Biomedical subjects

S Murakami

Publications and source records attributed to S Murakami.

At least 19 recordsLinked to original sources

PubMed2000

Prejunctional alpha-adrenoceptors regulate nitrergic neurotransmission in the rabbit urethra.

We evaluated the effects of prejunctional alpha-adrenoceptors on nitric oxide (NO)-mediated urethral relaxation in rabbits using a muscle bath technique and high-performance liquid chromatography coupled with a microdialysis procedure. The amount of NO(2)(-)/NO(3)(-) released during electrical field stimulation was measured by an NO(2)(-)/NO(3)(-) analyzer based on the Griess method. Pretreatment with phenylephrine (0.01 microM) and yohimbine (0.1-10 microM) significantly reduced the relaxation responses induced by electrical field stimulation. In contrast, pretreatment with clonidine (0.01 microM) and prazosin (0.01-1 microM) enhanced the relaxation responses. Cys-NO-induced relaxations of rabbit urethral smooth muscle were not affected by pretreatment with alpha-adrenoceptor agonists and antagonists. The amount of NO(2)(-)/NO(3)(-) released by electrical field stimulation increased after pretreatment with clonidine (0.01 microM) and prazosin (0.01-1 microM), but decreased after pretreatment with phenylephrine (0.01 microM) and yohimbine (0.1-10 microM). The results suggest that the release of NO from nitrergic nerves in the rabbit urethra is reduced and increased by stimulation of prejunctional alpha(1)- and alpha(2)-adrenoceptors, respectively.

Adrenergic alpha-Agonists↗

PubMed2000

Telomerase activity reconstituted in vitro with purified human telomerase reverse transcriptase and human telomerase RNA component.

Telomerase is a specialized reverse transcriptase that catalyzes elongation of the telomeric tandem repeat, TTAGGG, by addition of this sequence to the ends of existing telomeres. Human telomerase reverse transcriptase (hTERT) has been identified as a catalytic enzyme involved in telomere elongation that requires telomerase RNA, human telomerase RNA component (hTR), as an RNA template. We established a new method to express and purify soluble insect-expressed recombinant hTERT. The partially purified FLAG-hTERT retained the catalytic activity of telomerase in a complementation assay in vitro to exhibit telomerase activity in telomerase-negative TIG3 cell extract and in a reconstitution assay with FLAG-hTERT and purified hTR in vitro. FLAG-hTERT (D712A) with a mutation in the VDV motif exhibited no telomerase activity, confirming the authentic catalytic activity of FLAG-hTERT. The reconstituted complex of FLAG-hTERT and hTR in vitro was detected by electrophoretic mobility shift assay, and its activity was stimulated by more than 30-fold by TIG3 cell extract. This suggested that some cellular component(s) in the extract facilitated the reconstituted telomerase activity in vitro. Geldanamycin had no effect on the reconstituted activity but partially reduced the stimulated activity of the reconstituted telomerase by the TIG3 cell extract, suggesting that Hsp90 may contribute to the stimulatory effect of the cellular components.

Benzoquinones↗

PubMed2000

Enzymatic removal of polysialic acid from neural cell adhesion molecule perturbs the migration route of luteinizing hormone-releasing hormone neurons in the developing chick forebrain.

During development in the chick embryo, luteinizing hormone-releasing hormone (LHRH) neurons migrate along the olfactory nerve from the olfactory epithelium to the forebrain. At embryonic day 5.5 (E5.5) to E6.0, the majority of LHRH neurons begin to enter the medial forebrain and then course dorsocaudally along the forebrain substance just beneath the pia matter in association with the somatostatin (SST)-positive fibers, which branch medially from the SST-positive olfactory nerve. By E6.5, the neurons and SST-positive medial branch of the olfactory nerve have proceeded toward the septal area. Intense immunoreactivity for the polysialylated form of neural cell adhesion molecule (PSA-NCAM) on both the LHRH neurons and the SST-positive fibers during this period suggests that this less adhesive form of NCAM is involved in the migratory process. This possibility was examined by using a polysialic acid (PSA)-specific endoneuraminidase. PSA removal did not alter the behavior or appearance of the SST-positive olfactory fibers within the migration pathway. However, it induced a significant deviation of migrating LHRH neurons from the regular path in the forebrain. The effect of PSA removal is more likely to involve changes in the interaction of the migrating neurons with a subset of the SST-positive olfactory fibers and/or other elements in the forebrain rather than an alteration in the pattern of their axonal substrate. On the basis of these results, it is suggested that PSA contributes to the specific pattern of LHRH neuronal migration in the forebrain by limiting interaction of these LHRH neurons with their surrounding environment.

Animals↗

PubMed2000

Protease activity of CND41, a chloroplast nucleoid DNA-binding protein, isolated from cultured tobacco cells.

CND41 is a 41 kDa DNA-binding protein isolated from chloroplast nucleoids of cultured tobacco cells. The presence of the active domain of aspartic protease in the deduced amino acid sequence of CND41 suggests that it has proteolytic activity. To confirm this, CND41 was highly purified from cultured tobacco cells and its proteolytic activity was characterized with fluorescein isothiocyanate-labeled hemoglobin as the substrate. The purified CND41 had strong proteolytic activity at an acidic pH (pH 2-4). This activity was inhibited by various chemicals, including the nucleoside triphosphates, NADPH, Fe(3+) and sodium dodecyl sulfate.

Adenosine Triphosphate↗

PubMed2000

Human hepatitis B virus X protein is a possible mediator of hypoxia-induced angiogenesis in hepatocarcinogenesis.

The hepatitis B virus (HBV)-encoded transcriptional activator HBV-X protein (HBx) was known to be involved in hepatocarcinogenesis. Hepatocarcinogenesis generally included an active angiogenesis that was mainly considered to be due to a local hypoxia in liver tissues. However, the exact mechanisms of HBx-induced hepatocarcinogenesis were poorly understood. In this study, we examined the role of HBx in the increased angiogenesis and the possible regulating mechanisms of HBx by hypoxia. We demonstrated that HBx stimulated the transcription of vascular endothelial growth factor (VEGF), a potent angiogenic factor, in HBx-stable transfectants. HBx-induced angiogenesis was confirmed by in vivo tumor angiogenesis assay, resulting in that the HBx transfectants increased the formation of new blood vessels compared to the control transfectants. Then, we demonstrated that the expression of HBx was enhanced after incubating HBV-infected hepatoma cells under hypoxia. Moreover, the activity of HBV enhancer 1 (Enh1) was increased when hepatoma cells transfected with the reporter plasmid containing HBV Enh1 were exposed to hypoxic conditions. These results strongly suggest that HBx may play a critical role in the hypoxia-induced angiogenesis through transcriptional activation of VEGF during hepatocarcinogenesis.

Cell Hypoxia↗

PubMed2000

Importance of the carboxy-terminal 25 amino acid residues of lung collectins in interactions with lipids and alveolar type II cells.

Surfactant proteins A and D (SP-A and SP-D) are structurally related members of the collectin family found in the alveolar compartment of the lung. SP-A binds dipalmitoylphosphatidylcholine (DPPC) and galactosylceramide (GalCer), induces liposome aggregation, and regulates the uptake and secretion of surfactant lipids by alveolar type II cells in vitro. SP-D binds phosphatidylinositol (PI) and glucosylceramide. The purpose of this study was to identify a critical stretch of primary sequence in the SP-A region Cys(204)-Phe(228) and the SP-D region Cys(331)-Phe(355) that is involved in protein-specific lipid and type II cell interactions. Chimeras ad1 and ad2 were constructed with rat SP-A/SP-D splice junctions at Cys(218)/Gly(346) and Lys(203)/Cys(331), respectively. Chimera ad1 but not ad2 retained DPPC liposome binding activity. Both chimeras retained significant binding to GalCer liposomes. Chimera ad1 did not bind to PI, whereas chimera ad2 acquired a significant PI binding. Both chimeras failed to induce liposome aggregation and to interact with alveolar type II cells. In addition, monoclonal antibody 1D6 that blocks specific SP-A functions did not recognize either chimera. From these results, we conclude that (1) the SP-A region Leu(219)-Phe(228) is required for liposome aggregation and interaction with alveolar type II cells, (2) the SP-A region Cys(204)-Cys(218) is required for DPPC binding, (3) the SP-D region Cys(331)-Phe(355) is essential for minimal PI binding, and (4) the epitope for mAb 1D6 is located at the region contiguous to the SP-A region Leu(219)-Phe(228).

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2000

Energy confinement time and heat transport in initial neutral beam heated plasmas on the large helical device

The confinement characteristics of large net-current-free plasmas heated by neutral-beam injection have been investigated in the Large Helical Device (LHD). A systematic enhancement in energy-confinement times from the scaling derived from the medium-sized heliotron/torsatron experiments have been observed, which is attributed to the edge pedestal. The core confinement is scaled with the Bohm term divided by the square root of the gyro radii. The comparative analysis using a dimensionally similar discharge in the Compact Helical System indicates gyro-Bohm dependence in the core and transport improvement in the edge region of LHD plasmas.

Journal Article↗

PubMed2000

Potent inhibition of the master chondrogenic factor Sox9 gene by interleukin-1 and tumor necrosis factor-alpha.

The inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) strongly inhibit the expression of genes for cartilage extracellular matrix proteins. We have recently obtained genetic evidence indicating that the high mobility group domain containing transcription factor Sox9 is required for cartilage formation and for expression of chondrocyte-specific genes including the gene for type II collagen (Col2a1). We show here that IL-1 and TNF-alpha cause a marked and rapid decrease in the levels of Sox9 mRNA and/or protein in chondrocytes. A role for the transcription factor NFkappaB in Sox9 down-regulation was suggested by the ability of pyrrolidine dithiocarbamate, an inhibitor of the NFkappaB pathway, to block the effects of IL-1 and TNF-alpha. This role was further supported by the ability of a dominant-negative mutant of IkappaBalpha to block the IL-1 and TNF-alpha inhibition of Sox9-dependent Col2a1 enhancer elements. Furthermore, forced expression of the NFkappaB subunits p65 or p50 also inhibited Sox9-dependent Col2a1 enhancer. Because Sox9 is essential for chondrogenesis, the marked down-regulation of the Sox9 gene by IL-1 and TNF-alpha in chondrocytes is sufficient to account for the inhibition of the chondrocyte phenotype by these cytokines. The down-regulation of Sox9 may have a crucial role in inhibiting expression of the cartilage phenotype in inflammatory joint diseases.

Animals↗

PubMed2000

Up-regulation of the chondrogenic Sox9 gene by fibroblast growth factors is mediated by the mitogen-activated protein kinase pathway.

Recent experiments have established that Sox9 is required for chondrocyte differentiation. Here, we show that fibroblast growth factors (FGFs) markedly enhance Sox9 expression in mouse primary chondrocytes as well as in C3H10T1/2 cells that express low levels of Sox9. FGFs also strongly increase the activity of a Sox9-dependent chondrocyte-specific enhancer in the gene for collagen type II. Transient transfection experiments using constructs encoding FGF receptors strongly suggested that all FGF receptors, FGFR1-R4, can transduce signals that lead to the increase in Sox9 expression. The increase in Sox9 levels induced by FGF2 was inhibited by a specific mitogen-activated protein kinase kinase (MAPKK)/mitogen-activated protein kinase/ERK kinase (MEK) inhibitor U0126 in primary chondrocytes. In addition, coexpression of a dual-specificity phosphatase, CL100/MKP-1, that is able to dephosphorylate and inactivate mitogen-activated protein kinases (MAPKs) inhibited the FGF2-induced increase in activity of the Sox9-dependent enhancer. Furthermore, coexpression of a constitutively active mutant of MEK1 increased the activity of the Sox9-dependent enhancer in primary chondrocytes and C3H10T1/2 cells, mimicking the effects of FGFs. These results indicate that expression of the gene for the master chondrogenic factor Sox9 is stimulated by FGFs in chondrocytes as well as in undifferentiated mesenchymal cells and strongly suggest that this regulation is mediated by the MAPK pathway. Because Sox9 is essential for chondrocyte differentiation, we propose that FGFs and the MAPK pathway play an important role in chondrogenesis.

Animals↗

PubMed2000

Edge thermal transport barrier In LHD discharges

In LHD discharges a significant enhancement of the global energy confinement has been achieved for the first time in a helical device with an edge thermal barrier, which exhibits a sharp gradient at the edge of the temperature profile. Key features associated with the barrier are quite different from those seen in tokamaks: (i) almost no change in particle (including impurity) transport, (ii) a gradual formation of the barrier, (iii) a very high ratio of the edge temperature to the average temperature, and (iv) no edge relaxation phenomenon. These features are very attractive in applying the thermal barrier to future reactor grade devices.

Journal Article↗

PubMed2000

Tumor necrosis factor-alpha and its receptor in the corpus luteum of pregnant cows.

The objective of this study was to investigate the presence of tumor necrosis factor (TNF)-alpha mRNA and TNF-alpha receptors in the bovine corpus luteum (CL) during the gestation period. TNF-alpha mRNA and TNF-alpha receptors were determined on bovine CL from pregnant cows at three stages: trimester I (fetal crown-rump length; 6-20 cm), trimester II (25-45 cm) and trimester III (50-80 cm). TNF-alpha mRNA was detected by an RT-PCR analysis in the CL of all stages of gestation. A Scatchard analysis revealed the presence of a high-affinity binding site (Kd; 5.1-6.9 nM) in the CL membranes collected at each stage of gestation. Furthermore, the concentrations of TNF-alpha receptors in the CL of trimesters I (24. 0 +/- 1.95 pmol/mg protein) and III (21.6 +/- 2.39 pmol/mg protein) of gestation were significantly higher than the concentration in trimester II (14.9 +/- 2.07 pmol/mg protein) (P < 0.05). These results indicate that TNF-alpha is locally produced and that TNF-alpha receptors are present in bovine CL during the gestation period, and suggest that TNF-alpha plays one or more roles as a paracrine factor in regulating bovine CL function during the entire gestation period.

Animals↗

PubMed2000

Tumor necrosis factor-alpha stimulates prostaglandin F2alpha secretion by bovine luteal cells via activation of mitogen-activated protein kinase and phospholipase A2 pathways.

It has been well demonstrated that tumor necrosis factor-alpha (TNFalpha) stimulates prostaglandin (PG) F2alpha secretion by bovine corpus luteum (CL) in vitro. The objective of the present study was to clarify the intracellular signaling pathway of TNFalpha to stimulate PGF2alpha production in cultured bovine luteal cells. Bovine luteal cells that were obtained from mid- (days 8-12 after ovulation) CL were incubated with TNFalpha (0.6 nM) and/or various compounds as follows: U-73122 (an inhibitor of phospholipase [PL] C), ACA (an inhibitor of PL-A2), H-89 (an inhibitor of protein kinase [PK] A), calphostin C (an inhibitor of PK-C), L-NAME/L-NORG (inhibitors of nitric oxide synthase), and PD98059 (an inhibitor of mitogen-activated protein kinase [MAPK] kinase). Although U-73122 (0. 1-10 microM), H-89 (0.1-10 microM), calphostin C (0.01-1 microM) and L-NAME/L-NORG (1-100 microM) did not affect TNFalpha-induced PGF2alpha secretion by the cultured cells, ACA (1-100 microM) and PD98059 (0.1-100 microM) inhibited TNFalpha-stimulated PGF2alpha secretion by the cells in a dose-dependent fashion (P < 0.05 or lower). These findings suggest that TNFalpha activates the MAPK and PL-A2 pathways in bovine luteal cells to stimulate PGF2alpha secretion.

Animals↗

PubMed2000

Expression of Indian hedgehog during fracture healing in adult rat femora.

Indian hedgehog (Ihh) has recently been shown to be expressed in prehypertrophic and hypertrophic chondrocytes during embryonic development, and it has been implicated in the regulation of terminal differentiation of chondrocytes. In this paper we examined the expression of Ihh during fracture healing in an adult rat model. A transverse diaphyseal fracture was made in the right femur, and the expression of Ihh in the fracture callus was examined at 1, 2, and 3 weeks after fracture. Northern blot analysis demonstrated the expression of Ihh mRNA in these tissues. Immunohistological analysis detected hedgehog protein in prehypertrophic chondrocytes in the fracture callus at 1 week after fracture. From 2 weeks and on, positive staining was observed in hypertrophic chondrocytes as well. At 3 weeks, some of the osteoblasts close to the endochondral ossification front were also stained positive for hedgehog protein. Our data indicate that Ihh is expressed in chondrocytes and osteoblasts during the process of fracture healing in adult rat femora, suggesting that Ihh, a regulator of endochondral ossification in embryonic development, may also play a role in the regulation of bone formation during fracture repair in adult animals.

Animals↗

PubMed2000

Completion pneumonectomy for patients with recurrent lung cancer: the impact of microvessel density on outcome.

The effectiveness of performing completion pneumonectomy (CP) to treat recurrent lung cancer was assessed in 11 patients, 6 of whom had intrapulmonary recurrence, and 5, locoregional recurrence. The pathological types of cancer were adenocarcinoma in 5 patients, squamous cell carcinomas in 5 patients, and adenoid cystic carcinoma in 1 patient. The pathological stages of the recurrent lung cancer were stage I in 8 patients, stage II in 2 patients, and stage IV in 1 patient. The microvessels in the tumors were also examined and the results compared with those in the microvessels of 61 nonrecurrent tumors. The 1- and 3-year overall survival rates after CP were 54.5% and 27.3%, respectively. The vessel densities within primary and correspondent recurrent tumors were able to be assessed in 9 patients and found to be significantly greater compared with the mean value for the nonrecurrent tumors (P < 0.0001). The vessel densities in the primary and recurrent tumors of 2 of 3 long-term survivors were found to be relatively low. These results indicate the high-grade malignant potential of recurrent tumors, which led us to conclude that the use of CP should be carefully evaluated in patients with recurrence. Assessing the vasculature in primary tumors may be a useful indicator for determining which patients could benefit from CP.

Adenocarcinoma↗

PubMed2000

Selection of surgical approaches for small acoustic neurinomas.

BACKGROUND: The purpose of this study was to evaluate the results of surgery for small acoustic neurinomas at our institute via the middle cranial fossa and retrosigmoid approaches, and to determine the indications for each approach. METHODS: Fifty-three patients with unilateral tumors less than 2 cm in diameter were studied. Surgery was performed via the middle cranial fossa approach in 36 tumors and via the retrosigmoid approach in 17 tumors. RESULTS: The hearing preservation rate was 68% (36/53) in all patients, 93% (14/15) in patients with intracanalicular tumors, 79% (15/19) in patients with tumors less than 1 cm in diameter, and 43% (7/19) in patients with tumors between 1 and 2 cm in diameter. The facial nerve function was excellent or good in 80% (42/53), 74% (11/15), 84% (16/19), and 78% (15/19), respectively. Among the 19 patients with tumors between 1 and 2 cm in diameter, the frequencies of hearing preservation and of excellent or good facial nerve function (47% and 87%, respectively) in the 15 patients approached via the retrosigmoid approach were higher than those (0% and 50%, respectively) in the four patients approached via the middle cranial fossa approach. CONCLUSIONS: We conclude that tumors smaller than 2 cm should be removed because preservation of hearing as well as facial nerve function may be possible in almost all of these patients. Tumors larger than 1 cm should be surgically treated through the retrosigmoid approach.

Adult↗

PubMed2000

Gerontogenes mediate health and longevity in nematodes through increasing resistance to environmental toxins and stressors.

More than 40 mutants in Caenorhabditis elegans have been demonstrated to lead to increased life span (a rigorous, operational test for being a gerontogene) of 20% or more ("Age" mutants). Age mutants alter rate-limiting determinants of longevity; moreover, important genes are identified independent of prior hypotheses as to actual mode of gene action in extending longevity and/or "slowing" aging. Age mutants define as many as nine (possibly) distinct pathways and/or modes of action, as defined by primary phenotype. Three well-studied mutants (age-1, clk-1, and spe-26) alter age-specific mortality rates in characteristic fashions; in age-1 mutants, especially, the changes in mortality rates are quite dramatic. All Age mutants (so far without exception) increase response to several (but not all) stresses, including heat, UV, and reactive oxidants. We have used directed strategies, as well as random mutagenesis, to identify novel genes increasing the worm's ability to resist stress. Two genes (daf-16 and old-1) yield over-expression strains that are stress resistant and long-lived. A variety of approaches to assess transcriptional alterations associated with increased longevity are underway. We suggest that the role of the Age genes in both longevity and stress resistance indicates that a major evolutionary determinant of longevity is the ability to respond to stress.

Aging↗

PubMed2000

Transcriptional mechanisms of chondrocyte differentiation.

With the goal of identifying master transcription factors that control the genetic program of differentiation of mesenchymal cells into chondrocytes, we first delineated a 48-bp chondrocyte-specific enhancer element in the gene for proalpha1(II) collagen (Col2a1), an early and abundant marker of chondrocytes. Our experiments have demonstrated that the HMG-box-containing transcription factor, Sox9 which binds and activates this enhancer element, is required for chondrocyte differentiation and for expression of a series of chondrocyte-specific marker genes including Col2a1, Col9a2, Col11a2 and Aggrecan. In the absence of Sox9 the block in differentiation occurs at the stage of mesenchymal condensation, suggesting the hypothesis that Sox9 might also control expression of cell surface proteins needed for mesenchymal condensation. Since Sox9 also contains a potent transcription activation domain, it is a typical transcription factor. Two other members of the Sox family, L-Sox5 and Sox6, also bind to the 48-bp Col2a1 enhancer and together with Sox9 activate this enhancer as well as the endogenous Col2a1 and aggrecan genes. L-Sox5 and Sox6 have a high degree of sequence identity to each other and are likely to have redundant functions. Except for the HMG-box, L-Sox5 and Sox6 have no similarity to Sox9 and, hence, are likely to have a complementary function to that of Sox9. Our experiments suggest the hypothesis that, like Sox9, Sox5 and Sox6 might also be needed for chondrocyte differentiation. Other experiments, have provided evidence that the Sox9 polypeptide and the Sox9 gene are targets of signaling molecules that are known to control discrete steps of chondrogenesis in the growth plate of endochondral bones. Protein kinase A (PKA) phosphorylation of Sox9 increases its DNA binding and transcriptional activity. Since PKA-phosphorylated-Sox9 is found in the prehypertrophic zone of the growth plate, the same location where the gene for the receptor of the parathyroid hormone-related peptide (PTHrP) is expressed and since PTHrP signaling is mediated by cyclic AMP, we have hypothesized that Sox9 is a target for PTHrP signaling. Other experiments have also shown that fibroblast growth factors (FGFs) increase the expression of Sox9 in chondrocytes in culture and that this activation is mediated by the mitogen-activated protein kinase pathway. These results favor the hypothesis that in achondroplasia, a disease caused by activating mutations in FGF receptor 3, there might also be an abnormally high Sox9 expression.

Animals↗

PubMed2000

Enzymatic formation of ether linkage producing shoyuflavones from genistein and (+/-)-trans-epoxysuccinic acid.

The production mechanism of shoyuflavones, conjugated ethers of isoflavones with tartaric acid and isolated from fermented soy sauce, was studied. In the high molecular weight fraction of the culture extract of Aspergillus oryzae, genistein was transformed into shoyuflavone B in the presence of (+/-)-trans-epoxysuccinic acid but not in the low molecular one. Asp. sojae and Asp. tamarii showed high activity similar to Asp. oryzae but none of Asp. niger, Rhizopus oligosporus, and Mucor praini did. The contents of epoxysuccinic acids in the starting materials of soy sauce and the cultures of various Asp. fungi were determined as dimethyl 2-chloro-3-hydroxysuccinate derivatives by GC-MS. Although epoxysuccinic acids were contained in Asp. oryzae, Asp. sojae, and Asp. tamarii cultures, they were not found in soybeans and wheat. A possible producing mechanism for shoyuflavones by enzymatically conjugating isoflavones to (+/-)-trans-epoxysuccinic acid with ether linkage was suggested.

Aspergillus niger↗