dependent changes in GTP cyclohydrolase I activity in stimulated mononuclear blood cells measured by high-performance liquid chromatography.

GTP cyclohydrolase I (GCH1) activity in phytohemaglutinin (PHA)-stimulated mononuclear blood cells (MBCs) is a useful clinical marker for diagnosis of tetrahydrobiopterin (BH4)-related genetic disorders such as recessively inherited GCH1 deficiency and dominantly inherited dopa-responsive dystonia (Segawa's disease). Since the assay is complex, including isolation of MBCs from blood, stimulation of MBCs by PHA under culture, isolation of the protein fraction from the PHA-stimulated MBCs, and the subsequent activity measurement, the reproducibility is problematic in its application to clinical study. We established a sensitive and reproducible method by high-performance liquid chromatography with fluorescence detection for clinical assay of GCH1 in PHA-stimulated MBCs, and measured the normal values of 91 healthy males and females of various ages (1-74 years). The mean normal values were 19.1+/-0.9 pmol/mg protein per h (mean+/-S.E., n=91). There were no significant differences between males and females. The activity tends to be higher in the first decade and to be decreased from the second to third decade and becomes almost stable from the third decade.

Age Factors↗

PubMed2000

Premature chromatin condensation caused by loss of RCC1.

Hamster rcc1 mutant, tsBN2, prematurely enter mitosis during S phase. RCC1 is a guanine nucleotide exchanging factor for a small G protein Ran and localised on the chromatin, whereas RanGTPase activating protein is in the cytoplasm. Consistently, Ran shuttles between the nucleus and the cytoplasm, carrying out nucleus-cytosol exchange of macromolecules, which regulates the cell cycle. The finding that loss of RCC1 which disturbs nuclear protein export due to loss of RanGTP, abrogates the check point control suggests that RCC1 senses the status of the chromatin, such as replication, and couples it to the cell cycle progression through Ran.

Animals↗

PubMed2000

Isolation and characterisation of a mutation in the PMR1 gene encoding a Golgi membrane ATPase, which causes hypersensitivity to over-expression of Clb3 in Saccharomyces cerevisiae.

We screened for mutant strains of Saccharomyces cerevisiae that are sensitive to overexpression of specific cyclins, and identified mutations in two genes that caused growth inhibition in response to mild overexpression of Clb3. One was the ANP1 gene, which encodes a glycosyltransferase previously identified by a similar strategy using Clb2 instead of Clb3. This paper describes the second strain of S. cerevisiae that is hypersensitive to Clb3 expression. The gene mutated in this strain was identified as PMR1, which encodes a Ca2+-ATPase located in the Golgi membrane. The protein product of pmr1-1 was truncated at residue 409 and thus lacked the C-terminal ATPase domain. The pmr1-1 strain was hypersensitive to over-expression of Clb3, but not Cln2, Clb5 or Clb2. The lethality due to Clb3 expression in pmr1-1 could be suppressed by adding Ca2+ ions to the medium. The pmr1-1 strain proved to be defective in glycosylation, and the defects in glycosylation were exacerbated by high levels of Clb3. On induction of Clb3 expression in the pmr1-1 strain, the cells arrested at anaphase with an elongated daughter bud. We discuss possible interpretations of this synthetic lethal phenotype.

Calcium↗

PubMed2000

Development of a host-vector system for Lactobacillus plantarum L137 isolated from a traditional fermented food produced in the Philippines.

Lactobacillus plantarum NC13, a strain derived from the L. plantarum strain L137 isolated from a traditional fermented food produced in the Philippines, contains 9 of the 15 plasmids in the parental strain. To construct a shuttle vector between L. plantarum and Escherichia coli for genetic manipulation of L137 and its derivatives, recombinant plasmids were prepared by using the 9-plasmid DNA mixture and an E. coli vector, pBluescript II SK+. The resultant recombinant plasmids were re-transferred to L. plantarum NCL21, an NC13-derived strain cured of 3 of the 9 plasmids, and 3 recombinant plasmids were obtained. The smallest plasmid, pRN14, contained a small cryptic plasmid, pLTK2, which is one of the plasmids in L. plantarum L137. Thus, the complete nucleotide sequence of pLTK2 was determined. The pLTK2 is 2295 bp in length, and has a major open reading frame of 951 bp. An encoded sequence of 317-amino acids showed extensive similarity with genes encoding replication protein (repA). A putative replication origin in pLTK2 also showed high homology to those of other gram-positive bacterial plasmids that replicate by the rolling circle mechanism. The shuttle vector pRN14 contained the erythromycin resistance gene and the ColE1 and pLTK2 replication origins. Transformation of L. plantarum strains with pRN14 by electroporation was optimized to give a transformation efficiency of 2 x 10(4) transformants/ mug plasmid. Plasmid pRN14 was stably maintained in strain NCL21, as well as in L. casei K95-5.

Journal Article↗