Biomedical subjects

S Nishikawa

Publications and source records attributed to S Nishikawa.

At least 145 records · Page 8Linked to original sources

PubMed1998

Mechanism of endothelin-1 release from endothelial cells in pregnancy-induced hypertension.

We investigated the mechanism of vasoconstrictor-induced endothelin-1 (ET-1) release from human umbilical vein endothelial cells (HUVECs) in serum from women with pregnancy-induced hypertension (PIH). We obtained serum samples from seven women with PIH, seven healthy nonpregnant women (NP), and seven normal pregnant women (NPIH). ET-1 and inositol 1,4,5-trisphosphate (IP3) were assayed by ET-1 ELISA and an IP3 3H assay system, respectively. ET-1 release from HUVECs incubated with 10% serum (NP, NPIH, and PIH) was greater than that without human serum. Angiotensin II (Ang II)- and epinephrine (Epi)-induced ET-1 release were significantly increased by PIH serum. IP3 production in HUVECs incubated with 10% serum (NP, NPIH, and PIH) was greater than that without human serum. Ang II- or Epi-induced IP3 production in HUVECs incubated with PIH serum was increased but not significantly compared to that with other sera. Our results suggest that increased ET-1 release from HUVECs incubated with human serum may be mediated by IP3 production, but that Ang II- or Epi-induced ET-1 release from HUVECs incubated with PIH serum may be mediated by another mechanism.

Endothelin-1↗

PubMed1998

Systemic dissemination by intrarectal infection with Listeria monocytogenes in mice.

Orally ingested Listeria monocytogenes is known to penetrate into Peyer's patches (PP) and translocate to the spleen and liver. Herein, extraintestinal dissemination of the bacterium independent of PP was investigated. Dissemination of Listeriae to the spleen and liver was observed in intrarectally infected mice as well as in intragastrically infected animals in spite that no Listeriae were detected in the small intestines of mice infected intrarectally. Decreased numbers of intestinal intraepithelial lymphocytes (iIEL) and increased numbers of lymphocytes in the contents of the small and large intestines were observed after intragastric infection and in the large intestine after intrarectal infection, giving the assumption that the leakage of iIEL caused by injury of epithelial layers in intestines might occur during infection. These results suggest that L. monocytogenes might be able to disseminate through small and large intestines in part by a PP-independent mechanism.

Animals↗

PubMed1998

Neuropeptides in the livers of mice during bacterial infections.

Neuropeptides such as substance P (SP) and vasoactive intestinal peptide (VIP) are known to act as immunomodulators. We investigated the induction of SP and VIP in the livers of mice infected with Listeria monocytogenes or injected with Tsukamurella paurometabolum. VIP was detected in the livers of mice after L. monocytogenes infection by an immunohistochemical technique and preproVIP mRNA, which was detected by reverse transcription-polymerase chain reaction (PCR), was induced post infection. However, no SP was detected. In contrast, SP, but not VIP was detected within granulomas in the livers of T. paurometabolum-injected mice, suggesting VIP and SP might be selectively induced in the liver by different bacterial infections.

Actinomycetales↗

PubMed1998

Cloning and sequencing of the Sphingomonas (Pseudomonas) paucimobilis gene essential for the O demethylation of vanillate and syringate.

Sphingomonas (Pseudomonas) paucimobilis SYK-6 is able to grow on 5,5'-dehydrodivanillic acid (DDVA), syringate, vanillate, and other dimeric model compounds of lignin as a sole carbon source. Nitrosoguanidine mutagenesis of S. paucimobilis SYK-6 was performed, and two mutants with altered DDVA degradation pathways were isolated. The mutant strain NT-1 could not degrade DDVA, but could degrade syringate, vanillate, and 2,2',3'-trihydroxy-3-methoxy-5,5'-dicarboxybiphenyl (OH-DDVA). Strain DC-49 could slowly assimilate DDVA, but could degrade neither vanillate nor syringate, although it could degrade protocatechuate and 3-O-methylgallate. A complementing DNA fragment of strain DC-49 was isolated from the cosmid library of strain SYK-6. The minimum DNA fragment complementing DC-49 was determined to be the 1.8-kbp insert of pKEX2.0. Sequencing analysis showed an open reading frame of 1,671 bp in this fragment, and a similarity search indicated that the deduced amino acid sequence of this open reading frame had significant similarity (60%) to the formyltetrahydrofolate synthetase of Clostridium thermoaceticum.

Amino Acid Sequence↗

PubMed1998

Cloning of a Sphingomonas paucimobilis SYK-6 gene encoding a novel oxygenase that cleaves lignin-related biphenyl and characterization of the enzyme.

Sphingomonas paucimobilis SYK-6 transforms 2,2'-dihydroxy-3,3'-dimethoxy-5,5'-dicarboxybiphenyl (DDVA), a lignin-related biphenyl compound, to 5-carboxyvanillic acid via 2,2',3-trihydroxy-3'-methoxy-5,5'-dicarboxybiphenyl (OH-DDVA) as an intermediate (15). The ring fission of OH-DDVA is an essential step in the DDVA degradative pathway. A 15-kb EcoRI fragment isolated from the cosmid library complemented the growth deficiency of a mutant on OH-DDVA. Subcloning and deletion analysis showed that a 1.4-kb DNA fragment included the gene responsible for the ring fission of OH-DDVA. An open reading frame encoding 334 amino acids was identified and designated ligZ. The deduced amino acid sequence of LigZ had 18 to 21% identity with the class III extradiol dioxygenase family, including the beta subunit (LigB) of protocatechuate 4,5-dioxygenase of SYK-6 (Y. Noda, S. Nishikawa, K.-I. Shiozuka, H. Kadokura, H. Nakajima, K. Yano, Y. Katayama, N. Morohoshi, T. Haraguchi, and M. Yamasaki, J. Bacteriol. 172:2704-2709, 1990), catechol 2,3-dioxygenase I (MpcI) of Alcaligenes eutrophus JMP222 (M. Kabisch and P. Fortnagel, Nucleic Acids Res. 18:3405-3406, 1990), the catalytic subunit of the meta-cleavage enzyme (CarBb) for 2'-aminobiphenyl-2,3-diol from Pseudomonas sp. strain CA10 (S. I. Sato, N. Ouchiyama, T. Kimura, H. Nojiri, H. Yamane, and T. Omori, J. Bacteriol. 179:4841-4849, 1997), and 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) of Escherichia coli (E. L. Spence, M. Kawamukai, J. Sanvoisin, H. Braven, and T. D. H. Bugg, J. Bacteriol. 178:5249-5256, 1996). The ring fission product formed from OH-DDVA by LigZ developed a yellow color with an absorption maximum at 455 nm, suggesting meta cleavage. Thus, LigZ was concluded to be a ring cleavage extradiol dioxygenase. LigZ activity was detected only for OH-DDVA and 2,2',3,3'-tetrahydroxy-5,5'-dicarboxybiphenyl and was dependent on the ferrous ion.

Amino Acid Sequence↗

PubMed1998

Protective role of nitric oxide in Staphylococcus aureus infection in mice.

This study was carried out to determine the role of nitric oxide (NO) in Staphylococcus aureus infection in mice. NO production in spleen cell cultures was induced by heat-killed S. aureus. Expression of mRNA of the inducible isoform of NO synthase (iNOS) was induced in the spleens and kidneys of S. aureus-infected mice. When mice were treated with monoclonal antibodies (MAbs) against tumor necrosis factor alpha (TNF-alpha) or gamma interferon (IFN-gamma) before S. aureus infection, the induction of iNOS mRNA expression in the kidneys was inhibited. These MAbs also inhibited NO production in spleen cell cultures stimulated with heat-killed S. aureus. NO production in the spleen cell cultures and levels of urinary nitrate plus nitrite were suppressed by treatment with aminoguanidine (AG), a selective inhibitor of iNOS. The survival rates of AG-treated mice were significantly decreased by either lethal or sublethal S. aureus infections. However, an effect of AG administration on bacterial growth was not observed in the spleens and kidneys of mice during either type of infection. Production of TNF-alpha and IFN-gamma was not affected by AG treatment in vitro and in vivo. These results suggest that NO plays an important role in protection from lethality by the infection, but the protective role of NO in host resistance against S. aureus infection was not proved. Moreover, our results show that TNF-alpha and IFN-gamma regulate NO production while NO may not be involved in the regulation of the production of these cytokines during S. aureus infection.

Animals↗

PubMed1998

Transgene expression of steel factor in the basal layer of epidermis promotes survival, proliferation, differentiation and migration of melanocyte precursors.

Mutations at the murine dominant white spotting (KitW) and steel (MgfSl) loci, encoding c-Kit receptor kinase and its ligand respectively, exert developmental defects on hematopoietic cells, melanocytes, germ cells and interstitial cells of Cajal. The expression patterns of steel factor (SLF) observed in the skin and gonads suggest that SLF mediates a migratory or a chemotactic signal for c-Kit-expressing stem cells (melanocyte precursors and primordial germ cells). By targeting expression of SLF to epidermal keratinocytes in mice, we observed extended distribution of melanocytes in a number of sites including oral epithelium and footpads where neither melanocytes nor their precursors are normally detected. In addition, enlarged pigmented spots of KitW and other spotting mutant mice were observed in the presence of the SLF transgene. These results provide direct evidence that SLF stimulates migration of melanocytes in vivo. We also present data suggesting that SLF does not simply support survival and proliferation of melanocytes but also promotes differentiation of these cells. Unexpectedly, melanocyte stem cells independent of the c-Kit signal were maintained in the skin of the SLF transgenic mice. After the elimination of c-Kit-dependent melanoblasts by function-blocking anti-c-Kit antibody, these stem cells continued to proliferate and differentiate into mature melanocytes. These melanoblasts are able to migrate to cover most of the epidermis after several months. The SLF transgenic mice described in this report will be useful in the study of melanocyte biology.

Animals↗

PubMed1998

Stage-specific expression of the Kit receptor and its ligand (KL) during male gametogenesis in the mouse: a Kit-KL interaction critical for meiosis.

The Kit receptor and its ligand KL, which together constitute an essential effector at various stages of embryonic development, are both present during adult gametogenesis. In the testis, KL is expressed in Sertoli cells, and Kit in germ cells, starting at the premeiotic stages. A series of observations indicated previously a role in spermatogonia survival, without excluding a possible function at later stages. We identified a complex pattern of expression of the two components in the adult murine testis, suggestive of a role in the meiotic progression of spermatocytes. At stages VII-VIII of the cycle of the seminiferous epithelium, the time when spermatocytes enter meiosis, the membrane-associated form of KL extends on the Sertoli cell from the peripheral to the adluminal compartment of the tubule. We also found that the receptor is present on the surface of germ cells up to the pachytene stage. The availability of differentiated Sertoli cell lines, which express the KL protein and support part of the maturation of germ cells in coculture, allowed us to ask whether, in the in vitro reconstructed system, transit of spermatocytes through meiosis requires the Kit-KL interaction. Addition of a blocking monoclonal antibody against the Kit receptor (ACK2) inhibited extensively the appearance of haploid cells and the expression of a haploid-phase-specific gene (Prm1). Recognition of the supporting Sertoli cell by germ cells was not affected, indicating a requirement for the activity of the receptor for either entering or completing meiosis. Involvement of the membrane-associated form of the ligand was suggested by the observation that addition of the soluble form of KL was equally inhibitory.

Animals↗

PubMed1998

Progressive lineage analysis by cell sorting and culture identifies FLK1+VE-cadherin+ cells at a diverging point of endothelial and hemopoietic lineages.

Totipotent murine ES cells have an enormous potential for the study of cell specification. Here we demonstrate that ES cells can differentiate to hemopoietic cells through the proximal lateral mesoderm, merely upon culturing in type IV collagen-coated dishes. Separation of the Flk1+ mesoderm from other cell lineages was critical for hemopoietic cell differentiation, whereas formation of the embryoid body was not. Since the two-dimensionally spreading cells can be monitored easily in real time, this culture system will greatly facilitate the study of the mechanisms involved in the cell specification to mesoderm, endothelial, and hemopoietic cells. In the culture of ES cells, however, lineages and stages of differentiating cells can only be defined by their own characteristics. We showed that a combination of monoclonal antibodies against E-cadherin, Flk1/KDR, PDGF receptor(alpha), VE-cadherin, CD45 and Ter119 was sufficient to define most intermediate stages during differentiation of ES cells to blood cells. Using this culture system and surface markers, we determined the following order for blood cell differentiation: ES cell (E-cadherin+Flk1-PDGFRalpha-), proximal lateral mesoderm (E-cadherin-Flk1+VE-cadherin-), progenitor with hemoangiogenic potential (Flk1+VE-cadherin+CD45-), hemopoietic progenitor (CD45+c-Kit+) and mature blood cells (c-Kit-CD45+ or Ter119+), though direct differentiation of blood cells from the Flk1+VE-cadherin- stage cannot be ruled out. Not only the VE-cadherin+CD45- population generated from ES cells but also those directly sorted from the yolk sac of 9.5 dpc embryos have a potential to give rise to hemopoietic cells. Progenitors with hemoangiogenic potential were identified in both the Flk1+VE-cadherin- and Flk1+VE-cadherin+ populations by the single cell deposition experiment. This line of evidence implicates Flk1+VE-cadherin+ cells as a diverging point of hemopoietic and endothelial cell lineages.

Animals↗

PubMed1998

Flt3/Flk-2 and c-Kit are not essential for the proliferation of B lymphoid progenitor cells in the bone marrow of the adult mouse.

The receptor tyrosine kinase Flk-2/Flt3 was originally cloned from hematopoietic stem cell-enriched fetal liver and placenta and is believed by some investigators to play a role in the regulation of the hematopoietic stem cell. However, targeted disruption of the flt3 gene results in a specific deficiency in early B cell progenitors. Using an antagonistic monoclonal antibody developed against the extracellular domain of Flt3, we investigated the expression and function of the molecule on B lymphoid lineage cells in the bone marrow (BM) of adult mice. Approximately 10% of B220+ cells in the BM expressed Flt3 on the cell surface, and most of the cells belonged to a pro-B cell fraction when judged by an expression pattern of CD43, heat-stable antigen, and BP-1. However, B lymphoid precursor cells that are clonable in vitro could not be enriched in the B220+/Flt3+ cell fraction sorted by flow cytometry. Furthermore, proliferation of B lymphoid precursor cells in the adult BM was not blocked by administration of the antagonistic monoclonal antibodies against Flt3 and c-Kit, suggesting that signalings mediated by Flt3 and c-Kit receptors are not essential for the proliferation of B cell progenitors in adult mouse BM.

Animals↗

PubMed1998

Problem in prolonged levodopa administration of Parkinson disease patients--from the standpoint of the autonomic nervous function.

We studied 50 patients with Parkinson's disease to determine any relation of epidemiologic factors (such as the duration of illness), the duration of levodopa therapy, the total amount of administered levodopa to degree of autonomic dysfunction. Autonomic dysfunction was evaluated using the composite autonomic scoring scale (CASS). The CASS scores were significantly higher among patients with greater administration amount of levodopa regardless of the duration of illness (p < 0.05). There was a definite positive correlation between cardiovascular heart rate index, which was one of the parameters of CASS and reflected parasympathetic neuron system function of cardiac origin, and the duration of levodopa therapy (r = 0.44, p < 0.01) or the total administered amount of levodopa (r = 0.43, p < 0.01). We conclude that long-term levodopa therapy negatively affects autonomic nervous function.

Aged↗

PubMed1997

Mutation of the mouse klotho gene leads to a syndrome resembling ageing.

A new gene, termed klotho, has been identified that is involved in the suppression of several ageing phenotypes. A defect in klotho gene expression in the mouse results in a syndrome that resembles human ageing, including a short lifespan, infertility, arteriosclerosis, skin atrophy, osteoporosis and emphysema. The gene encodes a membrane protein that shares sequence similarity with the beta-glucosidase enzymes. The klotho gene product may function as part of a signalling pathway that regulates ageing in vivo and morbidity in age-related diseases.

Aging↗

PubMed1997

Inhibition of osteoblastic cell differentiation by conditioned medium derived from the human prostatic cancer cell line PC-3 in vitro.

Human prostatic carcinoma frequently metastasizes to bone tissue and activates bone metabolism, especially bone formation, at the site of metastasis. It has been reported that an extract of prostatic carcinoma and conditioned medium (CM) of a human prostatic carcinoma cell line, PC-3, established from a bone metastastic lesion, stimulate osteoblastic cell proliferation. However, there is little information about the effect of PC-3 CM on the differentiation of osteoblastic cells. In this study, we investigated the effect of PC-3 CM on the differentiation of two types of osteoblastic cells, primary fetal rat calvaria (RC) cells containing many undifferentiated osteoprogenitor cells, and ROS 17/2.8, a well-differentiated rat osteosarcoma cell line. PC-3 CM inhibited bone nodule formation and the activity of alkaline phosphatase (ALPase), an osteoblastic marker enzyme, on days 7, 14, and 21 (RC cells) or 3, 6, and 9 (ROS 17/2.8 cells) in a dose-dependent manner (5-30% CM). However, the CM did not affect cell proliferation or cell viability. PC-3 CM was found to markedly block the gene expression of ALPase and osteocalcin (OCN) mRNAs but had no effect on the mRNA expression of osteopontin (OPN), the latter two being noncollagenous proteins related to bone matrix mineralization. These findings suggest that PC-3 CM contains a factor that inhibits osteoblastic cell differentiation and that this factor may be involved in the process of bone metastasis from prostatic carcinoma.

Alkaline Phosphatase↗

PubMed1997

Isolation of RNA aptamers specific to the NS3 protein of hepatitis C virus from a pool of completely random RNA.

Hepatitis C virus (HCV) is a single-stranded RNA virus and its genome is translated into a single large polyprotein. The viral-encoded NS3 protein possesses protease, nucleoside triphosphatase, and helicase activities. Since these activities appear to be important for viral replication, efforts are being made to identify compounds that might inhibit the enzymatic activities of NS3 and serve as potential anti-HCV agents. We used a genetic selection strategy in vitro to isolate, from a pool of completely random RNA (120 random bases), those RNA aptamers that could bind to NS3. After six cycles of selection and amplification, 14% of the pooled RNAs could bind specifically to the NS3 protein. When the aptamers in the pool (cycle 6) were analyzed for binding and inhibition of the proteolytic activity of NS3 with the NS5A/NS5B peptide as substrate (S1), two aptamers, designated G6-16 and G6-19 RNA, were found to inhibit NS3 in vitro. Kinetic studies of the inhibition revealed that the aptamer G6-16 inhibited the NS3 protease with an inhibitory constant (Ki) of 3 microM. We also analyzed aptamers G6-16 and G6-19 for their action with a longer protein substrate (amino acid region 2203-2506) and found that these aptamers efficiently inhibited the proteolytic activity of NS3. In addition, both G6-16 and G6-19 aptamers were found to inhibit the helicase activity of NS3. Since these aptamers possesses dual inhibitory function for NS3, they could prove to be useful as anti-HCV drug leads.

Base Sequence↗

PubMed1997

Inhibition of transcription by the TAR RNA of HIV-1 in a nuclear extract of HeLa cells.

Regulation of transcription of human immunodeficiency virus type-1 (HIV-1) requires specific interaction of Tat protein with the trans-activation response region (TAR). Inhibition of replication of HIV-1 has previously been achieved with a TAR decoy, namely a short RNA oligonucleotide that corresponded to the sequence of the authentic TAR RNA. Since TAR RNA has the potential to interact with cellular factors, we examined the effect of TAR RNA on efficiency of transcription in nuclear of HeLa cell extracts. We performed an in vitro transcription assay in the presence of authentic TAR RNA using a template that was driven by the CMV (cytomegalovirus) early promoter in a HeLa nuclear extract and found, for the first time, that TAR RNA inhibited transcription by approximately 60-70% independently of the Tat-TAR interaction. Furthermore, we evaluated inhibition of transcription by variants of TAR RNA and found that the TAR RNA loop, bases surrounding the loop, the triple base bulge and the 'lower' stem region of TAR RNA were responsible for the inhibition of transcription. Taken together, earlier reports on proteins that bind to TAR RNA and the present results suggest that integrity of TAR RNA is important for efficient binding to cellular transcription factors. As judged from the significant inhibition observed in this study, the TAR decoy might sequester transcription factors and thus it might potentially be able to inhibit transcription of housekeeping genes that are unrelated to Tat function.

Base Sequence↗

PubMed1997

Selection of RNA aptamers that bind specifically to the NS3 protease of hepatitis C virus.

The RNA genome of human hepatitis C virus (HCV) is translated into a large precursor polyprotein. The NS3 protease of HCV has a crucial role in the processing of the polyprotein into functional viral proteins. We have used an in vitro genetic-selection strategy to isolate high-affinity RNA aptamers that bind to the NS3 protein, especially to its protease domain. Starting from a RNA pool that had a random sequence core of 12-18 nucleotides, aptamers that bind specifically to the NS3 protein were selected after 10 rounds of selection and amplification. A single aptamer, 10G-1, was found predominantly (71%) in the selected pool. This aptamer could bind to the NS3 protein with a binding constant of 650 nM and inhibit the proteolytic activity in vitro. By phosphate-modification-interference analysis we showed that the phosphate residues that are critical for the binding of 10G-1 to NS3 lie within the selected regions of the aptamer and that binding involves electrostatic contacts with the phosphates of regions G28-U34 and A47-A55. The NS3-binding region in 10G-1 can serve as a basis for designing more potential inhibitors of the NS3 protein.

Base Sequence↗

PubMed1997

Analysis of the cleavage reaction of a trans-acting human hepatitis delta virus ribozyme.

The cleavage reaction catalyzed by the trans -acting genomic ribozyme of human hepatitis delta virus (HDV) was analyzed with a 13mer substrate (R13) and thio-substituted [SR13(Rp) and SR13(Sp)] substrates under single-turnover conditions. The cleavage of RNA by the trans -acting HDV ribozyme proceeded as a first order reaction. The logarithm of the rate of cleavage (kclv) increased linearly (with a slope of approximately 1) between pH 4.0 and 6.0, an indication that a single deprotonation reaction occurred. This result suggests that kclv reflects the rate of the chemical cleavage step, at least around pH 5. The amount of active complex with the SR13(Sp) substrate was almost as large as with R13 (60-80%), whereas the amount of the corresponding active complex formed with the SR13(Rp) substrate was, at most, 20% of this value (with 0.5-100 mM Mg2+ions) at pH 5.0. Nonetheless, the value of kclv for all substrates was almost the same (0.4-0.5 min-1). Neither a 'thio effect' nor a 'Mn2+rescue effect' were observed. These results suggest that Mg2+ions do not interact with pro-R oxygen directly but are essential to the formation of the active complex of the ribozyme and its substrate.

Base Sequence↗

PubMed1997

Analysis of the functional domain of the rat liver mitochondrial import receptor Tom20.

Tom20 is an outer mitochondrial membrane protein and functions as a component of the import receptor complex for the cytoplasmically synthesized mitochondrial precursor proteins. It consists of the N-terminal membrane-anchor segment, the tetratricopeptide repeat (TPR) motif, a charged amino acids-rich linker segment between the membrane anchor and the TPR motif, and the C-terminal acidic amino acid cluster. To assess the functional significance of these segments in mammalian Tom20, we cloned rat Tom20 and expressed mutant rat Tom20 proteins in Deltatom20 yeast cells and examined their ability to complement the defects of respiration-driven growth and mitochondrial protein import. Tom20N69, a mutant consisting of the membrane anchor and the linker segments, was targeted to mitochondria and complemented the growth and import defects as efficiently as wild-type Tom20, whereas a mutant lacking the linker segment did not. In vitro protein import into mitochondria isolated from the complemented yeast cells revealed that the precursor targeted to yeast Tom70 was efficiently imported into the mitochondria via rat Tom20N69. Thus the linker segment is essential for the function of rat Tom20, whereas the TPR motif and the C-terminal acidic amino acids are not.

Amino Acid Sequence↗