Biomedical subjects

T Murakami

Publications and source records attributed to T Murakami.

At least 865 records · Page 48Linked to original sources

PubMed1994

Structure-activity relationships of an anti-HIV peptide, T22.

T22 ([Tyr5,12,Lys7]-polyphemusin II) has been shown to have a strong anti-human immunodeficiency virus (HIV) activity comparable to that of 3'-azido-2',3'-dideoxythymidine (AZT). We studied the structure-anti-HIV activity relationships of T22 and determined the following information. The number of Arg residues in the N-terminal and C-terminal regions of T22 is closely related with anti-HIV activity. Disulfide rings, especially the major disulfide ring, are indispensable for anti-HIV activity and maintenance of the secondary structure. Between two repeats of Tyr-Arg-Lys, which are a characteristic structure contained in T22, Tyr-Arg-Lys in the N-terminal portion is more closely related with anti-HIV activity. We found some compounds having a higher selectivity index (50% cytotoxic concentration/50% effective concentration) than that of T22.

Amino Acid Sequence↗

PubMed1994

Decrease in Cu/Zn- and Mn-superoxide dismutase activities in brain and spinal cord of patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a degenerative disorder of motor neurons in the central nervous system (CNS). Mutation of the Cu/Zn-superoxide dismutase (SOD) gene on chromosome 21 has been found in some families with autosomal dominant familial ALS (FALS). We sought to determine whether there may be differences in the distribution and activity of SOD in the CNS of patients with sporadic ALS, and of control patients without neurological disorders. The frontal cortex, cerebellum, and spinal cord were obtained at autopsy on 5 patients with ALS and from 10 controls. Immunohistochemically, in the controls, the cytosols of the large pyramidal neurons of the cerebral cortex, anterior and posterior horn cells, and neurons of the nucleus thoracicus of spinal cord were stained homogeneously with anti-human Cu/Zn-SOD antibody, and in a granular manner with anti-human Mn-SOD antibody. Pia mater and epithelial cells of choroid plexus also stained well. Conversely, in the CNS of the ALS patients, most neurons were stained faintly, or not at all with both anti-Cu/Zn- and Mn-SOD antibodies, whereas the pia mater and the epithelial cells of choroid plexus stained intensely. There was no difference in total SOD activity in the entire CNS between ALS patients and controls, as determined by enzyme assay. Results suggest that, in cases of sporadic ALS, the activities of Cu/Zn- and Mn-SOD are decreased and superoxide produced within the neurons accumulates because of an insufficient elimination, leading to the development or acceleration of cell damage, ultimately producing neuronal degeneration and necrosis.

Adolescent↗

PubMed1994

The role of insulin in activation of two enhancers in the mouse GLUT1 gene.

We identified two enhancer elements of the mouse GLUT1 gene responsive to serum, growth factor, and oncogenes; the first enhancer element (enhancer-1) is located 2.7 kilobases upstream of the cap site of the gene, and the second one (enhancer-2) is located in the second intron of the gene (Murakami, T., Nishiyama, T., Shirotani, T., Shinohara, Y., Kan, M., Ishii, K., Kanai, F., Nakazuru, S., and Ebina, Y. (1992) J. Biol. Chem. 267, 9300-9306). In the present work, we describe the role of insulin in activation of these two enhancers. NIH/3T3 HIR3.5 cells, which express a large number of insulin receptors, were stably transformed by hybrid genes containing the enhancer(s) and promoter of GLUT1 gene and the coding region of chloramphenicol acetyltransferase (CAT) gene as a reporter gene. In stable transformants of the reporter gene without the enhancers, the CAT mRNA was not induced by insulin; however, in clones containing the reporter gene with enhancer-1, the CAT mRNA was induced by insulin at 30 min and reached a maximum at 1 h. In clones transfected by the reporter gene with enhancer-2, the CAT mRNA was induced at 1 h and reached a maximum at 3 h. To determine the early response element to insulin in enhancer-1, transformants of hybrid reporter genes containing truncated or mutated enhancer-1 were examined. The homologous sequence with the serum response element in enhancer-1 is essential for an early response to insulin.

3T3 Cells↗

PubMed1994

[Expression of nitric oxide synthase in enkephalin and dynorphin systems of the rat hypothalamus].

Nitric oxide (NO), a simple gas with free radical chemical properties, is synthesized by nitric oxide synthase (NOS) from arginine in neurons and acts as a neurotransmitter in the central and peripheral nervous systems. The immunohistochemical demonstration of NOS-immunoreactivity and its histochemical marker, NADPH-diaphorase activity in many neurons of the hypothalamus, suggest that NO plays a role in controlling the production and/or release of hypothalamic neuroendocrine peptides. In the present study, the expression of NOS in the enkephalin and dynorphin systems of the rat hypothalamus was examined by the combined method of the NADPH-diaphorase histochemistry and the immunocytochemistry of methionine enkephalin (M-Enk) or dynorphin B (Dyn-B). About 6 to 9% of M-Enk immunoreactive neurons in the paraventricular, arcuate and ventromedial nuclei expressed NADPH-diaphorase activity. Dyn-B immunoreactive neurons, however, showed NADPH-diaphorase activity in high ratio (37%-84%) in the supraoptic nucleus and the parvocellular and magnocellular paraventricular nucleus. These results revealed that a part of the enkephalin and dynorphin neurons in the rat hypothalamus have the ability to produce NO. The high ratio of expression of NO in magnocellular neurosecretory dynorphin containing neurons suggested that NO participates in controlling posterior pituitary hormone secretion together with dynorphin.

Amino Acid Oxidoreductases↗

PubMed1994

Flow-cytometric quantification in human gliomas of alpha satellite DNA sequences specific for chromosome 7 using fluorescence in situ hybridization.

Cell suspensions prepared from freshly frozen tissue specimens were used to examine aberrations in the number of chromosome 7 signals in 10 human gliomas. Nuclear DNA was hybridized in vitro with an alpha satellite DNA probe specific for the centromeric regions of chromosome 7, using fluorescence in situ hybridization (FISH). The intensity of the fluorescence signal from the hybridized probe was measured, together with the nuclear DNA content, by flow cytometry. The mean probe fluorescence of all nuclei was compared to the mean copy number per nucleus found with microscopic scoring. Moreover, the mean probe fluorescence ratio of DNA aneuploid nuclei relative to DNA diploid nuclei (FISHa/FISHd) was calculated to determine how the numerical aberration of chromosome 7 signals contributes to the DNA ploidy of the sample. The results from the flow-cytometric analysis and from microscopic evaluation were compatible. One of four tumors with DNA diploidy had a higher average intensity of FISH signal and a broader coefficient of variation in FISH signal than normal brain tissue; this was shown to be due to the gain of chromosome 7 signals. Although FISHa/FISHd correlated with DNA indices (P < 0.01), there were some disparities, probably due to other complex genotypic associations involving several gains or losses of chromosomes. Thus gain of chromosome 7 in gliomas is related to both DNA ploidy change and chromosome specific gain. It is concluded that flow-cytometric quantification of FISH is useful in investigating numerical aberrations of chromosomes and nuclear DNA content simultaneously.

Aneuploidy↗

PubMed1994

Synthesis of 1,3-di-O-alkyl-2-O-(beta-glycosyl)glycerols bearing oligosaccharides as hydrophilic groups.

A novel series of synthetic glycolipids, 1,3-di-O-alkyl-2-O-(beta-glycosyl)glycerols, and their efficient synthetic route were proposed. These glyceroglycolipids were synthesized in good overall yields and stereoselectivity in five steps via trichloroacetimidate glycosylation with 1,3-di-O-alkylglycerols. This route was applied to prepare the glycolipids bearing a cello- or malto-oligosaccharide with a definite number of sugar residues from one to six. Thin-layer chromatography, elemental analysis, nuclear magnetic resonance spectroscopy and infrared absorption spectroscopy confirmed that these glycolipids were chemically pure compounds.

Carbohydrate Sequence↗

PubMed1994

A high-level and regulatable production system for recombinant glycoproteins using a human interferon-alpha promoter-based expression vector.

A novel expression vector for human cells, pIFP, which expresses a cloned gene under the control of the human interferon-alpha-encoding gene (IFN-alpha) promoter (pIFN) was constructed. As a model of glycoprotein production, a human erythropoietin-encoding cDNA (EPO) inserted downstream from pIFN in pIFP was introduced into human B-cell leukemia-derived BALL-1 cells, and EPO-producing cells were established. Upon stimulation with Sendai virus, the cells produced human EPO at high levels. The highest production level and the highest inducibility were 872 IU/ml and 67-fold, respectively. Simultaneously, the transformed cells also produced IFN-alpha and tumor necrosis factor-alpha (TNF-alpha), as the parental BALL-1 cells did. Comparing the amounts of the substances produced, activity of the exogenous pIFN introduced seemed much higher than that of the endogenous one. Further, the transformed cells could be obtained in a large quantity by being applied to the 'in vivo cell proliferation method (hamster method)'. Human EPO produced by the transformed cells had a molecular mass range of 35 to 42 kDa, similar to that of EPO produced by CHO cells. The processing of EPO seemed to occur properly. The combination of the human pIFN, BALL-1 cells and the hamster method provides us with a useful production system for bioactive glycoproteins of human origin.

Animals↗

PubMed1994

An improved method for detecting cytostatic toxin (emetic toxin) of Bacillus cereus and its application to food samples.

We developed an improved HEp-2 cell assay method for the detection of Bacillus cereus toxin, which affects the proliferation of HEp-2 cells. The cytostatic toxin was stable upon exposure to heat, pH 2, pH 11 and trypsin, which suggests it is an emetic. Using the HEp-2 cell assay, we examined the distribution and contamination of B. cereus strains that produced an emetic toxin in various foods. Although there were 228 enterotoxin producers among 310 B. cereus strains obtained from foods, 16 of them produced the cytostatic type (emetic toxin). All of the strains that produced the cytostatic toxin were of the H.1 serotype.

Bacillus cereus↗

PubMed1994

Purification and partial characterization of 3-hydroxyisobutyryl-coenzyme A hydrolase of rat liver.

An unusual feature of valine catabolism is a reaction in which an intermediate of its catabolic pathway, (S)-3-hydroxyisobutyryl-CoA, is hydrolyzed to give the free acid and CoA-SH. The enzyme responsible for this reaction, 3-hydroxyisobutyryl-CoA hydrolase (EC 3.1.2.4), was purified 7200-fold from rat liver in this study. The purified enzyme consists of a single polypeptide with an M(r) of 36,000 in the native and denatured forms. The hydrolase is highly specific for (S)-3-hydroxyisobutyryl-CoA and 3-hydroxypropionyl-CoA (Km, 6 and 25 microM, respectively) with optimal activity around pH 8. The turnover rate of the enzyme for (S)-3-hydroxyisobutyryl-CoA is 270 s-1, which is high relative to other enzymes of the valine pathway. Likewise, activity of the enzyme expressed on a wet weight basis is also very high in the major tissues of the rat. These findings suggest that rapid destruction of (S)-3-hydroxyisobutyryl-CoA produced during valine catabolism is physiologically important. We propose that the need for a mechanism to protect cells against the toxic effects of methacrylyl-CoA, which is maintained in equilibrium with (S)-3-hydroxyisobutyryl-CoA by crotonase, explains why valine catabolism involves this enzyme and why its tissue activity is so high.

Acyl Coenzyme A↗

PubMed1994

Expression of the gene encoding the tyrosine kinase-deficient human insulin receptor in transgenic mice.

Defects in the insulin receptor (IR) in diabetic patients have been given much attention. To address the role of such defects, we generated a transgenic (TG) mouse carrying the cDNA encoding a tyrosine-kinase (TK)-deficient human IR (hIR), under the control of the native promoter. The TG mouse expressed the transgene (TG) mRNA in the liver, as identified in Northern blots. Analyses of various tissues by reverse transcription-polymerase chain reaction revealed that expressions of the TG mRNA in brain, heart, kidney, lung, stomach, skeletal muscle and adipose tissue were higher than those seen with the endogenous mouse IR (mIR), but expression in small intestine, colon, spleen, testis and ovary were approximately half those seen with the endogenous mIR. In the liver, the expression of the TG was about one tenth that of the endogenous mIR. In analyses of insulin binding and IR autophosphorylation, using a human-specific anti-IR antibody, the TK-deficient hIR was synthesized in the tissues of the TG mice. Despite the expression of TK-deficient hIRs in various tissues, including the major insulin-target tissues, muscle and adipose tissues, of the TG mice, no glucose intolerance was observed as assessed by the intraperitoneal glucose tolerance test, before and after sucrose feeding for 55 weeks. Our results suggest that a higher expression of the mutated IR, especially in the liver which is another major insulin-target tissue, or additional pathogenic factors, environmental or genetic, might be required for glucose intolerance.

Animals↗

PubMed1994

[CT evaluation of extravascular perfusion of contrast medium and its potential to a new method of diagnosis: an experimental study using macro, micro-molecular contrast media].

To evaluate the dynamics of extravascular perfusion, dynamic CT with two different molecular sized contrast media was performed on VX2 tumor of rabbit. The first dynamic CT was performed with a bolus injection of iopamidol (IP:120 mgI/ml, 5 ml). After ascertaining that the tumor attenuation had returned to the pre-contrast level, the second dynamic CT was performed on the same slice with bolus injection of iodoethylated starch (IES:120 mgI/ml). The time-density (T-D) curves of the same tumor area on the images obtained by two contrast media were compared. The T-D curve with IP showed definitely higher level than that with IES. This occurrence can be explained that IP, 13 A in size, has higher permeability distributing not only in the intravascular space, but also into the extravascular space. On the other hand, IES, 200 A in size, will stay mostly in the intravascular space. From this, we consider that the attenuation difference between the two curves will be an indicator for the dynamics of extravascular perfusion, suggesting to become a new method for CT diagnosis.

Animals↗

PubMed1994

[Usefulness of 3DFT-FISP MR imaging in differential diagnosis of liver tumor].

Dynamic 3-dimensional Fourier transformation (3 DFT)-FISP MR imaging was performed in 30 patients with 49 liver tumors (hepatocellular carcinoma 26, hemangioma 13, cholangiocellular carcinoma 2, metastatic liver tumor 8) to evaluate characteristic enhancement patterns. MR images of 3 DFT-FISP (TR/TE/flip angle/slab thickness/partition/slice thickness, 20 msec/8 msec/30 degree/21-75 mm/7 or 15/3-5 mm) were obtained before, just after (early phase), 1 min after (late phase 1) and 2-4 min after (late phase 2) the administration of 0.1 mmol/kg of Gd-DTPA. Twenty-three of the 26 (88%) hepatocellular carcinomas showed total hyperintense enhancement relative to surrounding liver parenchyma in the early phase, and isointense or total hypointense enhancement in the late phase. Eleven of 13 (85%) hemangiomas showed peripheral hyperintense enhancement in the early phase, and peripheral or total hyperintense enhancement in the late phase. Cholangiocellular carcinomas were enhanced gradually from the early to the late phase. Metastatic liver tumors showed doughnut-like ring enhancement in all 8 lesions in the early phase. We concluded that 3 DFT-FISP dynamic MR imaging was useful in the differential diagnosis of liver tumor.

Adult↗

PubMed1994

[Experimental study on vascular permeability and hemodynamics of nonionic dimeric contrast media (Iotrolan): a comparison to nonionic monomer on dynamic CT and angiography].

The difference of vascular permeability and hemodynamics between Iotrolan and Iopamidol was experimentally studied on dynamic CT and angiography. A hemodialyzer with 60 A pores were applied as a phantom of a target organ with capillaries and extravascular space. Dynamic CT scans and DSA of the phantom were performed with injection of each contrast medium and time-density curves obtained from several regions of interest were compared. The dynamic CT scans performed at 40 seconds after the completion of injection, showed that Iopamidol was mostly distributed at the site of inlet, while Iotrolan was mostly at the center of the phantom. Time-density curves on DSA with Iotrolan revealed lower and shorter peak density and time than did Iopamidol. In conclusion, Iotrolan has lower permeability than that of Iopamidol because of its larger molecular size. Iotrolan has slower flow through catheters and capillaries than that of Iopamidol because of its higher viscosity. One should be aware the difference between the two contrast media and be careful to the analysis of time-density curves of dynamic CT and DSA.

Angiography, Digital Subtraction↗

PubMed1994

Functioning of the intrathoracic stomach after esophagectomy.

OBJECTIVE: To investigate acid secretion and motility of the intrathoracic stomach with truncal vagotomy after esophagectomy. DESIGN: Case-control study of patients undergoing esophagectomy, with average follow-up of 3.2 months, compared with health controls. SETTING: Yamaguchi University School of Medicine, Kogushi, Japan. PATIENTS: A random sample of 21 patients selected from 79 patients with esophageal cancer during the period from May 1988 to August 1993 received the intrathoracic stomach after subtotal esophagectomy. A control sample consisted of 14 men. INTERVENTIONS AND MAIN OUTCOME MEASURES: For acid secretion, a pH probe was placed in the intrathoracic stomach to measure the average pH value (average of total monitoring values) and pH holding time (ratio of the period during which the pH value was 3 or higher based on the entire continuous monitoring periods). For motility, digestive tract scintigraphy was performed to obtain the time activity curve from the analog image observed when patients ate meals labeled with indium 111. In addition, hemorrhagic ulcers in the intrathoracic stomachs of three patients were reported. RESULTS: A pH value below 3 was recognized after surgery, and there was no significant difference between pH values in patients and normal volunteers (average pH value, 3.9 +/- 1.5 vs 3.3 +/- 1.9; pH holding time, 52.2 +/- 20.2% vs 57.1 +/- 24.4%, respectively). The time activity curve of the intrathoracic stomach observed by scintigraphy underwent no change after surgery. Among three patients with hemorrhagic ulcers, two exhibited improvements after receiving a histamine blocker, while in the other, it was not effective. CONCLUSION: Acid secretion ability and motility of the intrathoracic stomach with truncal vagotomy may be unchanged after esophagectomy.

Case-Control Studies↗

PubMed1994

Single muscle fiber analysis of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS).

We examined muscle sections from 3 patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), using single-fiber polymerase chain reaction, histochemistry, and in situ hybridization. Most type 1 ragged-red fibers showed positive cytochrome c oxidase activity at the subsarcolemmal region, while type 2 ragged-red fibers had little cytochrome c oxidase activity. However, there was no difference in the amount of total (mutant and wild-type) mitochondrial DNAs (mtDNAs) and the proportion of mutant mtDNA between type 1 and type 2 ragged-red fibers. These observations suggest that mitochondrial proliferation and nuclear factors affect muscle pathology, including cytochrome c oxidase activity, in MELAS. Total mtDNAs were greatly increased in ragged-red fibers (about 5-17 times over those in non-ragged-red fibers). The proportion of mutant mtDNA was significantly higher in ragged-red fibers (88.1 +/- 5.5%) than in non-ragged-red fibers (63.2 +/- 21.6%). Thus, the amount of wild-type mtDNA as well as mutant mtDNA was increased in ragged-red fibers in MELAS, failing to support the contention of a replicative advantage of mutant mtDNA. The proportion of mutant mtDNA was significantly higher in the strongly succinate dehydrogenase-reactive blood vessels (83.2 +/- 4.2%) than in non-succinate dehydrogenase-reactive blood vessels (38.8 +/- 16.2%). It seems likely that systemic vascular abnormalities involving cerebral vessels lead to the evolution of stroke-like episodes in MELAS.

Base Sequence↗

PubMed1994

Dose-dependent plasma clearance of human epidermal growth factor in rats.

Exogenously administered human epidermal growth factor (hEGF) shows a marked dose-dependent plasma disposition in rats. In the present study, total plasma clearance (CLtotal) of hEGF was examined from the viewpoint of hepatic blood flow and accessible EGF receptors in a dosing range of hEGF from 30 to 1000 micrograms/kg. In rats in which down-regulation of EGF receptors caused a reduction in their number (50% below the normal level) (group 1), the CLtotal of hEGF was decreased only at a medium dose (100 micrograms/kg) compared to those in normal rats. In rats in which hepatic intoxication with carbon tetrachloride caused 50% reduction of both EGF receptor number and hepatic blood flow (group 2), CLtotal was decreased at low and medium hEGF doses. The decrease at a low hEGF dose was proportional to the decrease in the hepatic blood flow. In rats with reduced hepatic blood flow caused by hypothermia (group 3), CLtotal was decreased at all hEGF doses examined, and the decrease at a low hEGF dose (50 micrograms/kg) was proportional to the hepatic blood flow. The decreases of CLtotal at a medium hEGF dose in groups 1, 2 and 3 were well accounted for by the decrease of hepatic blood flow and/or EGF receptor number in a well-stirred model.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

High-dose chemotherapy and blood stem cell autografts for children with first relapsed acute lymphoblastic leukemia: a pilot study of the Children's Cancer and Leukemia Study Group of Japan (CCLSG).

This study was performed to determine the value of high-dose chemotherapy and peripheral blood stem cell autografts (PBSCT) in the treatment of children with first relapsed acute lymphoblastic leukemia (ALL). Eighteen children underwent PBSCT during the second complete remission (CR) and had a minimum 10 month follow-up. The median age of the patients was 11 yr (range, 2-17 yr). Fifteen patients received the "MCVAC" regimen, one received high-dose MCNU+busulfan therapy, one received high-dose melphalan+VP-16, and one received melphalan+carboplatin+cytosine arabinoside+MCNU. None of these regimens included total body irradiation. Eight patients developed recurrence of the disease at 1 to 19 mo (median, 3 mo) after PBSCT. Patients in whom the first relapse occurred sooner, that is, within 16 mo of initial therapy, tended to have a better survival rate than those who developed relapse after 30 mo (six of seven survived versus four of 11; not significant). Although the preliminary data provided little conclusive information, it did suggest that incorporation of PBSCT in the salvage protocol of relapsed childhood ALL can be justified.

Adolescent↗

PubMed1994

A complete culture system for avian transgenesis, supporting quail embryos from the single-cell stage to hatching.

We report here a method to produce quail hatchlings by culture in vitro from the single-cell stage. The culture is composed of three steps. In the first step, the fertilized ovum surrounded by thick albumen obtained from the magnum is cultured for 24 hr at 41.5 degrees C in a tightly sealed 20-ml plastic cup with chicken thin albumen added to the equator level of the ovum (System Q1). In the second step, a quail egg shell, cut horizontally and emptied, is used as a bed shell. After the thick albumen is removed, the embryo with egg yolk is transferred to the bed shell and thin albumen from chicken eggs is added to fill the shell. Then, the embryo is cultured for an additional 52 hr at 37.5 degrees C while being rocked at an angle of 90 degrees at 30-min intervals (System Q2). The embryo is transferred again to a chicken bed shell and cultured at 37.5 degrees C with rocking at a 30-degree angle (System Q3). Just before hatching, the rocking of embryos is stopped. The procedure yielded a hatchability of 25%. For transgenesis, a plasmid construct containing a beta-actin-lacZ hybrid gene (pMiwZ) is microinjected into the ovum at the single-cell stage, which is cultured in vitro for 85-90 hr using Systems Q1 and Q2 consecutively. Seven out of 17 surviving embryos exhibited lacZ gene expression in embryonic tissues as detected by histochemistry. The procedure described here should be highly applicable for the production of transgenic birds.

Actins↗