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Biomedical subjects

K Murakami

Publications and source records attributed to K Murakami.

At least 325 records · Page 18Linked to original sources
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

ATF-like element contributes to hepatic activation of human angiotensinogen promoter.

Angiotensinogen is the precursor protein of angiotensin II that is involved in regulating blood pressure and electrolyte homeostasis, and it is mainly synthesized in the liver. In the present study, we analyzed the human angiotensinogen proximal promoter region by means of Chloramphenicol acetyltransferase assays, and suggested that the region from -106 to +44 is sufficient for hepatoma cell line (HepG2)-specific expression. Electrophoretic mobility shift assays using ALE (ATF-like element, -102 to -87) fragment identified CREB/ATF family nuclear factors and novel ones, ALF (ALE-binding factor). The deletion and in vivo competition of ALE decreased the human angiotensinogen promoter activity. Furthermore, the heterologous promoter analysis demonstrated that ALE acts as a HepG2-dependent activating element. These results indicate that ALE plays an important role in hepatic expression of human angiotensinogen gene.

Activating Transcription Factors↗
PubMed1997

Differential action of AGCF2 upon cell type-dependent expression of human angiotensinogen gene.

To investigate the regulatory mechanisms of human angiotensinogen (ANG) gene expression in the brain, we analyzed the 1.3-kb promoter by transfection studies and gel shift assays. The region from -106 to +44 was sufficient for promoter activity in glioblastoma cells, and multiple nuclear factors including AGCF2 (human ANG core promoter binding factor 2) bound within this 150-bp region. The mutations within AGCF2-binding elements decreased the transcriptional activity in glioblastoma cells but rather increased it in hepatoma cells. These results indicate that AGCF2 has a differential function between these cells and contributes to the glia-dependent angiotensinogen promoter activity.

Angiotensinogen↗
PubMed1997

9,13-di-cis-Retinoic acid induces the production of tPA and activation of latent TGF-beta via RAR alpha in a human liver stellate cell line, LI90.

We studied the mechanism by which 9,13-di-cis-retinoic acid (9,13dcRA), a novel and endogenous stereoisomer of all-trans-RA, induces TGF-beta formation in a human liver stellate cell line, LI90. 9,13dcRA induced the expression of RAR alpha and RARbeta, enhanced the production of tissue-type plasminogen activator (tPA), thereby, surface plasmin levels, and induced the activation of latent TGF-beta. Similar effects were obtained with RAR alpha-selective retinoid, but not with RARbeta- or RARgamma-selective retinoid, and the induction was inhibited by RAR alpha-selective antagonist. These results suggest that 9,13dcRA up-regulates tPA expression, resulting in the formation of TGF-beta by LI90 cells, at least in part, via induction and activation of RAR alpha.

Animals↗
PubMed1997

A novel proximal element mediates the regulation of mouse Ren-1C promoter by retinoblastoma protein in cultured cells.

The protein product of the retinoblastoma susceptibility gene, RB, is a nuclear phosphoprotein that modulates transcription of genes involved in growth control via interactions with transcription factors. Renin is a rate-limiting enzyme of the renin-angiotensin system that regulates blood pressure and water-electrolyte balance. Renin gene expression is regulated in a tissue-specific and developmentally linked manner. Similarly, the expression of RB is controlled in a differentiation-linked manner. Thus, to investigate whether RB is involved in the regulation of renin gene expression, we examined the effects of RB on transcriptional activity of the mouse renin (Ren-1C) promoter. The Ren-1C promoter contains two transcriptionally important elements; the RU-1 (-224 to -138) and RP-2 (-75 to -47) elements. RB activated the Ren-1C promoter in human embryonic kidney cells. The promoter element responsible for RB-mediated transcriptional regulation was the RP-2 element. The results of DNA-protein binding experiments showed that RB increased nuclear binding activity to the RP-2 element, and site-directed mutation which disrupted binding of nuclear factors to the RP-2 element markedly reduced RB-mediated activation of Ren-1C promoter in human embryonic kidney cells. These results indicate that the RP-2 element plays an important role in RB-mediated transcriptional regulation of Ren-1C promoter activity in human embryonic kidney cells, thereby suggesting an interesting mechanism by which RB may modulate the renin-angiotensin system.

Activating Transcription Factors↗
PubMed1997

Stretch-induced MAP kinase activation in cardiomyocytes of angiotensinogen-deficient mice.

The renin-angiotensin system plays an important role in the hypertrophic responses in cardiac myocytes through the activation of signal transduction pathways and expression of oncogenes. In the present study, we examined mechanical stretch-induced activation of mitogen-activated protein kinases (MAP kinases) using cultured cardiac myocytes derived from neonatal angiotensinogen gene deficient mice (Agt-/-) and neonatal wild type mice (Agt+/+). Within 2 minutes of being added to cardiac myocytes, angiotensin II activated MAP kinases and the response was completely blocked by pretreatment of the cardiac myocytes with CV-11974, a selective antagonist of angiotensin II type 1 receptors. Interestingly, mechanical stretch resulted in significantly greater activation of MAP kinases in Agt-/- cardiac myocytes than in Agt+/+ cardiac myocytes. CV-11974 failed to suppress the stretch-induced activation of MAP kinases in Agt-/- cardiac myocytes while it inhibited the activation in Agt+/+ cardiac myocytes. BQ123, an endothelin type A receptor antagonist, had no effect on stretch-induced activation of MAP kinases in cardiac myocytes from either mouse strain. These results suggest that cardiac RAS is important for stretch-induced MAP kinase activation in Agt+/+ cardiac myocytes; however, angiotensin II is not indispensable for mechanical stretch-induced activation of MAP kinases in Agt-/- cardiac myocytes.

Angiotensin II↗
PubMed1997

Reduction of CuZn-superoxide dismutase activity exacerbates neuronal cell injury and edema formation after transient focal cerebral ischemia.

Apoptotic neuronal cell death has recently been associated with the development of infarction after cerebral ischemia. In a variety of studies, CuZn-superoxide dismutase (CuZn-SOD) has been shown to protect the brain from ischemic injury. A possible role for CuZn-SOD-related modulation of neuronal viability is suggested by the finding that CuZn-SOD inhibits apoptotic neuronal cell death in response to some forms of cellular damage. We evaluated this possibility in the model of transient focal cerebral ischemia in mice bearing a disruption of the CuZn-SOD gene (Sod1). Homozygous mutant (Sod1 -/-) mice had no detectable CuZn-SOD activity, and heterozygous mutants (Sod1 +/-) showed a 50% decrease compared with wild-type mice. Sod1 -/- mice showed a high level of blood-brain barrier disruption soon after 1 hr of middle cerebral artery occlusion and 100% mortality at 24 hr after ischemia. Sod1 +/- mice showed 30% mortality at 24 hr after ischemia, and neurological deficits were exacerbated compared with wild-type controls. The Sod1 +/- animals also had increased infarct volume and brain swelling, accompanied by increased apoptotic neuronal cell death as indicated by the in situ nick-end labeling technique to detect DNA fragmentation and morphological criteria. These results suggest that oxygen-free radicals, especially superoxide anions, are an important factor for the development of infarction by brain edema formation and apoptotic neuronal cell death after focal cerebral ischemia and reperfusion.

Animals↗
PubMed1997

Identification and characterization of receptor for cytokine-induced neutrophil chemoattractant-3 on rat neutrophils.

Cytokine-induced neutrophil chemoattractants (CINCs), members of alpha-chemokines, are considered to be major neutrophil chemotactic factors in rats. Recombinant CINC-3/rat macrophage inflammatory protein-2 (MIP-2) having an additional tyrosine residue at the carboxyl terminus (CINC-3-Tyr) was constructed, purified, radiolabeled with 125I, and used for binding studies. The specific binding of 125I-labeled CINC-3-Tyr (125I-CINC-3-Tyr) to rat neutrophils reached a plateau after approximately 60 min at 4 degrees C. This binding of 125I-CINC-3-Tyr could be reversed by adding an excess amount of unlabeled CINC-3-Tyr. Scatchard analysis revealed approximately 12,000 binding sites per cell on rat neutrophils with an apparent Kd value of 120 pM. Chemical cross-linking experiments suggested that the rat neutrophil CINC-3 receptor is a mass of approximately 69 kDa. Taken together, our results strongly suggest that rat neutrophils express a high affinity receptor for CINC-3.

Animals↗
PubMed1997

Reperfusion following focal cerebral ischemia alters distribution of neuronal cells with DNA fragmentation in mice.

To clarify the role of reperfusion in DNA damage following focal cerebral ischemia, we determined the distribution of cells with DNA fragmentation following permanent or transient focal ischemia. Less DNA-damaged cells were observed in the permanent than in the transient group in the ischemic core, but there was no difference in the boundary zone. We conclude that reperfusion following transient ischemia may exacerbate neuronal death following DNA damage.

Animals↗
PubMed1997

Surface modifications of titanium in calcium-ion-containing solutions.

The surface of titanium was modified in calcium-ion-containing solutions to improve bone conductivity. Three kinds of aqueous solutions for the modification were prepared using calcium nitrate, calcium chloride, and calcium oxide. The pH values of the solutions were 3.9, 7.4, and 12.6, respectively. At first, anatase powder was immersed in the solutions of 7 days to confirm the effect of the solutions on the modification. As a result, calcium titanate was formed on the anatase powder, indicating that the titanium surface was possibly modified by the solutions. Titanium plates were then immersed in the solutions at ambient temperature for 7 days, washed by deionized water, and dried. X-ray photo-electron spectroscopy of surface-modified titanium revealed that the surface-modified layer contained calcium hydroxide and/or calcium titanate. The surface-modified layer in which titanium was modified was thickest in this order: calcium oxide, calcium chloride, and calcium nitrate solutions. Apatite was formed on the surface-modified titanium in Hanks' solution while apatite was undetected on unmodified titanium. Therefore, the surface modification of titanium may improve bone conductivity. The amount of apatite corresponded to the thickness of the surface-modified layer and the amount of calcium in the layer.

Apatites↗
PubMed1997

The two alpha subunits of Escherichia coli RNA polymerase are asymmetrically arranged and contact different halves of the DNA upstream element.

RNA polymerase core enzyme of Escherichia coli is composed of two alpha subunits and one each of the beta and beta' subunits. The C-terminal domain of the RNA polymerase alpha subunit plays a key role in molecular communications with class I transcription factors and upstream (UP) elements of promoter DNA, using the same protein surface. To identify possible differences in the functional roles of the two alpha subunits, we have developed a reconstitution method for hybrid RNA polymerases containing two distinct alpha subunit derivatives in a defined orientation ("oriented alpha-heterodimer"). The binding sites of two alpha C-terminal domains on the UP element DNA were determined by hydroxyl radical-based DNA cleavage mediated by (p-bromoacetamidobenzyl)-EDTA x Fe, which was bound at Cys-269 on the UP recognition surface of one or both alpha subunits. The results clearly indicated that the two alpha subunits bind in tandem to two helix turns of the rrnBP1 UP element, and that the beta'-associated alpha subunit is bound to the promoter-distal region.

Binding Sites↗
PubMed1997

Leukocyte depletion and ONO-5046, a specific inhibitor of granulocyte elastase, prevent a stress-induced decrease in gastric prostaglandin I2 in rats.

To examine whether activated leukocytes may impair the endothelial production of prostaglandin (PG) I2, an important cytoprotective agent in gastric mucosa, we investigated the effects of leukocyte depletion and ONO-5046, a specific inhibitor of granulocyte elastase, on the gastric level of this prostaglandin and gastric mucosal injury in rats subjected to water-immersion restraint stress (WIR). Gastric 6-keto-PGF1 alpha was increased after 30 min of WIR, followed by a decrease to below baseline after 6 h of stress. Gastric levels of 6-keto-PGF1 alpha in leukopenic animals or animals pretreated with ONO-5046 after 1 h of stress were significantly higher than those of controls, levels after 6 h of stress were not lower than those preceding stress. Leukocytopenia or ONO-5046 significantly inhibited WIR-induced gastric mucosa lesion formation. Iloprost, a stable derivative of PGI2, prevented stress-induced lesions. These results suggest that activated leukocytes may play an important role in stress-induced gastric mucosal lesion formation by inhibiting production of PGI2.

6-Ketoprostaglandin F1 alpha↗
PubMed1997

Identification of the cDNA for human red blood cell-specific hexokinase isozyme.

A unique cDNA for hexokinase (HK) was identified from poly(A)+ RNA of human reticulocytes by anchored polymerase chain reaction. This appeared to represent the cDNA for the red blood cell (RBC)-specific HK isozyme (HKR) described in our previous study (Murakami et al: Blood 75:770, 1990). Its nucleotide sequence was identical to HKI cDNA except for the 5' extreme end. It lacked the first 62 nucleotides of the HKI coding region: instead, it contained a unique sequence of 60 nucleotides at the beginning of the coding sequence as well as another unique sequence upstream of the putative translation initiation site. It lacked the porin-binding domain which facilitates binding to the mitochondria, thus explaining the exclusive cytoplasmic localization of HKR. It was the major cDNA derived from reticulocytes, consistent with the observation that HKR activity is predominant in reticulocytes. Northern blot analysis showed that it was expressed in the reticulocytes and in the K562 erythroleukemic cell line, but not in a lymphocytic cell line. In the extract of K562 cells, HKR activity co-eluted with the HKR of human RBCs on a MonoQ column (Pharmacia, Piscataway, NJ) chromatography, using a salt gradient elution. The separate genetic control of the RBC-specific HK isozyme explains the clinical reports of two types of HK deficiency, one in which the HK activity was reduced exclusively in the RBC (HKR defect) and another with general decrease of HK activity in several tissues (HKI defect).

Amino Acid Sequence↗
PubMed1997

Apoptosis in the striatum of rats following intraperitoneal injection of 3-nitropropionic acid.

The present study investigated the mechanism of cellular degeneration within the striatum following administration of the mitochondrial toxin, 3-nitropropionic (3-NP) acid. Internucleosomal fragmentation typical of apoptosis was present in the DNA of cells from the striatum of 3-NP-treated rats. DNA fragmentation was also evident in this region by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling. The data suggest that striatal cells die by apoptosis following administration of 3-NP.

Animals↗
PubMed1997

Effect of human urinary thrombomodulin on endotoxin-induced intravascular coagulation and pulmonary vascular injury in rats.

Adult respiratory distress syndrome (ARDS) and disseminated intravascular coagulation (DIC) are serious complications of sepsis. Thrombomodulin, an important endothelial anticoagulant, binds thrombin to generate activated protein C (APC). To determine whether thrombomodulin purified from human urine (urinary thrombomodulin, UTM) is useful for the treatment of DIC and ARDS in sepsis, we examined the effect of UTM on endotoxin (ET)-induced coagulation abnormalities and pulmonary vascular injury in rats. Intravenous administration of UTM prevented the ET-induced pulmonary accumulation of leukocytes and the increase in pulmonary vascular permeability, as well as ET-induced histological changes such as leukocyte infiltration and pulmonary interstitial edema. On the other hand, dansyl-Glu-Gly-Arg-chloromethyl ketone-treated factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, did not prevent these effects of ET. UTM did not prevent ET-induced pulmonary accumulation of leukocytes and pulmonary vascular injury in rats pretreated with DEGR-Xa. Our findings suggest that UTM attenuates ET-induced coagulation abnormalities and pulmonary vascular injury. Furthermore, the latter effect may be dependent on the capacity of UTM to activate protein C.

Amino Acid Chloromethyl Ketones↗
PubMed1997

Plasma levels of soluble E-selectin in patients with disseminated intravascular coagulation.

The plasma level of soluble E-selectin (sE) reflects the activation of endothelial cells induced by cytokines such as tumor necrosis factor-alpha and interleukin-1 in vitro. These cytokines are important in the development of coagulation abnormalities in patients with sepsis. We compared the plasma levels of sE in patients with infections suspected of having disseminated intravascular coagulation (DIC) (n = 33) and in patients with underlying disorders other than infections, including solid tumors (n = 28), obstetric disorders (n = 13), hematologic malignancies (n = 13), and liver disease (n = 9), to clarify the involvement of cytokines in the development of coagulation abnormalities in patients with sepsis. Plasma levels of sE in patients with infection were significantly higher than in patients with the other underlying disorders. The plasma level of sE was also significantly higher in patients with infection with DIC (114.6 +/- 77.9 ng/ml, n = 21) than in patients with infection without DIC (54.5 +/- 53.1 ng/ml, n = 12, P < 0.02). There was no significant difference in sE level between patients with the other underlying disorders with and without DIC. The plasma level of sE was significantly correlated with the serum level of FDP(E) in patients with infection. The plasma level of sE was significantly higher in patients with infection with organ failure compared to patients without organ failure. There was no significant difference between patients with the other underlying disorders with and without organ failure. Plasma levels of tumor necrosis factor-alpha and interleukin-6 were detected in only 12.1% and 20.0% of patients with infections, respectively. These observations strongly suggest that plasma levels of sE reflect the activation of endothelial cells induced by cytokines, which may lead to DIC and organ failure in the presence of sepsis. Furthermore, determination of plasma level of sE may be useful for detecting the endothelial activation induced by cytokines in the pathologic conditions of sepsis, even when plasma levels of cytokines cannot be detected.

Biomarkers↗
PubMed1997

Early bone formation around calcium-ion-implanted titanium inserted into rat tibia.

Rat tibia tissue into which calcium ion (Ca2+)-implanted titanium was surgically placed was histologically analyzed to investigate the performance of the Ca(2+)-implanted titanium as a biomaterial. Calcium ions were implanted into only one side of titanium plates at 10(17) ions/cm2 and the Ca(2+)-treated titanium was surgically implanted into rat tibia for 2, 8, and 18 days. Tetracycline and calcein were used as hard-tissue labels. After excision of the tibia, the tissues were fixed, stained, embedded in polymethyl methacrylate, and sliced. The specimens were observed using a fluorescence microscope. A larger amount of new bone was formed on the Ca(2+)-treated side than on the untreated side, even at 2 days after surgery. In addition, part of the bone made contact with the Ca2(+)-treated surface. On the other hand, bone formation on the untreated side was delayed and the bone did not make contact with the surface. Mature bone with bone marrow formed in 8 days. Neither macrophage nor inflammatory cell infiltration was observed. The results indicated that Ca(2+)-implanted titanium is superior to titanium alone for bone conduction.

Animals↗