Biomedical subjects

N Sato

Publications and source records attributed to N Sato.

At least 361 records · Page 20Linked to original sources

PubMed1999

CDC7 kinase complex as a molecular switch for DNA replication.

Cdc7 kinase and its activator Dbf4 protein, originally identified in budding yeast Saccharomyces cerevisiae, are widely conserved in eukaryotes including fission yeast and human. Dbf4-related activators bind and stimulate kinase activity of Cdc7-like catalytic subunit. Its kinase activity is cell cycle-regulated, mainly through availability of the activation subunit whose level increases at G1/S boundary and is maintained at a high level throughout S phase. MCM2 protein is among physiologically important substrates. Genetic studies in fission yeast indicate that Cdc7-related kinase complex also functions in meiosis, uninduced mutagenesis, DNA replication checkpoint signaling and maintenance of chromatin structures during S phase.

Animals↗

PubMed1999

Enhancement of fcgamma receptor-mediated phagocytosis by transforming mutants of Cbl.

Phagocytosis mediated by FcgammaR plays an important role in host defense. The molecular events involved in this process have not been completely defined. The adapter protein Cbl has been implicated in FcgammaR signaling, but the function of Cbl in phagocytosis is unknown. Here we show that overexpression of the transforming mutants of Cbl, Cbl-70Z, and v-Cbl, but not wild-type (wt) Cbl, enhance phagocytosis mediated by FcgammaR in COS cells. Cbl-70Z, but not Cbl-wt, also enhanced FcgammaR-mediated phagocytosis in P388D1 murine macrophage cells. Cbl-70Z did not affect tyrosine phosphorylation or in vitro kinase activity of Syk, indicating that Syk may not be the direct target of Cbl-70Z in the enhancement of phagocytosis. A point mutation (G306E) in the phosphotyrosine domain of Cbl-70Z, as well as a C-terminal 67-aa deletion, partially abolished the enhancing effect on FcgammaR-mediated phagocytosis. A double mutant of Cbl-70Z containing both the G306E mutation and the C-terminal deletion completely lacked the ability to enhance phagocytosis. Thus, both the phosphotyrosine binding domain and the carboxyl-terminal tail were required for optimal enhancement of phagocytosis by Cbl-70Z. Functional phosphatidylinositol 3-kinase was required for Cbl-70Z to enhance phagocytosis, since wortmannin, a phosphatidylinositol 3-kinase inhibitor, inhibited FcgammaR-mediated phagocytosis in the presence of Cbl-70Z. These studies demonstrate that mutants of Cbl can modulate the phagocytic pathway mediated by FcgammaR and imply a functional involvement of c-Cbl in Fcgamma receptor-mediated phagocytosis.

Androstadienes↗

PubMed1999

Different time course between scene processing and face processing: a MEG study.

Using magnetoencephalography (MEG), the neural response to scenes was recorded and compared with that to faces. The prominent MEG signals in response to scenes appeared 200-300 ms after the stimulus presentation while those in response to faces appeared between 150 and 200 ms. Source locations of the signals were estimated in the right parahippocampal and parieto-occipital regions with a latency of 300 ms for the scene response, whereas those were estimated in the lingual or fusiform gyri bilaterally with a latency of 160 ms for the face response. These data suggest that both the temporal and parietal regions process scenes, while the occipito-temporal regions process faces, and that scene processing takes a longer time than face processing.

Adult↗

PubMed1999

Defects in the generation of IFN-gamma are overcome to control infection with Leishmania donovani in CC chemokine receptor (CCR) 5-, macrophage inflammatory protein-1 alpha-, or CCR2-deficient mice.

We investigated the immune responses in mice lacking CCR2, CCR5, or macrophage inflammatory protein-1 alpha (MIP-1 alpha), a ligand for CCR5, in two situations: following T cell stimulation or after challenge with Leishmania donovani, an intracellular microbe whose control is dependent on a Th1 immune response. Mice deficient in CCR5, MIP-1 alpha, or CCR2 had reduced IFN-gamma responses following ligation of the TCR. Reduced IFN-gamma responses following PMA and ionomycin were also observed in CD8+ T cells of CCR5-/- and CCR2-/- mice. During the early phases of infection, all three knockout mice had low Ag-specific IFN-gamma responses. However, this reduced IFN-gamma response was overcome during a state of persistent Ag stimulation (chronic infection), and was not associated with an adverse parasitologic outcome in any of the gene-targeted mouse strains. To the contrary, during the late phase of infection, an exaggerated Ag-specific IFN-gamma response was evident in CCR5-/- and MIP-1 alpha-/- mice, and this correlated with an enhanced control of parasite replication. Although granuloma formation was abnormal in each of the knockout mice, there was no correlation between the number or architecture of the granulomas and parasite burden. Collectively, these findings indicate an important role for CCR5, MIP-1 alpha, and CCR2 in granulomatous inflammation, and that CCR5 and MIP-1 alpha, possibly acting through CCR5, might play a deleterious role in the outcome of chronic L. donovani infection. Our data also suggest that there might be cross-talk between TCR and chemokine receptor signaling pathways.

Animals↗

PubMed1999

Synthesis and pharmacological evaluation of carboxamide derivatives as selective serotoninergic 5-HT(4) receptor agonists.

A number of new carboxamide derivatives were synthesized. The affinity of these compounds for the serotoninergic 5-HT(4) receptor was evaluated by use of radioligand-binding techniques. The agonistic activity was evaluated as the contractile effect of the ascending colon isolated from guinea-pigs. Among these compounds, 4-amino-5-chloro-2-methoxy-N-[1-[2-[(methylsulfonyl)amino]ethly]-4-piperidinylmethyl]benzamide (24) showed a high affinity for the 5-HT(4) receptor (Ki = 9.6 nM). Compound 24 displayed a higher affinity for 5-HT(4) receptors than the other receptors, including, 5-HT(3) and dopamine D(2) receptors. In addition, compound 24 was confirmed to be a potent 5-HT(4) receptor agonist (ED(50) = 7.0 nM). An interaction model between compound 24 and 5-HT(4) receptor was proposed.

Journal Article↗

PubMed1999

Adenoviral GM-CSF Gene Transduction into Breast Cancer Cells Induced Long-Lasting Antitumor Immunity in Mice.

BACKGROUND: Immunogene therapy is regarded as a novel treatment that overcomes the limitation of preexisting adjuvant therapies and demonstrates the potential for the total elimination of cancer cells by affecting concealed tumor cells. The aim of this study was to examine the enhancement of antitumor immunity of irradiated granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-transduced mouse breast cancer cells. METHODS: To study prophylactic vaccine effects, Balb/c mice were vaccinated subcutaneously with saline or irradiated mouse breast cancer cells (BalbMC, 1 x10&sup6; /mouse) infected with or without recombinant adenovirus harboring the GM-CSF gene (Adv/GM-CSF) on day-7. Mice were challenged with parental cells (1x10&sup5;/mouse) on day 0 in the flank opposite the vaccination site. RESULTS: BalbMC cells secreted GM-CSF after infection with Adv/GM-CSF. Vaccination with irradiated GM-CSF-secreting BalbMC completely protected syngeneic mouse challenged with live parental cells. On the other hand, vaccination with irradiated BalbMC protected 60% of mice from tumor development after challenge with parental cells. None of the tumor-free mice initially vaccinated with irradiated GM-CSF-producing BalbMC cells developed tumor upon repeated challenge with parental cells during the entire observation period. CONCLUSION: Our study demonstrated the feasibility of this immunotherapeutic approach as a novel adjuvant therapy after surgery for breast cancer.

Journal Article↗

PubMed1999

Race-specific HIV-1 disease-modifying effects associated with CCR5 haplotypes.

Genetic variation in CC chemokine receptor 5 (CCR5), the major HIV-1 coreceptor, has been shown to influence HIV-1 transmission and disease progression. However, it is generally assumed that the same CCR5 genotype (or haplotype) has similar phenotypic effects in different populations. To test this assumption, we used an evolutionary-based classification of CCR5 haplotypes to determine their associated HIV-1 disease-modifying effects in a large well-characterized racially mixed cohort of HIV-1-seropositive individuals. We demonstrate that the spectrum of CCR5 haplotypes associated with disease acceleration or retardation differs between African Americans and Caucasians. Also, we show that there is a strong interactive effect between CCR5 haplotypes with different evolutionary histories. The striking population-specific phenotypic effects associated with CCR5 haplotypes emphasize the importance of understanding the evolutionary context in which disease susceptibility genes are expressed.

Acquired Immunodeficiency Syndrome↗

PubMed1999

Dendritic cell (DC)-based anti-infective strategies: DCs engineered to secrete IL-12 are a potent vaccine in a murine model of an intracellular infection.

Infections with intracellular pathogens such as Leishmania donovani and Mycobacterium tuberculosis pose serious health problems worldwide. Effective vaccines for these pathogens are not available. Furthermore, despite optimal therapy, disease progression is often seen with several intracellular infections. For these reasons, we initiated studies to develop novel anti-infective vaccine and treatment strategies that couple the potent Ag-presenting capacity of dendritic cells (DC) with paracrine delivery of potent anti-infective cytokines such as IL-12 to local immune response sites. We tested this strategy in a murine model of visceral leishmaniasis. Adoptive transfer of DCs pulsed ex vivo with soluble L. donovani Ags (SLDA) to naive mice induced the Ag-specific production of IFN-gamma, and increased the percentage of activation markers on spleen lymphocytes. SLDA-pulsed DCs engineered by retroviral gene transfer techniques to secrete high levels of biologically active murine IL-12 augmented this immune response further. In several different vaccination and immunotherapy protocols, compared with sham-treated mice, animals receiving SLDA-pulsed DCs either before or following infection had 1-3 log lower parasite burdens, and this protection was associated with a pronounced enhancement in the parasite-specific IFN-gamma response. The augmentation of this protection by IL-12-engineered DCs was striking. First, live parasites were not detected in the liver of mice vaccinated with IL-12-transduced, SLDA-pulsed DCs. Second, this parasitological response was associated with a nearly normal liver histology. In contrast, parasites and granulomas were found in mice vaccinated with SLDA-pulsed, nontransduced DCs. Collectively, these studies provide the rationale for the development of potent DC-based immunotherapies.

3T3 Cells↗

PubMed1999

Identification of natural antigenic peptides of a human gastric signet ring cell carcinoma recognized by HLA-A31-restricted cytotoxic T lymphocytes.

Peptides of human melanomas recognized by CD8+ CTLs have been identified, but the nature of those of nonmelanoma tumors remains to be elucidated. Previously, we established a gastric signet ring cell carcinoma HST-2 and HLA-A31 (A*31012)-restricted autologous CTL clone, TcHST-2. In the present study, we determined the natural antigenic peptides of HST-2 cells. The purified preparation of acid-extracted Ags was submitted to the peptide sequencer, and one peptide, designated F4.2 (Tyr-Ser-Trp-Met-Asp-Ile-Ser-Cys-Trp-Ile), appeared to be immunogenic. To confirm the antigenicity of F4.2 further, we constructed an expression minigene vector (pF4.2ss) coding adenovirus E3, a 19-kDa protein signal sequence plus F4.2. An introduction of pF4.2ss minigene to HST-2 and HLA-A31(+) allogeneic tumor cells clearly enhanced and induced the TcHST-2 reactivity, respectively. Furthermore, when synthetic peptides of F4.2 C-terminal-deleted peptides were pulsed to HST-2 cells, F4.2-9 (nonamers), but not F4.2-8 or F4.2-7 (octamer or heptamer, respectively), enhanced the reactivity of TcHST-2, suggesting that the N-terminal ninth Trp might be a T cell epitope. This was confirmed by lack of antigenicity when using synthetic substituted peptides as well as minigenes coding F4.2 variant peptides with Ala or Arg at the ninth position of F4.2. Meanwhile, it was indicated that the sixth position Ile was critically important for the binding to HLA-A31 molecules. Thus, our data indicate that F4.2 may work as an HLA-A31-restricted natural antigenic peptide recognized by CTLs.

Amino Acid Sequence↗

PubMed1999

Unsaturated glucuronyl hydrolase of Bacillus sp. GL1: novel enzyme prerequisite for metabolism of unsaturated oligosaccharides produced by polysaccharide lyases.

The bacterium Bacillus sp. GL1 assimilates two kinds of heteropolysaccharides, gellan and xanthan, by using extracellular gellan and xanthan lyases, respectively, and produces unsaturated saccharides as the first degradation products. A novel unsaturated glucuronyl hydrolase (glycuronidase), which was induced in the bacterial cells grown on either gellan or xanthan, was found to act on the tetrasaccharide of unsaturated glucuronyl-glucosyl-rhamnosyl-glucose produced from gellan by gellan lyase, and the enzyme and its gene were isolated from gellan-grown cells. The nucleotide sequence showed that the gene contained an ORF consisting of 1131 base pairs coding a polypeptide with a molecular weight of 42,859. The purified enzyme was a monomer with a molecular mass of 42 kDa and was most active at pH 6.0 and 45 degrees C. Because the enzyme can act not only on the gellan-degrading product by gellan lyase, but also on unsaturated chondroitin and hyaluronate disaccharides produced by chondroitin and hyaluronate lyases, respectively, it is considered that the unsaturated glucuronyl hydrolase plays specific and ubiquitous roles in the degradation of oligosaccharides with unsaturated uronic acid at the nonreducing terminal produced by polysaccharide lyases.

Amino Acid Sequence↗

PubMed1999

Quantitative determination of apoptotic death in cultured human pancreatic cancer cells by propidium iodide and digitonin.

We present here the efficacy of an in vitro cytotoxicity assay which can measure rapidly both apoptotic dead cells and cell growth rate, quantitatively. Using a multi-well plate reader, the fluorescence intensity of propidium iodide (PI) corresponding to dead cells and to total cells after digitonin treatment were measured in cultured human pancreatic cancer cells following exposure to etoposide. The percentage of dead cells measured by this assay was well correlated to that determined by Trypan blue staining. Furthermore, the cell growth rate determined simultaneously was also correlated to the cell number counted directly using a microscope. We demonstrate that this method, which was originally established for evaluating necrosis, could be applied to measure apoptotic cell death. Taken together, this simple assay is useful for testing the efficacy of anti-cancer agents and for investigating the molecular mechanisms of apoptosis in the cultured cells.

Antineoplastic Agents, Phytogenic↗

PubMed1999

Characterization of alpha-L-rhamnosidase of Bacillus sp. GL1 responsible for the complete depolymerization of gellan.

A bacterium, Bacillus sp. GL1, depolymerizes a heteropolysaccharide (gellan) to a tetrasaccharide (unsaturated glucuronyl-glucosyl-rhamnosyl-glucose) by extracellular gellan lyase. The resultant tetrasaccharide was degraded to the constituent monosaccharides by subsequent reactions of unsaturated glucuronyl hydrolase, beta-d-glucosidase, and alpha-l-rhamnosidase. alpha-l-Rhamnosidase was substantially induced in the bacterial cells when grown in a medium containing gellan as a carbon source. The purified enzyme from the cells was a monomer with a molecular mass of about 100 kDa and was most active at pH 7.0 and 50 degrees C. The enzyme acted on the gellan-degrading product (rhamnosyl-glucose) formed after successive reactions catalyzed by gellan lyase, unsaturated-glucuronyl hydrolase and beta-d-glucosidase, and released rhamnose from the disaccharide. Therefore, the alpha-l-rhamnosidase is found to be responsible as the final enzyme for the complete depolymerization of gellan.

Bacillus↗

PubMed1999

Conservation of structure and cold-regulation of RNA-binding proteins in cyanobacteria: probable convergent evolution with eukaryotic glycine-rich RNA-binding proteins.

The rbp gene family of the cyanobacterium Anabaena variabilis strain M3 consists of eight members that encode small RNA-binding proteins containing a single RNA recognition motif (RRM). Similar genes are found in the genomes of Synechocystis sp. PCC6803, Helicobacter pylori and Treponema pallidum, but are absent from the other completely sequenced prokaryotic genomes. The expression of the rbp genes of Anabaena is induced by low temperature, with the exception of the rbpD gene. We found four stretches of conserved sequences in the 5'-untranslated region of the cyanobacterial rbp genes that are known to be induced by low temperature. The cold-regulated Rbp proteins contain a short C-terminal glycine-rich domain. In this respect, these proteins are similar to plant and mammalian glycine-rich RNA-binding proteins (GRPs), which also contain a single RRM domain with a C-terminal glycine-rich domain and are highly expressed at low temperature. Detailed phylogenetic analysis showed, however, that the cyanobacterial Rbp proteins and the eukaryotic GRPs do not belong to a single lineage, but that the glycine-rich domains are likely to have been added independently. The cold-regulation of both types of proteins is also likely to have evolved independently. Furthermore, the chloroplast RNA-binding proteins are not likely to have originated from the Rbp proteins of endosymbiont cyanobacterium, but are supposed to have diverged from the GRPs. These results suggest that the cyanobacterial Rbp proteins and the eukaryotic GRPs are similar in both structure and regulation, but that this apparent similarity has resulted from convergent evolution.

Amino Acid Sequence↗

PubMed1999

Reciprocal gene expression of osteoclastogenesis inhibitory factor and osteoclast differentiation factor regulates osteoclast formation.

Osteoblasts/stromal cells support the formation of osteoclast-like cells (OCL) from osteoclast progenitor cells via expressing a membrane-associated protein, osteoclast differentiation factor (ODF), in the presence of osteotropic factors, whereas the cells secrete a substantial amount of osteoclastogenesis inhibitory factor (OCIF) in the unstimulated state. There are both OCL formation-supporting and the nonsupporting cell lines in osteoblasts/stromal cell lineages. The mechanism that divides osteoblasts/stromal cell lines into the two types is not known. The present study reports that OCL formation-supporting cell line ST2 showed a greatly increased level of ODF mRNA, whereas their OCIF mRNA was drastically diminished in the presence of 1alpha, 25(OH)2-dihydroxyvitamin D3 or prostaglandin E2. In contrast, MC3T3-E1 cells lacking OCL formation-supporting ability did not show a decrease in OCIF mRNA in response to the factors, despite a similar increase in ODF mRNA as ST2 cells. However, inactivated MC3T3-E1 cells secreting nothing supported OCL formation in coculture with human promyelocytic cells, HL60. On the contrary, ST2 cells did not support OCL formation from HL60 cells when cocultured in medium conditioned by 1alpha, 25(OH)2 vitamin D3-treated MC3T3-E1. These findings indicate that reciprocal gene expression of ODF and OCIF in osteoblasts/stromal cells is essential for supporting OCL formation.

Animals↗

PubMed1999

Activated stellate (Ito) cells possess voltage-activated calcium current.

We previously reported stellate (Ito) cells possess voltage-activated Ca2+ current. The activation of stellate cells has been indicated to contribute to liver fibrosis and the regulation of hepatic hemodynamics. The aim of this study was to investigate the relationship between voltage-activated Ca2+ current and activation of stellate cells. Voltage-activated Ca2+ current in stellate cells isolated from rats were studied using whole-cell patch clamp technique. L-type voltage-activated Ca2+ current was hardly detected in stellate cells cultured for less than 9 days. Ca2+ current was detected 12.5 and 69% of cells at the 10th and 14th day of culture, respectively. BrdU incorporation indicated cell proliferation was recognized over 50% of cells at the 3rd and 5th day of culture, respectively, then decreased significantly in a time-dependent manner. On the other hand, the expression of alpha-smooth muscle actin indicated cell activation increased from 7th day of culture and collagen type I mRNA appeared remarkably in cells cultured for more than 10 days. In this study, we concluded L-type voltage-activated Ca2+ current was recognized in activated stellate (myofibroblast-like) cells.

Actins↗

PubMed1999

Interaction of the sarcin/ricin domain of 23 S ribosomal RNA with proteins L3 and L6.

We investigated interaction of an RNA domain covering the target site of alpha-sarcin and ricin (sarcin/ricin domain) of Escherichia coli 23 S rRNA with ribosomal proteins. RNA fragments comprising residues 2630-2788 (Tox-1) and residues 2640-2774 (Tox-2) of 23 S rRNA were transcribed in vitro and used to analyze the binding proteins by gel shift and filter binding. Protein L6 bound to both Tox-1 (Kd: 0.31 microM) and Tox-2 (Kd: 0.18 microM), and L3 bound only to Tox-1 (Kd: 0.069 microM) in a solution containing 10 mM MgCl2 and 175 mM KCl at 0 degreesC. Footprinting studies were performed using the chemical probe dimethyl sulfate on full-length 23 S rRNA. Binding of L6 protected a single base, A-2757, and strongly enhanced reactivity of C-2752. A direct role of A-2757 in the L6 binding was verified by site-directed mutagenesis; replacements of A-2757 with G and C impaired the L6 binding. On the other hand, binding of L3 protected A-2632, A-2634, A-2635, A-2675, A-2726, A-2733, A-2749, and A-2750. Interestingly, binding of L6 and L3 together protected additional bases A-2657, A-2662, C-2666, and C-2667 in the sarcin/ricin loop, in addition to A-2740, A-2741, A-2748, A-2753, A-2764, A-2765, and A-2766 in the other stem-loop. This appears to be due to cooperative interaction of L3 and L6 with the RNA. The results are discussed with respect to conformational modulation of the sarcin/ricin domain by the protein binding.

Base Sequence↗

PubMed1999

"Neuro-sweet disease": benign recurrent encephalitis with neutrophilic dermatosis.

OBJECTIVE: To describe benign recurrent encephalitis in a case of Sweet syndrome that also showed clinical features of Behçet disease. CASE REPORT: A 37-year-old Japanese man developed relapsing and remitting encephalitis and mucocutaneous symptoms mimicking Behçet disease. Magnetic resonance images showed at least 5 episodes of transient abnormal signal intensity in various cerebral regions over a period of 5 years. A skin biopsy specimen of the cutaneous edematous erythematous plaques revealed neutrophilic dermatitis compatible with Sweet syndrome. HLA typing showed B54, which is frequent in Sweet syndrome but rare in Behçet disease. Oral prednisolone therapy (10-60 mg/d) was remarkably effective for the encephalitis as well as for the mucocutaneous symptoms. CONCLUSION: We propose that there is an entity that is like Sweet disease, but with recurrent encephalitis characterized by an association with HLA-B54 and a high responsiveness to corticosteroid therapy, which we have tentatively named neuro-Sweet disease, that is distinct from the classic central nervous system involvement of Behçet disease.

Adult↗

PubMed1999

Differential expression of C-protein isoforms in developing and degenerating mouse striated muscles.

With the aim of clarifying the roles of C-protein isoforms in developing mammalian skeletal muscle, we cloned the complementary DNA (cDNAs) encoding mouse fast (F) and slow (S) skeletal muscle C-proteins and determined their entire sequences. Northern blotting with these cDNAs together with mouse cardiac (C) C-protein cDNA was performed. It revealed that in adult mice, C, F, and S isoforms are expressed in a tissue-specific fashion, although the messages for both F and S isoforms are transcribed in extensor digitorum longus muscle, which has been categorized as a fast muscle. In addition, although C isoform is expressed first and transiently during development of chicken skeletal muscles, C isoform is not expressed in mouse skeletal muscles at all through the developmental stages; S isoform is first expressed, followed by the appearance of F isoform. Finally, in dystrophic mouse skeletal muscles, the expression of S isoform is increased as it is in dystrophic chicken muscle. These observations suggest that mutations in C isoform (MyBP-C) do not lead to any disturbance in skeletal muscle, although they may lead to familial hypertrophic cardiomyopathy. We also suggest that the expression of S isoform may be stimulated in degenerating human dystrophic muscles.

Amino Acid Sequence↗