Search PubMed⌕ Search

Biomedical subjects

M Murakami

Publications and source records attributed to M Murakami.

At least 199 records · Page 11Linked to original sources
PubMed2000

Analysis of radiation damage of DNA by atomic force microscopy in comparison with agarose gel electrophoresis studies.

DNA damage induced with ionizing radiation is considered one of the main causes of cell inactivation. Several methods including gel electrophoresis, pulsed-field gel electrophoresis, neutral filter elution method, neutral sedimentation and electron microscopy have been applied to analyze this type of DNA damage. A new method employing an atomic force microscope (AFM) for nanometer-level-structure analysis of DNA damage induced with gamma-irradiation is introduced in this report. Structural changes of plasmid DNA on a molecular size scale of about 3 kbp were visually analyzed by AFM after irradiation with 60Co gamma-rays at doses of 1.9, 5.6, and 8.3 kGy. Three forms of plasmid DNA, closed circular (intact DNA), open circular (DNA with a single strand break) and linear form (DNA with a double strand break) were visualized by dynamic force mode AFM after gamma-irradiation. The torsional feature of the plasmid DNA was visualized better with AFM than with a transmission electron microscope (TEM). All three forms of plasmid DNA were observed in the sample irradiated with gamma-rays at the dose of 1.9 kGy. Open circular and linear forms were observed in the samples irradiated with gamma-rays at doses of 5.6 and 8.3 kGy, though no closed circular form was observed. A shortening of the length of a linear form of DNA irradiated with 5.6 and 8.3 kGy gamma-rays was observed by AFM. Structural changes of DNA after gamma-irradiation were visualized by AFM at nanometer level resolution. In addition, shortening of the length of the linear form of DNA after radiation exposure was observed by AFM.

Cobalt Radioisotopes↗
PubMed2000

Functional crosstalk between phospholipase D(2) and signaling phospholipase A(2)/cyclooxygenase-2-mediated prostaglandin biosynthetic pathways.

We performed reconstitution analyses of functional interaction between phospholipase A(2) (PLA(2)) and phospholipase D (PLD) enzymes. Cotransfection of HEK293 cells with cytosolic (cPLA(2)) or type IIA secretory (sPLA(2)-IIA) PLA(2) and PLD(2), but not PLD(1), led to marked augmentation of stimulus-induced arachidonate release. Interleukin-1-stimulated arachidonate release was accompanied by prostaglandin E(2) production via cyclooxygenase-2, the expression of which was augmented by PLD(2). Conversely, activation of PLD(2), not PLD(1), was facilitated by cPLA(2) or sPLA(2)-IIA. Thus, our results revealed functional crosstalk between signaling PLA(2)s and PLD(2) in the regulation of various cellular responses in which these enzymes have been implicated.

Animals↗
PubMed2000

Antinociceptive effect of cilnidipine, a novel N-type calcium channel antagonist.

We investigated the antinociceptic effects of cilnidipine, a dihydropyridine derivative which acts on both L- and N-type voltage-dependent calcium channels, in mice. Intrathecally injected cilnidipine showed significant analgesic effect in formalin test. Cilnidipine significantly suppressed N-type currents in dorsal root ganglion (DRG) cells. Our findings apparently support the idea that cilnidipine attenuates synaptic neurotransmission by inhibiting N-type calcium channels in DRG neurons.

Animals↗
PubMed2000

Distinct roles of two intracellular phospholipase A2s in fatty acid release in the cell death pathway. Proteolytic fragment of type IVA cytosolic phospholipase A2alpha inhibits stimulus-induced arachidonate release, whereas that of type VI Ca2+-independent phospholipase A2 augments spontaneous fatty acid release.

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha; type IVA), an essential initiator of stimulus-dependent arachidonic acid (AA) metabolism, underwent caspase-mediated cleavage at Asp(522) during apoptosis. Although the resultant catalytically inactive N-terminal fragment, cPLA(2)(1-522), was inessential for cell growth and the apoptotic process, it was constitutively associated with cellular membranes and attenuated both the A23187-elicited immediate and the interleukin-1-dependent delayed phases of AA release by several phospholipase A(2)s (PLA(2)s) involved in eicosanoid generation, without affecting spontaneous AA release by PLA(2)s implicated in phospholipid remodeling. Confocal microscopic analysis revealed that cPLA(2)(1-522) was distributed in the nucleus. Pharmacological and transfection studies revealed that Ca(2+)-independent PLA(2) (iPLA(2); type VI), a phospholipid remodeling PLA(2), contributes to the cell death-associated increase in fatty acid release. iPLA(2) was cleaved at Asp(183) by caspase-3 to a truncated enzyme lacking most of the first ankyrin repeat, and this cleavage resulted in increased iPLA(2) functions. iPLA(2) had a significant influence on cell growth or death, according to cell type. Collectively, the caspase-truncated form of cPLA(2)alpha behaves like a naturally occurring dominant-negative molecule for stimulus-induced AA release, rendering apoptotic cells no longer able to produce lipid mediators, whereas the caspase-truncated form of iPLA(2) accelerates phospholipid turnover that may lead to apoptotic membranous changes.

Apoptosis↗
PubMed2000

Plasma endothelin-1 and thrombomodulin levels in coronary sinus during right atrial pacing and percutaneous transluminal coronary angioplasty.

Increases in the levels of plasma endothelins (ETs) have been reported after percutaneous transluminal coronary angioplasty (PTCA). To examine the mechanism of this increase, we measured plasma endothelin-1 (ET-1) and thrombomodulin (TM) levels in both the Valsalva sinus (VAL) and the great cardiac vein (GCV) together with oxygen saturation of the GCV (Sat.GCVO2) during right atrial pacing and PTCA. Thirty patients with stenoses in the left anterior descending coronary arteries were enrolled. A fiberoptic pulmonary artery catheter was placed in GCV for monitoring Sat.GCVO2, and blood sampling was repeated before and after each procedure. ET-1 did not increase during pacing, but after PTCA it significantly increased from basal levels to 24.4+/- 8.3 pg/ml in GCV (P<0.01) and 19.3 +/-7.4 in VAL (P<0.05). Basal TM levels in GCV and VAL were significantly higher in diabetic than in non-diabetic patients, but TM did not change during pacing and PTCA. Sat.GCVO2 significantly decreased from the basal level during pacing and PTCA. We speculate that direct endothelial cell damage is more responsible for the increase of ET-1 during PTCA than myocardial ischemia. Our data indicate that ET-1 may be a useful marker for acute endothelial damage, while TM reflects only chronic and general damage of endothelial cells.

Aged↗
PubMed2000

Control of homeostasis of CD8+ memory T cells by opposing cytokines.

Memory T cells maintain their numbers for long periods after antigen exposure. Here we show that CD8+ T cells of memory phenotype divide slowly in animals. This division requires interleukin-15 and is markedly increased by inhibition of interleukin-2 (IL-2). Therefore, the numbers of CD8+ memory T cells in animals are controlled by a balance between IL-15 and IL-2.

Animals↗
PubMed2000

Involvement of protein kinase C in taxol-induced polyploidization in a cultured sarcoma cell line.

Taxol was found to inhibit the proliferation and to induce the polyploidization of cultured methylcholanthrene-induced sarcoma cells (Meth-A cells). To investigate whether protein kinase C is involved in taxol-induced polyploidization, phorbol 12-myristate 13-acetate (PMA), which regulates the activity of protein kinase C, was used along with taxol to treat the cells. We found that PMA did not interfere with the proliferation and did not induce polyploidization by itself. However, at low concentration, taxol, which by itself did not induce polyploidization, clearly induced polyploidization in the presence of PMA. To explore the mechanism by which PMA potentiates polyploidization, the levels of the G1 checkpoint-related proteins cyclin E and cdk2, and those of the G2 checkpoint-related proteins cyclin B and cdc2 were determined by flow cytometry. We found that both G1 and G2 checkpoint-related proteins increased during the induction of polyploidization. To verify the relationship between protein kinase C and tubulin polymerization, flow cytometry was used to determine the total content of tubulin protein, and morphological observation was used to examine spindle organization. PMA did not affect the taxol-induced increase in tubulin protein, but markedly potentiated taxol-induced spindle disorganization. These findings suggest that protein kinase C plays an important role in regulating the induction of polyploidization in Meth-A cells.

Animals↗
PubMed2000

P2 Purinoceptor expression and functional changes of hypoxia-activated cultured rat retinal microglia.

P(2) purinoceptors appear to modulate microglia function, but their role in hypoxic microglia has not been investigated. We examined in postnatal rat retinal microglia cultured under hypoxic (1% oxygen) condition, their P2 expression, proliferation and cytokine release in the presence or absence of the P2 receptor agonists and antagonists. Fura-2 fluorescence measurements of intracellular Ca(2+) rises to P2 receptor agonists and antagonists indicated that both P(2U) and P(2Z) were expressed in hypoxic microglia. Hypoxia induced BrdU incorporation and release of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) as well. The P(2U) agonist, UTP, maintained the BrdU incorporation, whereas the P(2Z) agonist, BzATP, suppressed it, but significantly enhanced IL-1beta and TNF-alpha release, suggesting that the P(2U) response may underlie the mitotic activity, and that of P(2Z), the IL-1beta and TNF-alpha release of hypoxia-activated microglia.

Animals↗
PubMed2000

Blocking the CD28-B7 T-cell costimulatory pathway abrogates the development of obliterative bronchiolitis in a murine heterotopic airway model.

BACKGROUND: CTLA4IgG that binds to B7 effectively inhibits the signaling of CD28/B7 pathway and induces antigen-specific T-cell unresponsiveness in vitro and in vivo. We examined whether the development of obliterative bronchiolitis in a murine heterotopic airway transplantation model is T cell dependent and whether CTLA4IgG abrogates the development of obliterative bronchiolitis. METHODS: Tracheae with main bronchi from C3H/He (H2k), BALB/C (H2d), or C57BL/6 (H2b) mice were transplanted heterotopically into subcutaneous pockets on the backs of BALB/C or BALB/C nu/nu mice on day 0. Recipient mice were untreated or intraperitoneally treated with either CTLA4IgG or human IgG with different time and dose schedules. RESULTS: The development of obliterative bronchiolitis, which leads to luminal obliteration by fibrous tissue in a murine heterotopic airway transplantation model, was T cell dependent and the development of obliterative bronchiolitis was significantly abrogated by the CTLA4IgG treatment. However, the normal ciliated columnar respiratory epithelial cells in allografts were lost and replaced by flattened attenuated epithelial cells even after the CTLA4IgG treatment. We further demonstrated that CTLA4IgG treatment did not result in the induction of donor-specific unresponsiveness. CONCLUSIONS: We demonstrated that the development of obliterative bronchiolitis in a murine heterotopic airway model involves both CD28/B7-dependent and -independent processes. The luminal obliteration by fibrous tissue is clearly CD28/B7 dependent and can be inhibited by CTLA4IgG. The luminal obliteration of allografted trachea by fibrous tissues and the loss of ciliated columnar respiratory epithelial cells represent distinct disease processes.

Abatacept↗
PubMed2000

Impurity-induced suppression of core turbulence and transport in the DIII-D tokamak

Turbulence is significantly reduced in a tokamak plasma as a result of neon seeding of an L-mode discharge. Correspondingly, confinement is improved and cross-field ion thermal transport reduced. Fully saturated turbulence in the range 0.1</=k( perpendicular)rho(s)</=0. 6 is measured at rho = 0.7 and exhibits a factor of 5 reduction in total power after neon injection, with almost complete suppression for k( perpendicular)rho(s)>0.35. These observations are consistent with a reduction in the calculated linear growth rate for k( perpendicular)rho(s)>0.5 and an increase in the measured ExB flow shearing rate.

Journal Article↗
PubMed2000

Exogenously added human group X secreted phospholipase A(2) but not the group IB, IIA, and V enzymes efficiently release arachidonic acid from adherent mammalian cells.

Mammalian secreted phospholipases A(2) (sPLA2s) comprise a group of at least eight enzymes, including the recently identified group X sPLA2. A bacterial expression system was developed to produce human group X sPLA2 (hGX). Inhibition studies show that the sPLA2 inhibitor LY311727 binds modestly more tightly to human group IIA sPLA2 than to hGX and that a pyrazole-based inhibitor of group IIA sPLA2 is much less active against hGX. The phospholipid head group preference of vesicle-bound hGX was determined. hGX binds tightly to phosphatidylcholine vesicles, which is thought to be required to act efficiently on cells. Tryptophan 67 hGX makes a significant contribution to interfacial binding to zwitterionic vesicles. As little as 10 ng/ml hGX releases arachidonic acid for cyclooxygenase-2- dependent prostaglandin E(2) generation when added exogenously to adherent mammalian cells. In contrast, human group IIA, rat group V, and mouse group IB sPLA2s are virtually inactive at releasing arachidonate when added exogenously to adherent cells. Dislodging cells from the growth surface enhances the ability of all the sPLA2s to release fatty acids. Studies with CHO-K1 cell mutants show that binding of sPLA2s to glycosaminoglycans is not the basis for poor plasma membrane hydrolysis by group IB, IIA, and V sPLA2s.

Animals↗
PubMed2000

Carbachol-induced [Ca2+]i increase, but not activation of protein kinase C, stimulates exocytosis in rat parotid acini.

1. A column perfusion system was applied to rat parotid acinar cells to clarify the roles of Ca2+ and protein kinase C (PKC) in the mechanisms of carbachol (CCh)-induced amylase secretion. 2. CCh evoked a biphasic response of amylase secretion with an initial rapid and large peak that reached maximum at about 10 s followed by a sustained plateau. The time profile and the dose-response relationship paralleled with those of cytosolic free Ca2+ concentration ([Ca2+]i). 3. The CCh-induced sustained response of amylase secretion maintained by Ca2+ influx into cells was ATP dependent, while the initial peak response regulated by Ca2+ released from InsP3-sensitive stores was relatively ATP independent. 4. Restoration of extracellular Ca2+ during continuous stimulation with CCh in Ca(2+)-free medium evoked a second rapid and large peak of amylase secretion. 5. Phorbol 12,13-dibutyrate (PDBu) potentiated the CCh-induced amylase secretion in both the initial peak and the sustained plateau without enhancing CCh-induced [Ca2+]i changes. 6. PKC inhibitors such as Ro 31-8220 inhibited the potentiating effect of PDBu but only slightly reduced amylase secretion induced by CCh alone. 7. These results suggest that a CCh-induced rise in [Ca2+]i triggers the final fusion and/or exocytosis of amylase secretion. CCh also has some ability to promote ATP-dependent priming of secretory granules that, together with Ca2+ influxed into cells, contributes to the CCh-induced sustained plateau of amylase secretion. PDBu-induced activation of PKC promotes the priming of secretory granules, thereby enhancing the efficacy for Ca2+ to trigger fusion/exocytosis.

Adenosine Triphosphate↗
PubMed2000

Determination of 18alpha-glycyrrhizin and 18beta-glycyrrhizin in dog plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic method has been developed for the separation and determination of 18alpha-glycyrrhizin (alpha-GZ) and 18beta-glycyrrhizin (beta-GZ) in dog plasma. The two compounds were separated on a reversed-phase column and detected by UV absorption at 254 nm. The mobile phase was a mixture of water-methanol-60% perchloric acid (45:55:0.5, v/v) and was adjusted to pH 8.0 with 25% ammonia solution. Indomethacin was added to the plasma as an internal standard. Methanol was selected for the extraction of both the compounds and internal standard. Alpha-GZ and beta-GZ could be precisely determined in concentration of 1 mg/ml in a 0.1 ml sample.

Ammonia↗
PubMed2000

Identification of a cellular protein that functionally interacts with the C2 domain of cytosolic phospholipase A(2)alpha.

Cytosolic phospholipase A(2) (cPLA(2)) alpha plays critical roles in lipid mediator synthesis. We performed far-Western analysis and identified a 60-kDa protein (P60) that interacted with cPLA(2)alpha in a Ca(2+)-dependent manner. Peptide microsequencing revealed that purified P60 was identical to vimentin, a major component of the intermediate filament. The interaction occurred between the C2 domain of cPLA(2)alpha and the head domain of vimentin. Immunofluorescence microscopic analysis demonstrated that cPLA(2)alpha and vimentin colocalized around the perinuclear area in cPLA(2)alpha-overexpressing human embryonic kidney 293 cells following A23187 stimulation. Forcible expression of vimentin in vimentin-deficient SW13 cells augmented A23187-induced arachidonate release. Moreover, overexpression of the vimentin head domain in rat fibroblastic 3Y1 cells exerted a dominant inhibitory effect on arachidonate metabolism, significantly reducing A23187-induced arachidonate release and attendant prostanoid generation. These results suggest that vimentin is an adaptor for cPLA(2)alpha to function properly during the eicosanoid-biosynthetic process.

Animals↗
PubMed2000

Characteristics of intestinal absorption and disposition of glycyrrhizin in mice.

As basic studies to apply an intestinal pressure-controlled colon delivery capsule (PCDC) for glycyrrhizin (GZ), the characteristics of intestinal absorption and disposition of GZ were investigated in mice. In the in vivo study, after intravenous (iv) administration of GZ, 10 mg/kg dose, plasma GZ disappeared from the systemic circulation with t(1/2(alpha)) of 0.0063 h, thereafter, it slowly declined with t(1/2(beta)) of 15.23 h. The area under the plasma drug concentration versus time curve (AUC) values of iv (10 mg/kg), intracolonic (50 mg/kg) and intraduodenum (50 mg/kg) administrations were 115.1, 16.7 and 2.7 microgh/mL, respectively. The AUC values of plasma glycyrrhetic acid (GA), a degradation product after intracolonic and intraduodenum administrations were 2.8 and 8.4 microgh/mL, respectively. In the in situ closed loop study, the concentrations of GZ in plasma and liver after intracolonic administration were significantly increased (p<0.05) in comparison with those after intrajejunum or intraileum administration, while the concentration of GA in plasma and liver after intracolonic administration had trends to increase. These observations clearly suggest that the intracolonic administration is a useful way to improve the oral bioavailability of GZ and to enhance its pharmacological efficacy. These pharmacokinetic results of GZ suggest that GZ is a subject drug to be applied for the PCDC system we previously developed. The PCDCs formulation of GZ will enable us to carry GZ to the colon and enhance the oral bioavailability of GZ.

Animals↗
PubMed2000

Abortive alloantigen presentation by donor dendritic cells leads to donor-specific tolerance: a study with a preoperative CTLA4lg inoculation.

Donor dendritic cells (DCs) within allografts initiate the induction of an allospecific T cell response, while an abortive alloantigen presentation by DCs may induce allospecific unresponsiveness. We thus investigated the tolerogenic effect of donor DCs that were made incompetent in alloantigen presentation by treatment of CTLA4Ig. When we treated rats with donor DCs (2 x 10(6)/rat i.v.) on the preoperative day, nine rejected allografts in an accelerated manner (5.0 +/- 2.2 vs. 8.2 +/- 1.6 days in the control group). Preoperative inoculation of DCs pulsed with CTLA4Ig, a procedure which suppresses an allogeneic mixed lymphocyte reaction (MLR), also provoked an accelerated rejection (5.6 +/- 1.7 days). When DCs and CTLA4Ig (500 microg/rat i.p. on days -9, -7 and -5) were concomitantly inoculated, allograft survival was significantly prolonged (>38.7 +/- 40.0 days); a preoperative CTLA4Ig inoculation alone failed to do so (7.5 +/- 1.2 days). Long-term graft survivors tolerated skin grafts from the donor but not from those from a third party. These results indicate that abortive alloantigen presentation by donor DCs, upon which an accessory signal pathway is suppressed by CTLA4Ig, leads to prolonged graft survival and donor-specific tolerance.

Abatacept↗
PubMed2000

Esophageal intramural pseudodiverticulosis associated with esophageal perforation.

We report a rare case of esophageal intramural pseudodiverticulosis with lower esophageal stricture which perforated into the peritoneal cavity after the patient vomited. A 61-year-old man was admitted with severe chest and epigastric pain after dysphagia and vomiting. Under a diagnosis of upper gastrointestinal perforation, laparotomy was performed. The anterior wall of the abdominal esophagus was found to have ruptured, and proximal gastrectomy with abdominal esophagectomy was performed. Histological examination revealed esophageal intramural pseudodiverticulosis with esophageal stricture distal to the site of rupture, and postoperative endoscopy showed diffuse pseudodiverticulosis in the remaining esophagus. The patient is free of symptoms 5 years after the surgery. This case suggests that careful treatment may be indicated in patients with esophageal intramural pseudodiverticulosis with stricture and elevated intraluminal pressure, to minimize the possibility of severe complications such as esophageal perforation.

Diverticulum, Esophageal↗