Biomedical subjects

S Murakami

Publications and source records attributed to S Murakami.

At least 73 records · Page 4Linked to original sources

PubMed2001

The OLD-1 positive regulator of longevity and stress resistance is under DAF-16 regulation in Caenorhabditis elegans.

Aging and limited life span are fundamental biological phenomena observed in a variety of species [1]. Approximately 55 genes have been identified that can extend longevity when altered in Caenorhabditis elegans [2-5]. These genes include an insulin-like receptor (daf-2) and a phosphatidylinositol 3-OH kinase (age-1) regulating a forkhead transcription factor (daf-16) [6, 7], as well as genes mediating metabolic throughput [8], sensory perception [9], and reproduction [10]. Moreover, these mutant alleles both extend life span and increase resistance to ultraviolet (UV) radiation [11], heat [12], and oxidative stress [13-15], though the stress resistance of clk-1 is controversial. With the exception of old-1 and perhaps some other genes [16-19], all of the life-extension alleles are hypomorphic or nullomorphic. Here, we show that the OLD-1 transmembrane tyrosine kinase (formerly TKR-1; [16, 20]) is expressed in a variety of tissues, is stress inducible, and is a positive regulator of longevity and stress resistance. The transcription of old-1 is upregulated in long-lived age-1 and daf-2 mutants and is upregulated in response to heat, UV light, and starvation. Both RT-PCR and analysis of an OLD-1::GFP tag suggest that old-1 expression is dependent on daf-16. Importantly, old-1 is required for the life extension of age-1 and daf-2 mutants. This study reveals a new system for specifying longevity and stress resistance and suggests possible mechanisms for mediating life extension by dietary restriction and hormesis.

Animals↗

PubMed2001

The effect of nitric oxide on acetylcholine release in the rabbit bladder.

We evaluated the effects of nitric oxide (NO) on acetylcholine release and the contractile response induced by electrical field stimulation in rabbit bladder smooth muscles using a muscle bath and high performance liquid chromatography coupled with microdialysis. Electrical field stimulation (supramaximum voltage, pulse duration 0.5 ms, frequency 5 and 20 Hz) was applied to a smooth muscle strip isolated from rabbit bladder. With low-frequency (5 Hz) stimulation, pretreatment with Nomega-nitro-L-arginine (L-NNA) (100 microM) significantly increased electrical field stimulation-induced acetylcholine release and contractile response, which were reduced by the addition of L-arginine. Pretreatment with sodium nitroprusside in the absence or presence of L-NNA significantly decreased electrical field stimulation-induced acetylcholine release and contractile response. In contrast, with high frequency (20 Hz) stimulation, pretreatment with L-NNA and sodium nitroprusside had no significant effect on either contractile response or acetylcholine release. Pretreatment with sodium nitroprusside caused no significant changes in carbachol and ATP-induced contractile responses. Sodium nitroprusside and L-NNA had no significant effects on the atropine-resistant part of the contraction induced by electrical field stimulation in rabbit bladder smooth muscles. The results suggest that there is a NO-mediated mechanism inhibiting acetylcholine release from cholinergic nerve endings in rabbit bladder, which may contribute to bladder function.

Acetylcholine↗

PubMed2001

Expression profile of active genes in human periodontal ligament and isolation of PLAP-1, a novel SLRP family gene.

Periodontal ligament (PDL) is one of the most important tissues in maintaining the homeostasis of tooth and tooth-supporting tissue, periodontium. In this study, we investigated the expression profile of active genes in the human PDL obtained by collecting sequences with a 3'-directed cDNA library, which faithfully represents the composition of the mRNA population. We succeeded in obtaining a total of 1752 cDNA sequences by sequencing randomly selected clones and identified a total of 1318 different species as gene signatures (GS) by their sequence identity, 344 of which were known genes in the GenBank, and 974 of which were new genes. The resulting expression profile showed that collagen type I and type III were the most abundant genes and that osteogenesis-related proteins, such as SPARC/osteonectin and osteoblast specific factor 2, were highly expressed. By comparing the expression profile of PDL with 44 profiles similarly obtained with unrelated human cell/tissue, nine novel genes, which are probably expressed specifically in PDL, were discovered. Among them, we cloned a full-length cDNA of GS5096, which is frequently expressed in freshly-isolated periodontal tissue. We found that it encodes a novel protein, which is a new member of the class I small leucine-rich repeat proteoglycan family, and designated it PLAP-1 (periodontal ligament associated protein-1). PLAP-1 mRNA expression was confirmed in in vitro-maintained PDL cells and was enhanced during the course of the cytodifferentiation of the PDL cells into mineralized tissue-forming cells such as osteoblasts and cementoblasts. These findings suggest the involvement of PLAP-1 in the mineralized matrix formation in PDL tissues.

Amino Acid Sequence↗

PubMed2001

Observation of the "self-healing" of an error field island in the large helical device.

It was observed that the vacuum magnetic island produced by an external error magnetic field in the large helical device shrank in the presence of plasma. This was evidenced by the disappearance of flat regions in the electron temperature profile obtained by Thomson scattering. This island behavior depended on the magnetic configuration in which the plasmas were produced.

Journal Article↗

PubMed2001

Extracellular Toll-like receptor 2 region containing Ser40-Ile64 but not Cys30-Ser39 is critical for the recognition of Staphylococcus aureus peptidoglycan.

Toll-like receptor 2 (TLR2) and CD14 function as pattern recognition receptors for bacterial peptidoglycan (PGN). TLRs and CD14 possess repeats of the leucine-rich motif. To address the role of the extracellular domain of TLR2 in PGN signaling, we constructed CD14/TLR2 chimeras, in which residues 1-356 or 1-323 of CD14 were substituted for the extracellular domain of TLR2, and five deletion mutants of TLR2, in which the progressively longer regions of extracellular TLR2 regions were deleted. PGN induced NF-kappaB activation in HEK293 cells expressing TLR2 but not in cells expressing CD14/TLR2 chimeras. The cells transfected with a deletion mutant TLR2(DeltaCys30-Ile64) as well as TLR2(DeltaCys30-Asp160) and TLR2(DeltaCys30-Asp305) failed to respond to PGN, indicating the importance of the TLR2 region Cys(30)-Ile(64). Although TLR2(DeltaCys30-Ser39) conferred cell responsiveness to PGN, the cells expressing TLR2(DeltaSer40-Ile64) failed to induce NF-kappaB activation. In addition, NF-kappaB activity elicited by PGN was significantly attenuated in the presence of synthetic peptide corresponding to the TLR2 region Ser(40)-Ile(64). From these results, we conclude that; 1) CD14 cannot functionally replace the extracellular domain of TLR2 in PGN signaling; 2) the TLR2 region Cys(30)-Ser(39) is not required for PGN recognition; 3) the TLR2 region containing Ser(40)-Ile(64) is critical for PGN recognition.

Amino Acid Sequence↗

PubMed2001

Identification of coding single-nucleotide polymorphisms in human taste receptor genes involving bitter tasting.

T2Rs comprise a G-protein-coupled receptor superfamily that contains functionally defined bitter taste receptors. Here we report the tissue expressions and coding single-nucleotide polymorphisms (cSNPs) in human T2R genes (hT2R3, hT2R4, and hT2R5) on chromosome 7q31. We first demonstrated that hT2R3, hT2R4, and hT2R5 are actually expressed in the circumvallate papillae of the human tongue by reverse transcription-polymerase chain reaction (RT-PCR). We identified six cSNPs within the T2R receptor genes. The hT2R4 and hT2R5 contained four and one cSNPs that cause missense mutations, respectively, while hT2R3 included one silent nucleotide mutation. However, we could not find any nonsense mutations that resulted in a frameshift or a premature stop codon within the open reading frames. Genotype frequencies of each cSNP were in Hardy-Weinberg equilibrium. The identification of nucleotide diversity and amino acid polymorphisms in human T2R receptors could help clarify individual differences in the acceptability and sensitivity to bitter compounds.

Base Sequence↗

PubMed2001

Distribution of various calcium channel alpha(1) subunits in murine DRG neurons and antinociceptive effect of omega-conotoxin SVIB in mice.

Immunohistological study revealed the differential localization of subtypes of voltage-dependent calcium channels in the dorsal root ganglion neurons. Intrathecal injection of omega-conotoxin SVIB, an analogue of omega-conotoxin GVIA, which acts on N-type voltage-dependent calcium channels, significantly shortened the licking time in the late phase of a formalin test.

Animals↗

PubMed2001

Reduction of ion thermal diffusivity associated with the transition of the radial electric field in neutral-beam-heated plasmas in the large helical device.

Recent large helical device experiments revealed that the transition from ion root to electron root occurred for the first time in neutral-beam-heated discharges, where no nonthermal electrons exist. The measured values of the radial electric field were found to be in qualitative agreement with those estimated by neoclassical theory. A clear reduction of ion thermal diffusivity was observed after the mode transition from ion root to electron root as predicted by neoclassical theory when the neoclassical ion loss is more dominant than the anomalous ion loss.

Journal Article↗

PubMed2001

Antinociceptive action of amlodipine blocking N-type Ca2+ channels at the primary afferent neurons in mice.

We investigated the antinociceptive action of amlodipine, a dihydropyridine derivative, which acts on both L- and N-type voltage-dependent Ca2+ channels (VDCCs), in mice. Intrathecal injection of amlodipine (300 nmol/kg) significantly shortened the licking time in the late phase of a formalin test, while no effect was found with another dihydropyridine derivative, nicardipine (300 nmol/kg). Cilnidipine and omega-conotoxin GVIA also showed marked analgesic effects under the same experimental conditions. Transcripts of alpha1A, alpha1B, alpha1E, alpha1F, alpha1H, beta3, and beta4 subunits were detected by polymerase-chain reaction (PCR) in the dorsal root ganglion, suggesting the existence of a variety of voltage-dependent Ca2+ channels. Electrophysiological experiments showed that amlodipine and cilnidipine inhibit N-type currents in the dorsal root ganglion cells. These results suggest that amlodipine, cilnidipine, and omega-conotoxin GVIA exert their antinociceptive actions by blocking N-type Ca2+ channels in the primary nociceptive afferent fibers. Blocking of the Ca2+ channels results in attenuation of synaptic transmission of nociceptive neurons. Furthermore, it is suggested that some N-type Ca2+ channel blockers might have therapeutic potential as analgesics when applied directly into the subarachnoidal space.

Amlodipine↗

PubMed2001

Effects of tumor necrosis factor-alpha on secretion of prostaglandins E2 and F2alpha in bovine endometrium throughout the estrous cycle.

To determine the physiological significance of tumor necrosis factor-alpha (TNFalpha) in the regulation of endometrial prostaglandin (PG) release in cattle, we investigated the effects of TNFalpha on the secretion of PGE2 and PGF2alpha by bovine endometrium during the estrous cycle. Bovine uteri were classified into six stages (estrus: Day 0, early luteal 1: Days 2 to 3, early luteal 11: Days 5 to 6, mid-luteal: Days 8 to 12, late luteal: Days 15 to 17 and follicular: Days 19 to 21). After 1 h of pre-incubation, endometrial tissues (20 to 30 mg) were exposed to 0 or 0.6 nM TNFalpha for 4 h. The PGE2 concentrations in the medium were higher in the luteal stages than in the follicular stage and in estrus. In contrast, PGF2alpha concentrations were higher in the follicular stage and in estrus than in the luteal stages. The ratio of the basal concentrations of PGE2 and PGF2alpha (PGE2/PGF2alpha ratio) was higher in the luteal stages than in the follicular stage and in estrus. Although TNFalpha stimulated both PGE2 and PGF2alpha secretion during the entire period of the estrous cycle, the level of stimulation of TNFalpha on PGE2 output by the bovine endometrium does not show the same cyclical changes as that shown on PGF2alpha output. The stimulation of TNFalpha resulted in a decrease in the PGE2/PGF2alpha ratio only in the late luteal stage. Furthermore, TNFalpha stimulated PGE2 secretion in stromal, but not epithelial cells. The overall results suggest that TNFalpha is a potent regulator of endometrial PGE2 secretion as well as PGF2alpha secretion during the entire period of estrous cycle, and that TNFalpha plays different roles in the regulation of secretory function of bovine endometrium at different phases of the estrous cycle.

Animals↗

PubMed2001

Direct interaction between the subunit RAP30 of transcription factor IIF (TFIIF) and RNA polymerase subunit 5, which contributes to the association between TFIIF and RNA polymerase II.

The general transcription factor IIF (TFIIF) assembled in the initiation complex, and RAP30 of TFIIF, have been shown to associate with RNA polymerase II (pol II), although it remains unclear which pol II subunit is responsible for the interaction. We examined whether TFIIF interacts with RNA polymerase II subunit 5 (RPB5), the exposed domain of which binds transcriptional regulatory factors such as hepatitis B virus X protein and a novel regulatory protein, RPB5-mediating protein. The results demonstrated that RPB5 directly binds RAP30 in vitro using purified recombinant proteins and in vivo in COS1 cells transiently expressing recombinant RAP30 and RPB5. The RAP30-binding region was mapped to the central region (amino acids (aa) 47-120) of RPB5, which partly overlaps the hepatitis B virus X protein-binding region. Although the middle part (aa 101-170) and the N-terminus (aa 1-100) of RAP30 independently bound RPB5, the latter was not involved in the RPB5 binding when RAP30 was present in TFIIF complex. Scanning of the middle part of RAP30 by clustered alanine substitutions and then point alanine substitutions pinpointed two residues critical for the RPB5 binding in in vitro and in vivo assays. Wild type but not mutants Y124A and Q131A of RAP30 coexpressed with FLAG-RAP74 efficiently recovered endogenous RPB5 to the FLAG-RAP74-bound anti-FLAG M2 resin. The recovered endogenous RPB5 is assembled in pol II as demonstrated immunologically. Interestingly, coexpression of the central region of RPB5 and wild type RAP30 inhibited recovery of endogenous pol II to the FLAG-RAP74-bound M2 resin, strongly suggesting that the RAP30-binding region of RPB5 inhibited the association of TFIIF and pol II. The exposed domain of RPB5 interacts with RAP30 of TFIIF and is important for the association between pol II and TFIIF.

Animals↗

PubMed2001

Uptake by vascular smooth muscle cells plays an important role in targeting of lipid microspheres incorporating prostaglandin E1 into a thickened intima.

A study was done to determine how lipid microspheres (LM) containing prostaglandin E1 (lipo PGE1) accumulate in injured arterial tissue. After administration of lipo PGE1 labeled with a fluorescence probe, 1,1'-dioctadecyl-3, 3, 3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-lipo PGE1) to rats 14 of days after balloon injury of the carotid artery, localization into the injured site was examined using a fluorescence confocal laser scanning microscope (CLSM). In contrast with the normal carotid artery, DiI-lipo PGE1 accumulated remarkably in neointima of the injured site which was occupied mainly by the migration of the proliferating and quiescent vascular smooth muscle cells (SMC). In vitro cellular uptake of DiI-lipo PGE1 was semi-quantitatively measured using CLSM, regarding differentiated and proliferative phenotypes of human vascular SMC, compared with the human endothelial cells (EC) and mouse fibroblasts. The differentiated SMC incorporated DiI-lipo PGE1 to equal or a higher level of the proliferative phenotype, and was significantly higher than EC and fibroblasts. The uptake of DiI-lipo PGE1 by both SMC and EC was inhibited at 4 degrees C, by dansylcadaverine and excessive LM, but was unaffected by cytochalasin B. These results suggest that the uptake of DiI-lipo PGE1 by SMC plays an important role in localization of DiI-lipo PGE1 at the injured site, and that the uptake seems to be a receptor mediated endocytosis.

3T3 Cells↗

PubMed2001

Expression and action of hepatocyte growth factor in bovine endometrial stromal and epithelial cells in vitro.

Hepatocyte growth factor (HGF) is a pleiotropic growth factor that acts on various epithelial cells. The objectives of this study were to determine whether HGF altered the proliferation and prostaglandin (PG) secretion of bovine endometrial stromal and epithelial cells in vitro. We also observed HGF and HGF receptor (c-met) mRNA expression in cultured bovine endometrial stromal and epithelial cells by RT-PCR. Stromal and epithelial cells obtained from cows in early stage of the estrous cycle (days 2-5) were cultured in DMEM/Ham's F-12 supplemented with 10% calf serum. The cells were exposed to HGF (0-10 ng/ml) for 2, 4, or 6 days. HGF significantly increased the total DNA in epithelial (P < 0.05), but not stromal cells. In another experiment, when the cells reached confluence, the culture medium was replaced with fresh medium with 0.1% BSA containing HGF 0-100 ng/ml and the cells were cultured for 24 hr. The HGF stimulated PGF2alpha secretion in epithelial, but not stromal cells. RT-PCR revealed that mRNA of HGF is expressed only in stromal cells, and that c-met mRNA is expressed in both stromal and epithelial cells. These results suggest that HGF plays roles in the proliferation and the regulation of secretory function of bovine endometrial epithelial cells in a paracrine fashion.

Animals↗

PubMed2001

Expression of adhesion molecules by cultured human glomerular endothelial cells in response to cytokines: comparison to human umbilical vein and dermal microvascular endothelial cells.

We investigated the expression of cell adhesion molecules on the surface of glomerular endothelial cells (GEC), dermal microvascular endothelial cells (MvE), and umbilical vein endothelial cells (HUVEC) that had or had not been stimulated by cytokines. PECAM-1 was constitutively expressed at a high level on HUVEC but its expression level decreased following stimulation by tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma). PECAM-1 was also constitutively expressed on microvascular endothelial cells MvE and GEC, but at lower levels than on HUVEC, and expression by these cells also decreased in response to TNF-alpha and IFN-gamma. There was no dose-dependent effect on MvE but there was a dose-dependent effect on the level of expression of cell adhesion molecules on GEC. TNF-alpha induced the expression of VCAM-1 on HUVEC and GEC, but not MvE, while IFN-gamma induced VCAM-1 expression only on HUVEC. TNF-alpha induced the expression of E-selectin on all three kinds of endothelial cells, but IFN-gamma had no effect on E-selectin expression. GEC therefore showed expression patterns of PECAM-1, VCAM-1, and E-selectin different from those seen in HUVEC and MvE upon treatment with TNF-alpha or IFN-gamma. The use of cultured human GEC allows us to study not only the inflammatory processes, but also the pathophysiological role of GEC in hemodynamic disturbances and their interaction with intrinsic mesangial cells at the molecular and subcellular levels.

Cell Adhesion Molecules↗

PubMed2001

Peripheral T/natural killer-cell lymphoma involving the female genital tract: a clinicopathologic study of 5 cases.

Malignant lymphoma of the female genital tract (FGT) is rare. In this study, 5 peripheral T/natural killer (NK)-cell lymphomas (PTCLs) involving the FGT are reported. They include 2 from the uterus and 1 each from ovary, uterus and ovary, and vagina, and were detected between 1996 and 2000. One of the 2 ovarian tumors was bilateral. In all cases, the FGT was the initial site of clinical presentation of disease. Age at presentation ranged from 21 to 52 years (median, 36 years). One case was stage I disease, 2 were stage II, and 2 were stage IV. All 5 tumors were positive for CD3epsilon, and 3 harbored the Epstein-Barr virus, although the detailed immunophenotypic profiles varied. Three were diagnosed as nasal type T/NK-cell lymphoma, 1 as anaplastic large-cell lymphoma (anaplastic lymphoma kinase [ALK]-positive), and 1 as unspecified PTCL of cytotoxic phenotype, according to the forthcoming World Health Organization classification. Four of 5 patients received laparotomy and chemotherapy. Four patients (in stages II and IV) died of disease within 16 months of the initial diagnosis, whereas only 1 patient (in stage I) is alive without disease at 39 months of follow-up. Our experience in this series provided clinically relevant information on diagnosis, treatment, and outcome for extremely rare tumors of the FGT.

Adult↗

PubMed2001

Prognostic value of DNA ploidy in 653 Japanese women with node-negative breast cancer.

BACKGROUND: The prognostic value of DNA ploidy has been extensively studied in breast cancer; however, the results remain controversial. Flow cytometry (FCM) studies of DNA ploidy on frozen sections have not yet been performed in a large series of Japanese women with node-negative breast cancer. METHODS: An FCM analysis of DNA ploidy was performed on frozen sections of node-negative breast cancer from 653 Japanese women, with a mean follow-up duration of 46 months. RESULTS: Three hundred and twenty-four (49.6%) patients showed a diploid tumor, while 329 (50.4%) showed an aneuploid tumor. There was a significant correlation between DNA ploidy and estrogen receptor (ER) status. Patients with an aneuploid tumor had a significantly worse outcome in terms of both disease-free survival (DFS; P = 0.0116) and overall survival (OS: P = 0.0492) than those with a diploid tumor, while the same effect, in terms of DFS, was also seen in ER-positive breast cancer. Multivariate analyses indicated DNA ploidy to be an independent prognostic factor for DFS, while DNA ploidy and tumor size were found to be independent prognostic factors for OS. DNA ploidy was also shown to be an independent prognostic factor for DFS in ER-positive breast cancer. CONCLUSION: Our findings demonstrated DNA ploidy, based on FCM, to have an important prognostic value in Japanese women with node-negative breast cancer.

Adult↗

PubMed2001

Expression and localization of connexin 43 in rat incisor odontoblasts.

We have examined the expression and localization of connexin 43 (CX43) in rat incisor odontoblasts using reverse transcriptase polymerase chain reaction, in situ hybridization and immunohistochemistry. The CX43 gene was expressed in odontoblasts, and levels of gene expression increased throughout the course of development. In contrast, CX43 was down-regulated at an incisal segment. In situ hybridization analysis showed no positive signal for CX43 RNA in the cytoplasm of differentiating dental papilla cells, but faint positive signals for CX43 RNA were observed in early pre-odontoblasts. Those signals were more intense in young and in old odontoblasts, but were less in short odontoblasts. CX43 could not be detected in differentiating dental papilla cells or in early pre-odontoblasts by immunohistochemical localization, but a positive reaction was found in the late pre-odontoblast stage where predentin had been produced. The positivity gradually increased during odontoblast maturation, and was highest in the layer of old odontoblasts. These results indicate that odontoblasts that secrete actively dentin matrix components are tightly in contact with each other by gap junctions as suggested by the intense CX43.

Animals↗

PubMed2001

Hepatitis B virus X protein: a multifunctional viral regulator.

Hepatitis B Virus (HBV) infection is one of the major causes of hepatocellular carcinoma (HCC). X protein (HBx) has been suspected to be oncogenic, although the precise role(s) remain uncertain. HBx is a multifunctional viral regulator that modulates transcription, cell responses to genotoxic stress, protein degradation, and signaling pathways. These modulations affect viral replication and viral proliferation, directly or indirectly. HBx also affects cell cycle checkpoints, cell death, and carcinogenesis. This article presents an overview of the progress in HBx research over the past several years.

Apoptosis↗