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Biomedical subjects

M Murakami

Publications and source records attributed to M Murakami.

At least 163 records · Page 9Linked to original sources
PubMed2001

The invasive features of glial and non-central nervous system tumor cells are different on organotypic brain slices from newborn rats.

On radiograms, glial tumors are usually seen to invade in a finger-like fashion, while non-central nervous system (CNS) tumors expand in a mass-like fashion. We prepared organotypic brain slice cultures from newborn rats to investigate the invasive behavior of human brain tumors using glial tumor cell lines (U-87MG, U-373MG, U-251MG, and SF-126) and of non-CNS tumors using cell lines; HT-1080 (human malignant fibrosarcoma), RFRF (human lung carcinoma), MIA-PICA (human pancreatic carcinoma), and Colo38 (human malignant melanoma). We selected an area that was centered at a junction between deep cortical layers and the striatum, punched a hole measuring 0.5-0.7 mm in diameter in this area, implanted different rhodamine-labeled tumor cells at that site, and observed their invasive behavior under an inverted fluorescent microscope. Over the course of several days, all glioma cells invaded in a finger-like fashion; U-373 MG cells invaded farthest. Non-CNS tumors expanded in mass-like fashion into adjacent areas. Using the slice cultures, we also investigated the regulatory effect on tumor invasion of forced expression of glial fibrillary acidic protein (GFAP) after gene transfection. The forced expression of GFAP rendered U-87MG and HT-1080 cells less invasive. Our results show that organotypic brain slice cultures are an excellent medium for studying the invasive features of glial and non-CNS tumors.

Animals↗
PubMed2001

Cytoskeletal-induced alterations in the adhesion of HT-1080 fibrosarcoma cells to extracellular matrix.

We have investigated the adhesion of the human fibrosarcoma cell line, HT-1080, transfected with glial fibrillary acidic protein (GFAP) to a variety of extracellular matrix macromolecules (ECM) including collagen type IV, laminin, and fibronectin. The GFAP-transfectants demonstrated altered adhesiveness to extracellular matrix substrates when compared to controls. GFAP-positive, heavy metal-induced fibrosarcoma cells were more adherent to plastic and collagen type IV than were the parental or uninduced cells. In contrast, GFAP-positive fibrosarcoma cells were less adherent to laminin- or fibronectin-coated dishes than controls. Time course adhesion studies over 9 days showed that the heavy metal-induced fibrosarcoma cells progressively became more adherent to collagen type IV and less adherent to laminin- or fibronectin-coated dishes than did uninduced cells. However, with the removal of heavy metal from the medium, the HT-1080 fibrosarcoma cells were restored to their original adhesive potential. By phase microscopy, uninduced and induced HT-1080 cells demonstrated different morphological features and remained viable in an anchorage-dependent fashion on collagen type IV as a substrate. By way of contrast, GFAP-induced HT-1080 cells were not particularly viable in monolayer culture and readily detached from laminin as a substrate. The expression of beta1 integrin in GFAP-positive fibrosarcoma cells was decreased following heavy metal induction by Western blot analyses. In contrast, the expression of alpha2 integrin was increased whereas alpha5 integrin was unchanged in HT-1080 cells following the induction of GFAP. Gelatin zymography showed that 72 kDa collagenase was less expressed in GFAP-induced clones than in controls. Our data suggest that the forced expression of the intermediate filament, GFAP, in HT-1080 cells may modulate cell adhesion to different ECM substrates through alterations in expression of integrins.

Blotting, Western↗
PubMed2001

The inhibitory effect of simvastatin on growth in malignant gliomas--with special reference to its local application with fibrin glue spray in vivo.

Simvastatin is one of the competitive inhibitors of HMG-CoA reductase. During clinical trials, it has shown the ability to lower serum cholesterol. We investigated the effect of simvastatin on the growth of malignant gliomas in vitro, semi-in vivo, and in vivo. An in-vitro MTT assay revealed that human malignant glioma cell lines: U-251MG, U-373MG, and U-87MG, and rat malignant glioma cell line C6 were well inhibited in growth in a dose-dependent fashion. An anchorage-independent growth assay showed that the number of colonies (more than 100 microM in size) of human (U-373MG) and rat malignant gliomas (C6) was markedly reduced in a dose-dependent fashion. A flow cytometry analysis revealed that simvastatin treatment led U-251MG cells to accumulate in sub G0-G1. Immunostaining by TUNEL method showed that most glioma cells treated by 10 microM simvastatin had nuclear immunostaining, suggesting apoptotic changes of the treated cells. The human umbilical vein endothelial cells and human lung fibroblasts were inhibited in growth by no more than 20% of controls even with a high dose (10 microM) of simvastatin. In the semi-in vivo model, using newborn rat brain slice cultures, the rhodamine-labeled glioma cells were abolished after 7 days of local simvastatin treatment with fibrin glue probably suggesting that simvastatin led the cells to apoptosis. In rat models using subcutaneously inoculated C6, the local application of simvastatin combined with fibrin glue (spray method) was quite effective in inhibiting the growth of the tumor. These data suggest that simvastatin may be a novel anti-glioma drug, and the local application of simvastatin combined with fibrin glue (by spray method) may be a crucial new clinical strategy against glioma growth.

Animals↗
PubMed2001

Characteristics of an Albumin Cobalt Binding Test for assessment of acute coronary syndrome patients: a multicenter study.

BACKGROUND: The ability of the N-terminal region of human albumin to bind cobalt is diminished by myocardial ischemia. The characteristics of an assay based on albumin cobalt binding were assessed in suspected acute coronary syndrome patients and in a control reference population. The ability of the Albumin Cobalt Binding (ACB) Test measurement at presentation to predict troponin-positive or -negative results 6-24 h later was also examined. METHODS: We enrolled 256 acute coronary syndrome patients at four medical centers. Blood specimens were collected at presentation and then 6-24 h later. The dichotomous decision limit and performance characteristics of the ACB Test for predicting troponin-positive or -negative status 6 h-24 h later were determined using ROC curve analysis. Results for 32 patients could not be used because the time of onset of ischemia appeared to have been >3 h before presentation or was uncertain. The reference interval was determined by parametric analysis to estimate the upper 95th percentile of a reference population (n = 109) of ostensibly healthy individuals. RESULTS: Increased cTnI was found in 35 of 224 patients. The ROC curve area for the ACB Test was 0.78 [95% confidence interval (CI), 0.70-0.86]. At the optimum decision point of 75 units/mL, the sensitivity and specificity of the ACB Test were 83% (95% CI, 66-93%) and 69% (95% CI, 62-76%). The negative predictive value was 96% (95% CI, 91-98%), and the positive predictive value was 33% (95% CI, 24-44%). The within-run CV of the ACB Test was 7.3%. Results for the reference population were normally distributed; the one-sided parametric 95th percentile was 80.2 units/mL. CONCLUSIONS: This exploratory study suggests that the ACB Test has high negative predictive value and sensitivity in the presentation sample for predicting troponin-negative or -positive results 6-24 h later.

Acute Disease↗
PubMed2001

[Evaluation of time dependency of the acetazolamide effect on cerebral hemodynamics as measured by 99mTc-ECD single-photon emission computed tomography].

PURPOSE: Kuwabara et al. have examined the cerebral artery dilation with acetazolamide (ACZ) challenge test using PET. And, they reported that ACZ reaction came out time dependently. We have developed a unique SPECT's method using Technetium-99m ethyl cysteinate dimer (99mTc-ECD) to verify the results obtained by Kuwabara et al. METHOD: 1000 MBq of 99mTc-ECD was exactly divided into three syringes. Each of which was intravenous infused (i.v.) at rest, 7.5, and 20 minutes after ACZ administration. Data collection was started using dynamic SPECT immediately after 99mTc-ECD i.v. at rest. Using necessary data only, SPECT images representing each of the three 99mTc-ECD i.v. was reconstructed. SPECT counts were obtained by the ROI method from each images to calculate relative CBF from rest to 7.5 and 20 minutes after ACZ administration. RESULT: The 18 hemispheres of nine patients in the negative control group in whom ACZ was not loaded. CBF was stable during the three evaluation. The measurement error our method was estimated as small. The 18 hemispheres of nine patients in the positive control group who has normal vasodilatory reserve, CBF was increased by 26.2 +/- 8.1% at 7.5 minutes and 29.3 +/- 13.1% at 20 minutes after ACZ administration. Seven patients with and chronic stage unilateral internal carotid artery severe stenosis and/or occlusion were evaluated as the test group. Case of unaffected side, CBF was increased by 17.6 +/- 6.9% at 7.5 minutes and 24.8 +/- 11.3% 20 minutes after ACZ administration. And, increase rate of CBF in the affected side was 2.8 +/- 1.6% at 7.5 minutes and 17.3 +/- 5.0% at 20 minutes after ACZ administration. In the affected side, timing of the maximum CBF increase caused by ACZ was remarkably delayed. CONCLUSION: Our method based on 99mTc-ECD SPECT also revealed delayed cerebral artery dilation in the affected side. It was suggested that ACZ reaction came out time dependently, as reported by Kuwabara et al.

Acetazolamide↗
PubMed2001

Effects of isoproterenol and butylscopolamine on the friction between an artificial stone and the intraureteral wall in anesthetized rabbits.

PURPOSE: We evaluated the effects of the nonselective beta-adrenoceptor agonist isoproterenol and the nonselective muscarinic antagonist butylscopolamine on ureteral wall tension, namely friction between an artificial stone and the intraureteral wall, in anesthetized rabbits. MATERIALS AND METHODS: The relaxing effect of the drugs on the KCl induced tonic contraction was examined in isolated rabbit ureters. The effect of the drugs on the applied force needed for the artificial stone to pass at a constant speed through the ureter, called sliding force, was evaluated in anesthetized rabbits. RESULTS: In a concentration dependent manner isoproterenol but not butylscopolamine reduced the KCl induced contraction in isolated ureter (mean pD2 7.35 +/- 0.06). Intravenous administration of 1 and 10 microg./kg. isoproterenol significantly decreased the friction between the artificial stone and intraureteral wall in anesthetized rabbits with sliding force at 15 minutes after drug administration decreased by 54.5% and 63.6%, respectively. In contrast, 100 and 1,000 microg./kg. butylscopolamine intravenously had no evident effect on ureteral wall tension. CONCLUSIONS: Our results strongly suggest that ureteral smooth muscle relaxation by beta-adrenergic stimulation reduces ureteral wall tension, thereby, diminishing mechanical effects impeding the movement of a ureteral stone down the ureter.

Adrenergic beta-Agonists↗
PubMed2001

17alpha-hydroxylase deficiency accompanied by adrenal myelolipoma.

A 45-year-old woman was admitted because of hypertension and hypokalemia. Primary amenorrhea from birth was noted. Plasma renin activity (PRA), 17alpha-hydroxyprogesterone and androgen levels were low, but progesterone, 11-deoxycorticosterone, corticosterone and adrenocorticotropic hormone (ACTH) were elevated, resulting in a diagnosis of 17alpha-hydroxylase deficiency. Abdominal magnetic resonance imaging revealed a round mass in the left adrenal region, the specimen of which was diagnosed as myelolipoma. After removal of the tumor, the blood pressure, serum potassium and hormone levels were unchanged, indicating an adrenal non-functioning tumor. Excessive ACTH secretion over a long period may stimulate the development of adrenal myelolipoma.

17-alpha-Hydroxyprogesterone↗
PubMed2001

[Morphological and histological studies of in-stent restenosis in seven types of stents implanted in porcine coronary arteries].

OBJECTIVES: Differences in the mechanism of restenosis after stenting between coil and tubular stents were examined in porcine coronary arteries using histological and immunohistochemical methods. METHODS: Twenty-four pigs underwent balloon-induced injury in the left anterior descending coronary artery. Two weeks later, seven different stents clinically available in Japan (Coil stents: GR I, GR II, Wiktor, Cordis; Tubular stents: gfx, Multilink, Palmaz-Schatz) were implanted in the injured site. Four weeks after the stent implantation, the pigs were sacrificed for histological examination and for morphometrical analysis of the lumen, neointima, media and adventitia. Immunohistochemical studies using anti-proliferating cell nuclear antigen (PCNA), anti-alpha-smooth muscle actin and anti-macrophage antibody were also performed. RESULTS: The coil stents formed eccentric, and the tubular stents formed concentric neointimal proliferation. Although there was no difference in the area of neointima between the stents, the area of lumen in the tubular stents was bigger than that in the coil stents (p < 0.01), because the vascular area was bigger in the tubular stents (p < 0.05). Immunohistochemical examination found many PCNA-positive cells in the proliferated neointima, especially in the area around the stent strut. Many of these cells around the stent strut were positively stained by anti-macrophage antibody. Other cells positively stained for PCNA were confirmed as smooth muscle cells. CONCLUSIONS: Tubular stents maintained a wider lumen than coil stents, because negative remodeling after stenting was less in the tubular stents. Implantation of stents with less negative remodeling is very important to prevent restenosis after stenting.

Actins↗
PubMed2001

[Clock drawing in dementia: its reliability and relation to the neuropsychological measures].

To examine the reliability and validity of clock drawing (CD) for evaluating dementia patients in Japan, we investigated the CD performance and its relation to several neuropsychological tests in 150 demented patients including 105 patients with Alzheimer disease and 30 age- and education-matched non-demented subjects (16 patients with mild cognitive impairment and 14 normals). Patients were also evaluated using the Mini-mental State Exam (MMSE), Wechsler Memory Scale (WMS), Kohs Block Design, and word fluency. CD was scored using the Shulman method. CD scores showed a high interrater reliability (r = 0.97). CD by non-demented subjects was essentially normal. As a screening test for Alzheimer disease (AD, mean MMSE = 18.0), CD had a sensitivity of 57.1% and a specificity of 96.7%. However, four of eight AD patients who showed normal MMSE score (> or = 24) drew abnormal clocks. CD scores in dementia were significantly correlated with performance on the Block Design (r = 0.68), MMSE (r = 0.56), and the Mental Control subtest in the WMS (r = 0.58). Stepwise regression analysis revealed that performance on the MMSE and the Block Design explain 53.8% of the variance in the CD scores. These findings indicate that low CD score by the Shulman method may be reflective of a constructional disability and general severity of dementia. CD is not so sensitive as the result of the original report when the patient group includes milder cases of AD. CD is, however, an efficient screening test for detecting and following the patients with dementia, especially combined with the MMSE.

Aged↗
PubMed2000

Wee1-regulated apoptosis mediated by the crk adaptor protein in Xenopus egg extracts.

Many of the biochemical reactions of apoptotic cell death, including mitochondrial cytochrome c release and caspase activation, can be reconstituted in cell-free extracts derived from Xenopus eggs. In addition, because caspase activation does not occur until the egg extract has been incubated for several hours on the bench, upstream signaling processes occurring before full apoptosis are rendered accessible to biochemical manipulation. We reported previously that the adaptor protein Crk is required for apoptotic signaling in egg extracts (Evans, E.K., W. Lu, S.L. Strum, B.J. Mayer, and S. Kornbluth. 1997. EMBO (Eur. Mol. Biol. Organ.) J. 16:230-241). Moreover, we demonstrated that removal of Crk Src homology (SH)2 or SH3 interactors from the extracts prevented apoptosis. We now report the finding that the relevant Crk SH2-interacting protein, important for apoptotic signaling in the extract, is the well-known cell cycle regulator, Wee1. We have demonstrated a specific interaction between tyrosine-phosphorylated Wee1 and the Crk SH2 domain and have shown that recombinant Wee1 can restore apoptosis to an extract depleted of SH2 interactors. Moreover, exogenous Wee1 accelerated apoptosis in egg extracts, and this acceleration was largely dependent on the presence of endogenous Crk protein. As other Cdk inhibitors, such as roscovitine and Myt1, did not act like Wee1 to accelerate apoptosis, we propose that Wee1-Crk complexes signal in a novel apoptotic pathway, which may be unrelated to Wee1's role as a cell cycle regulator.

Amino Acid Sequence↗
PubMed2000

Distinct arachidonate-releasing functions of mammalian secreted phospholipase A2s in human embryonic kidney 293 and rat mastocytoma RBL-2H3 cells through heparan sulfate shuttling and external plasma membrane mechanisms.

We analyzed the ability of a diverse set of mammalian secreted phospholipase A(2) (sPLA(2)) to release arachidonate for lipid mediator generation in two transfected cell lines. In human embryonic kidney 293 cells, the heparin-binding enzymes sPLA(2)-IIA, -IID, and -V promote stimulus-dependent arachidonic acid release and prostaglandin E(2) production in a manner dependent on the heparan sulfate proteoglycan glypican. In contrast, sPLA(2)-IB, -IIC, and -IIE, which bind weakly or not at all to heparanoids, fail to elicit arachidonate release, and addition of a heparin binding site to sPLA(2)-IIC allows it to release arachidonate. Heparin nonbinding sPLA(2)-X liberates arachidonic acid most likely from the phosphatidylcholine-rich outer plasma membrane in a glypican-independent manner. In rat mastocytoma RBL-2H3 cells that lack glypican, sPLA(2)-V and -X, which are unique among sPLA(2)s in being able to hydrolyze phosphatidylcholine-rich membranes, act most likely on the extracellular face of the plasma membrane to markedly augment IgE-dependent immediate production of leukotriene C(4) and platelet-activating factor. sPLA(2)-IB, -IIA, -IIC, -IID, and -IIE exert minimal effects in RBL-2H3 cells. These results are also supported by studies with sPLA(2) mutants and immunocytostaining and reveal that sPLA(2)-dependent lipid mediator generation occur by distinct (heparanoid-dependent and -independent) mechanisms in HEK293 and RBL-2H3 cells.

Amino Acid Sequence↗
PubMed2000

Serine 727 phosphorylation and activation of cytosolic phospholipase A2 by MNK1-related protein kinases.

We have previously reported that in thrombin-stimulated human platelets, cytosolic phospholipase A(2) (cPLA2) is phosphorylated on Ser-505 by p38 protein kinase and on Ser-727 by an unknown kinase. Pharmacological inhibition of p38 leads to inhibition of cPLA2 phosphorylation at both Ser-505 and Ser-727 suggesting that the kinase responsible for phosphorylation on Ser-727 is activated in a p38-dependent pathway. By using Chinese hamster ovary, HeLa, and HEK293 cells stably transfected with wild type and phosphorylation site mutant forms of cPLA2, we show that phosphorylation of cPLA2 at both Ser-505 and Ser-727 and elevation of Ca(2+) leads to its activation in agonist-stimulated cells. The p38-activated protein kinases MNK1, MSK1, and PRAK1 phosphorylate cPLA2 in vitro uniquely on Ser-727 as shown by mass spectrometry. Furthermore, MNK1 and PRAK1, but not MSK1, is present in platelets and undergo modest activation in response to thrombin. Expression of a dominant negative form of MNK1 in HEK293 cells leads to significant inhibition of cPLA2-mediated arachidonate release. The results suggest that MNK1 or a closely related kinase is responsible for in vivo phosphorylation of cPLA2 on Ser-727.

Animals↗