Biomedical subjects

H Nojima

Publications and source records attributed to H Nojima.

At least 19 recordsLinked to original sources

PubMed2001

A novel meiosis-specific protein of fission yeast, Meu13p, promotes homologous pairing independently of homologous recombination.

Meiotic homologous pairing is crucial to proper homologous recombination, which secures subsequent reductional chromosome segregation. We have identified a novel meiosis-specific protein of fission yeast Schizosaccharomyces pombe, Meu13p, to be a molecule that is required for proper homologous pairing and recombination. Rec12p (homologue of Saccharomyces cerevisiae Spo11p), which is essential for the initiation of meiotic recombination, is also shown for the first time to participate in the pairing process of S.pombe. Meu13p, however, contributes to pairing through a recombination-independent mechanism, as disruption of the meu13(+) gene reduces pairing whether the rec12(+) gene is deleted or not. We also demonstrate a dynamic nature of homologous pairing in living meiotic cells, which is markedly affected by meu13 deletion. Meu13p is not required for telomere clustering and the nuclear movement process, which are well known requirements for efficient pairing in S.pombe. Based on these results, together with the localization of Meu13p on meiotic chromatin, we propose that Meu13p directly promotes proper homologous pairing and recombination.

Amino Acid Sequence↗

PubMed2001

Comprehensive isolation of meiosis-specific genes identifies novel proteins and unusual non-coding transcripts in Schizosaccharomyces pombe.

In order to isolate meiosis-specific genes in Schizosaccharomyces pombe, we have constructed a subtracted cDNA library enriched in clones whose expression is enhanced during meiosis induced by nitrogen starvation. Using northern blot analysis, we isolated 31 kinds of clones whose expression was induced in a meiosis/sporulation-specific manner. We comprehensively named them meu after meiotic expression upregulated. The transcription of 20 meu genes was found to be dependent on the mei4(+) gene, which encodes a transcription factor required for the progression of meiosis. DNA sequencing indicated that most of the meu genes encode novel proteins. Notably, five of the meu genes harbor no apparent protein coding sequences, and the transcripts form stable hairpin structures, suggesting that they may generate non-coding RNAs or antisense RNAS: The results presented here imply that RNAs are also important for the comprehensive characterization of genomic expression.

Base Sequence↗

PubMed2001

Cyclin G1 is involved in G2/M arrest in response to DNA damage and in growth control after damage recovery.

Cyclin G1 is one of the target genes of the transcription factor p53, and is induced in a p53-dependent manner in response to DNA damage. Although cyclin G1 has been implicated in a range of biological phenomena, its precise function remains unclear. Here we present an analysis of the physiological role of cyclin G1 using mice homozygous for a targeted disruption of the cyclin G1 gene. In order to clarify the role of cyclin G1 in the p53 pathway, downstream events such as apoptosis, cell growth and cell cycle checkpoint control were analysed in thymocytes and embryonic fibroblasts derived from cyclin G1-disrupted mice. No difference was detected in induction of apoptosis between mouse embryo fibroblasts (MEFs) derived from cyclin G1+/+ and cyclin G1-/- mice. Following irradiation, cyclin G1-/- MEFs proliferated more slowly and reached lower cell densities in culture dishes than cyclin G1+/+ MEFs. Analysis of cell survival showed that cyclin G1-/- MEFs were about twice as sensitive as cyclin G1+/+ MEFs to gamma radiation or UV radiation. Cyclin G1-/- mice were more sensitive to gamma radiation than wild-type mice. Flow cytometeric analysis revealed that the number of cyclin G1-/- MEFs in G2/M phase after irradiation was reduced by 50% relative to cyclin G1+/+ MEFs. Our results demonstrate that cyclin G1 plays roles in G2/M arrest, damage recovery and growth promotion after cellular stress.

Animals↗

PubMed2001

Kcc4 associates with septin proteins of Saccharomyces cerevisiae.

Kcc4, a kinase of the budding yeast Saccharomyces cerevisiae, is homologous to the bud neck protein kinases Hsl1/Nik1 and Gin4. We report here that a GFP-Kcc4 fusion protein is localized at the bud neck and that the non-kinase domain is required for this localization. We also demonstrate that Kcc4 associates with septin proteins in vitro and in vivo by two-hybrid analysis, GST pull-down experiments, immunoprecipitation, and analysis of direct association with affinity-purified GST-Kcc4 and MBP-Septin proteins. From the results obtained here, we suggest that Cdc11 is the primary association partner of Kcc4.

Cell Cycle Proteins↗

PubMed2001

Increased expression of a nucleolar Nop5/Sik family member in metastatic melanoma cells: evidence for its role in nucleolar sizing and function.

F10 and BL6 cells of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells can metastasize to lungs after subcutaneous injection. Differences in gene expression between the two cell lines were examined, and a greater expression of the Sik-similar protein (Sik-SP) gene was found in BL6 cells. Structurally, Sik-SP belongs to the nucleolar Nop5/Sik family whose members play central roles in ribosome biogenesis; however, the function of Sik-SP has not been examined. Cytology with green fluorescent protein-fused proteins showed that Sik-SP was localized to the nucleolus. To examine whether Sik-SP is involved in ribosome biogenesis, two parameters were measured: magnitude of ribosomal RNA synthesis per nucleus and magnitude of protein production from the same amount of mRNA of an exogenous luciferase gene. Both values and, in addition, nucleolar size were larger in COS-7 monkey kidney cells overexpressing Sik-SP and BL6 cells than in mock-transfected COS-7 and F10 cells, respectively. Sik-SP seemed to promote ribosome biogenesis in the nucleolus. Furthermore, the expression of Sik-SP seemed to confer a greater cell growth response to serum, because such a response was greater in BL6 cells and F10 cells overexpressing Sik-SP than in untreated and mock-transfected F10 cells. Sik-SP may render melanoma cells more competent to survive through augmenting the activity of nucleolus.

Animals↗

PubMed2001

Induction by carrageenan inflammation of prepronociceptin mRNA in VR1-immunoreactive neurons in rat dorsal root ganglia.

Nociceptin (orphanin FQ) may act on primary afferents and be involved in the regulation of nociceptive processing. We have shown, using reverse transcription-polymerase chain reaction (RT-PCR), that carrageenan-produced peripheral inflammation induces the expression of prepronociceptin (PPN) mRNA in the dorsal root ganglia (DRG). The present experiments were conducted to determine the localization of PPN mRNA in primary sensory neurons after peripheral inflammation, using in situ hybridization. An intraplantar injection of carrageenan induced the expression of PPN mRNA in small and medium sized neurons in the DRG; the effect peaked 0.5 h after carrageenan and subsided by 6 h. All neurons positive for PPN mRNA were positive for vanilloid receptor subtype 1 (VR1)-like immunoreactivity and some VR1-immunoreactive neurons were negative for PPN mRNA. The results suggest that peripheral inflammation induces the production of nociceptin in a sub-population of VR1-positive primary sensory neurons and support the idea that nociceptin produced there is involved in the regulation of nociceptive processing.

Animals↗

PubMed2001

Characterization of itch-associated responses of NC mice with mite-induced chronic dermatitis.

To clarify the behavioral and pathological features of spontaneous scratching of NC mice with mite-induced chronic dermatitis, we investigated the spontaneous and pruritogen-evoked scratching of NC mice. Although the frequency of scratching of NC mouse did not increase under specific pathogen-free environment, it gradually and markedly increased from 3 to 6 weeks after transfer to conventional environment. The onset of increase in spontaneous scratching was similar to that of dermatitis development and the elevation of plasma concentration of immunoglobulin E. At chronic stage (16 weeks after environment change), the frequency of spontaneous scratching was roughly parallel to the degree of dermatitis, but not to the plasma concentration of immunoglobulin E. The spontaneous scratching of NC mice with dermatitis was inhibited by distraction and the opioid antagonist naltrexone, suggesting that the scratching is itch-associated response. An intradermal injection of serotonin, but not histamine and substance P, elicited scratching of the injected site. Methysergide and cyproheptadine inhibited the serotonin-induced scratching but not spontaneous scratching. The results suggest that marked elevation of plasma immunoglobulin E is not always the cause of spontaneous itch-associated response of NC mice with dermatitis. Serotonin, histamine and substance P may not play an important role in spontaneous itch-scratch response at a chronic stage.

Animals↗

PubMed2001

Characterization of rec7, an early meiotic recombination gene in Schizosaccharomyces pombe.

rec7 is involved in intra- and intergenic meiotic recombination in all tested regions of the genome of the fission yeast Schizosaccharomyces pombe. Segregational analysis in a rec7 gene disruption mutant revealed frequent occurrence of two-spored asci. Spores giving rise to diploid colonies were shown to derive from skipping of the second meiotic division. Nondisjunction of homologous chromosomes at the first meiotic division was also frequent. The cytological structures and processes, such as formation of linear elements, pairing of homologous chromosomes, and clustering of telomeres and centromeres, are regular in the mutant. Northern blot experiments revealed meiosis-specific expression of rec7. Screening of a meiotic cDNA library also identified transcripts from the opposite strand in the rec7 region. A Rec7-GFP fusion protein was localized in the nucleus of whole cells before karyogamy, during prophase, and after meiosis I. On spreads of prophase nuclei approximately 50 foci of Rec7-GFP were counted. Some of the observed phenotypes of the disruption mutant and the N-terminal sequence homology suggest that Rec7p is a functional homolog of Rec114p of Saccharomyces cerevisiae. The observed phenotypes of the disruption and the appearance of Rec7-GFP in mating haploid cells and after meiosis I are consistent with Rec7p functions before, during, and after meiotic prophase.

Blotting, Northern↗

PubMed2001

Structure, expression and chromosome mapping of MLZE, a novel gene which is preferentially expressed in metastatic melanoma cells.

We isolated a novel gene, termed MLZE, from a B16-BL6 cDNA library after subtraction of B16-F10 mRNA. Expression levels of mouse MLZE (mMLZE) increased in accordance with metastatic ability of B16 melanoma sublines. Human homolog of mMlze (hMlze) contained one leucine zipper structure and two potential nuclear localizing signals. Northern blot analysis of multiple human tissues showed that hMLZE was expressed primarily in trachea and spleen. We mapped the hMLZE gene (by fluorescence in situ hybridization) to 8q24.1 - 2, which contains the c-myc gene and is often amplified in malignant melanoma. Immunohistochemistry revealed that the number of hMlze-positive cases was significantly larger in Clark levels III, IV and V melanomas (6 / 11 = 55%) than in Clark levels I and II melanomas (2 / 15 = 13%). In two cases of hMlze-positive melanomas, the strength of hMlze staining increased substantially in the deep component of the tumor. Considering that melanomas above Clark level II are more metastatic than those below Clark level III, these findings suggested that MLZE is one of the genes whose expression is upregulated during the course of acquisition of metastatic potential in melanoma cells.

Adult↗

PubMed2001

Inhibition of itch-scratch response by fruits of Cnidium monnieri in mice.

We previously screened the anti-itching activities of 33 herbal medicines in substance P (SP)-induced itching model mice. One of the most potent antipruritogenic extracts, the methanol extract of fruits of Cnidium monnieri (Cnidii Fructus) was studied further. The chloroform-soluble fraction of the methanol extract markedly inhibited SP-induced scratching. Among 10 subfractions of the chloroform-soluble fraction, the CS-3 fraction had the most potent inhibitory effect on scratching. Each of 3 subfractions of CS-3 showed significant anti-scratching activities. However, inhibitory potencies were not different among the three and weaker than that of CS-3 itself at a same dose. These 3 subfractions of CS-3 mainly contained xanthotoxin, isopimpinellin, bergapten, imperatorin and osthol. Single administration of osthol did not inhibit SP-induced scratching, and imperatorin very weakly subsided scratching. These results suggest that the strong antipruritic action was focused on the CS-3 fraction of the C. monnieri methanol extract, and it might result from the combined effects of these coumarin derivatives, or by undetermined minor compounds.

Animals↗

PubMed2001

Roles of mast cells and histamine in mosquito bite-induced allergic itch-associated responses in mice.

We investigated itch-associated responses (scratching) to mosquito bites and the role of histamine and mast cells in mosquito-induced itching in mice. Although the first bites of mosquito Aedes albopictus did not increase scratching, repeated bites increased scratching. The response was not diminished even after an interval of 2 months. Similarly, repeated intradermal (i.d.) injections of salivary gland extract (SGE) from Aedes albopictus increased scratching after SGE injection itself and mosquito bites. The scratching peaked within 10 min and almost subsided by 60 min. The opioid antagonist naloxone (1 mg/kg, s.c.) inhibited scratching following SGE injection. Although the non-sedative H1-histamine-receptor antagonist terfenadine (30 mg/kg, p.o.) significantly suppressed scratching induced by histamine (100 nmol/site, i.d.) in either naive or mosquito-sensitized mice, it did not affect mosquito-induced scratching in mosquito-sensitized mice. Repeated injections of SGE increased scratching in mast cell-deficient (WBB6F1-W/Wv) mice as well as in normal (WBB6F1-+/+) littermates. Repeated exposure to mosquito bites roughly doubled serum concentrations of total IgE and IgG1, but not IgG2a. Repeated injections of SGE markedly increased plasma extravasation induced by mosquito bites and such an increase was almost completely suppressed by terfenadine (30 mg/kg, p.o.). The results show the presence of histamine-mediated and histamine-independent mechanisms in cutaneous itching and suggest that histamine probably released from mast cells does not play an important role in itching in immediate allergic reaction. Our murine model of mosquito itching may be useful for studying the mechanisms of immediate allergic itching.

Aedes↗

PubMed2001

Gabapentin antinociception in mice with acute herpetic pain induced by herpes simplex virus infection.

The effects of systemic and local injections of gabapentin, a novel anticonvulsant agent, were tested on nociceptive behaviors in mice with acute herpetic pain. Transdermal infection with herpes simplex virus type-1 (HSV-1) produced nociceptive hypersensitivity of the infected hind paw to innocuous (allodynia) and noxious mechanical stimulation (hyperalgesia) with von Frey filaments. Systemic administration of gabapentin (10-100 mg/kg, peroral) produced a dose-dependent inhibition of both allodynia and hyperalgesia; gabapentin (30-300 mg/kg) did not affect locomotor activity. Intrathecal injection of gabapentin (10-100 microg/animal) also attenuated dose dependently both nociceptive hypersensitivities. In contrast, intraplantar, intracisternal, and intracerebroventricular administration of gabapentin (10-100 microg/animal) had no effect on the HSV-1-induced nociceptive hypersensitivities. Pretreatment with naltrexone (1 mg/kg) inhibited antinociceptive effect of morphine (5 mg/kg), but not gabapentin (100 mg/kg). Repeated administration of morphine (5 mg/kg, four times) led to tolerance of antinociceptive action, whereas gabapentin (100 mg/kg, four times) had antinociceptive effect even after the forth administration. The present results suggest that gabapentin is effective in the treatment of acute herpetic pain without apparent adverse effects, and analgesic action of gabapentin is mainly mediated by actions on the spinal cord.

Acetates↗

PubMed2000

Dmc1 of Schizosaccharomyces pombe plays a role in meiotic recombination.

We report here a Schizosaccharomyces pombe gene (dmc1(+)) that resembles budding yeast DMC1 in the region immediately upstream of the rad24(+) gene. We showed by northern and Southern blot analysis that dmc1(+) and rad24(+) are co-transcribed as a bicistronic mRNA of 2.8 kb with meiotic specificity, whereas rad24(+) itself is constitutively transcribed as a 1.0-kb mRNA species during meiosis. Induction of the bicistronic transcript is under the control of a meiosis-specific transcription factor, Ste11. Disruption of both dmc1(+) and rad24(+) had no effect on mitosis or spore formation, and dmc1Delta cells displayed no change in sensitivity to UV or gamma irradiation relative to the wild type. Tetrad analysis indicated that Dmc1 is involved in meiotic recombination. Analysis of gene conversion frequencies using single and double mutants of dmc1 and rhp51 indicated that both Dmc1 and Rhp51 function in meiotic gene conversion. These observations, together with a high level of sequence identity, indicate that the dmc1(+) gene of S. POMBE: is a structural homolog of budding yeast DMC1, sharing both similar and distinct functions in meiosis.

Cell Cycle Proteins↗

PubMed2000

Electron microscopic observation and single-stranded DNA binding activity of the Mcm4,6,7 complex.

Mcm2-7 proteins that play an essential role in eukaryotic DNA replication contain DNA-dependent ATPase motifs in a central domain that, from yeast to mammals, is highly conserved. Our group has reported that a DNA helicase activity is associated with a 600 kDa human Mcm4, 6 and 7 complex. The structure of the Mcm4,6,7 complex was visualized by electron microscopy after negative staining with uranyl acetate. The complex contained toroidal forms with a central channel and also contained structures with a slit. Gel-shift analysis indicated that the level of affinity of the Mcm4,6,7 complex for single-stranded DNA was comparable to that of SV40 T antigen, although the Mcm4,6,7 complex required longer single-stranded DNA for the binding than did SV40 T antigen. The nucleoprotein complexes of Mcm4,6,7 and single-stranded DNA were visualized as beads in a queue or beads on string-like structures. The formation of these nucleoprotein complexes was erased by Mcm2 that is a potential inhibitor of the Mcm4,6,7 helicase. We also found that the DNA helicase activity of Mcm4,6,7 complex was inhibited by the binding of Mcm3,5 complex. These results support the notion that the Mcm4,6,7 complex functions as a DNA helicase and the formation of 600 kDa complex is essential for the activity.

Animals↗

PubMed2000

Rad24 is essential for proliferation of diploid cells in fission yeast.

The rad24(+) gene of Schizosaccharomyces pombe encodes a ubiquitously expressed 14-3-3 protein. We report here that Deltarad24 cells displayed a defect in diploid colony formation, although they conjugated efficiently. We found that a cumulative deletion of mei2(+) gene almost completely suppressed this defect, and demonstrated using two-hybrid analysis that Rad24 protein directly associates with Mei2 protein by recognizing Ser-438 which is a phosphorylation target of Pat1 kinase. We conclude that constitutive progression to meiosis, caused by lack of Mei2 inhibition due to the absence of Rad24 protein, is the primary cause of the proliferative deficiency observed in Deltarad24 cells.

Cell Cycle Proteins↗

PubMed2000

A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation.

Both F10 and BL6 sublines of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells are metastatic after subcutaneous injection. Retrotransposon insertion was found to produce an N-terminally truncated form (Deltagamma1) of the B56gamma1 regulatory subunit isoform of protein phosphatase (PP) 2A in BL6 cells, but not in F10 cells. We found an interaction of paxillin with PP2A C and B56gamma subunits by co-immunoprecipitation. B56gamma1 co-localized with paxillin at focal adhesions, suggesting a role for this isoform in targeting PP2A to paxillin. In this regard, Deltagamma1 behaved similarly to B56gamma1. However, the Deltagamma1-containing PP2A heterotrimer was insufficient for the dephosphorylation of paxillin. Transfection with Deltagamma1 enhanced paxillin phosphorylation on serine residues and recruitment into focal adhesions, and cell spreading with an actin network. In addition, Deltagamma1 rendered F10 cells as highly metastatic as BL6 cells. These results suggest that mutations in PP2A regulatory subunits may cause malignant progression.

3T3 Cells↗

PubMed2000

Structure, expression, and chromosome mapping of LATS2, a mammalian homologue of the Drosophila tumor suppressor gene lats/warts.

We have cloned and characterized LATS2, a novel mammalian homologue of the Drosophila tumor suppressor gene lats/warts. Northern blot analysis showed ubiquitous expression of mouse LATS2 (MmLATS2) mRNA, whereas expression of human LATS2 (HsLATS2) mRNA was enhanced in skeletal muscle and heart. Immunoblotting analysis of fractionated cell lysates showed HsLats2 to be a nuclear protein. We mapped the MmLATS2 gene to mouse chromosome 14 by interspecific backcross analysis. We also mapped the HsLATS2 gene (by fluorescence in situ hybridization) to the 13q11-q12 region, in which a loss of heterozygosity has been frequently observed in many primary cancers and to which the tumor suppressor genes RB and BRCA2 have also been mapped.

Amino Acid Sequence↗

PubMed2000

Role of cyclin G-associated kinase in uncoating clathrin-coated vesicles from non-neuronal cells.

Auxilin is a brain-specific DnaJ homolog that is required for Hsc70 to dissociate clathrin from bovine brain clathrin-coated vesicles. However, Hsc70 is also involved in uncoating clathrin-coated vesicles formed at the plasma membrane of non-neuronal cells suggesting that an auxilin homolog may be required for uncoating in these cells. One candidate is cyclin G-associated kinase (GAK), a 150-kDa protein expressed ubiquitously in various tissues. GAK has a C-terminal domain with high sequence similarity to auxilin; like auxilin this C-terminal domain consists of three subdomains, an N-terminal tensin-like domain, a clathrin-binding domain, and a C-terminal J-domain. Western blot analysis shows that GAK is present in rat liver, bovine testes, and bovine brain clathrin-coated vesicles. More importantly, liver clathrin-coated vesicles, which contain GAK but not auxilin, are uncoated by Hsc70, suggesting that GAK acts as an auxilin homolog in non-neuronal cells. In support of this view, the clathrin-binding domain of GAK alone induces clathrin polymerization into baskets and the combined clathrin-binding domain and J-domain of GAK supports uncoating of AP180-clathrin baskets by Hsc70 at pH 7 and induces Hsc70 binding to clathrin baskets at pH 6. Immunolocalization studies suggest that GAK is a cytosolic protein that is concentrated in the perinuclear region; it appears to be highly associated with the trans-Golgi where the budding of clathrin-coated vesicles occurs. We propose that GAK is a required cofactor for the uncoating of clathrin-coated vesicles by Hsc70 in non-neuronal cells.

Adaptor Proteins, Vesicular Transport↗