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Biomedical subjects

K Murakami

Publications and source records attributed to K Murakami.

At least 577 records · Page 32Linked to original sources
PubMed1994

Molecular cloning of a gene under control of the circadian clock and light in the rodent SCN.

We recently found a mouse unusual per repeat genomic gene showing circadian expression in the suprachiasmatic nucleus (SCN) of rat brain. As an initial step to the better understanding of biological functions of mammalian per repeat family, we isolated a new cDNA clone that encodes for the putative open reading frame of 133 amino acids, designating as mp41, having a per repeat sequence of (ACAGC)32 which lacks one base pair from a mouse unusual per repeat sequence (ACAGGC)n. In situ hybridization showed that the mRNA of mp41 gene expression is detected in the rat pancreas, uterus, ovary, liver, adrenal glands, kidney, intestine, spleen and brain. In brain, daily fluctuations of mp41 mRNA levels were found in the SCN under light and dark cycles--high during the day time and lower during the night time, even in constant darkness for 15 days. After exposing rats to light, mp41 mRNA increased only during the subjective night of the circadian cycle when light also induced the c-fos mRNA expression in the SCN. These results suggest that the transcriptional control of mp41 gene is regulated by light and a circadian clock and indicate that mp41 is a new marker gene for a cycling transcript in the SCN.

Amino Acid Sequence↗
PubMed1994

Chronic gliclazide treatment affects basal and post-ischemic cardiac function in diabetic rats.

1. A 6-week gliclazide treatment improved left ventricular developed pressure and left ventricular end-diastolic pressure, left ventricular pressure-rate products in isolated working hearts from streptozotocin-induced diabetic rats. 2. Post-ischemic recovery in heart rate, left ventricular developed pressure, left ventricular end-diastolic pressure, left ventricular pressure-rate products and cardiac work were also shown in gliclazide-treated diabetic rats. 3. Gliclazide treatment did not modify the degree of insulinopenia and hyperglycemia, nor the myocardial energy metabolism during ischemia-reperfusion. 4. The results suggest that the gliclazide treatment has a cardioprotective effect on basal and post-ischemic cardiac functions of chronic diabetes.

Adenosine Triphosphate↗
PubMed1994

An immunohistochemical study of an equine B-cell lymphoma.

The tissues of an 8-year-old thoroughbred castrated male horse with equine lymphoma were examined immunohistochemically. Neoplastic masses were observed in the mediastinum, mesenteric lymph nodes, gastric mucosa and serosa, liver capsule, and spleen capsule with associated lymph nodes. Histopathologically, the neoplastic cells were seen to consist predominantly of a mixture of well differentiated small and large types. Immunohistochemically, the small lymphoid cells were MHC class IIlow+ and PanT- and the large lymphoid cells were MHC class IIhigh+ and PanT-. These findings revealed that the neoplastic cells were of B-lymphocyte lineage.

Animals↗
PubMed1994

Nor-cucurbitacin glucosides from Caputo nigri.

Three new nor-cucurbitacin glucosides were isolated from the root of Caputo nigri (Cabeça-de-negro) and their structures established as 29-nor-1,2,3,4,5,10-dehydro-2,3,16 alpha,20R, 22 zeta,25-hexahydroxy-11-oxocucurbita-6,23-diene 2-O-beta-D-glucopyranoside and 2-O-beta-D-glucopyranosyl(1-2)beta-D- glucopyranoside(sophorose), and 1,2,3,4,5,10-dehydro-2,3,16 alpha-trihydroxy-4, 9,14-trimethyl-19-norpregn-6-ene-11,20-dione 2-O-beta-D-glucopyranoside on the basis of detailed spectroscopic analysis.

Glucosides↗
PubMed1994

Cortical expression of the human angiotensinogen gene in the kidney of transgenic mice.

We have previously generated "Tsukuba hypertensive mice" with elevated blood pressure by cross-mating separate lines of transgenic animals carrying either 15 kb of the human renin gene including its native 3-kb promoter or 14 kb of the human angiotensinogen gene along with its 1.3-kb promoter, the former of which is expressed predominantly in the kidney and the latter of which is also expressed in the kidney to levels comparable to those found in the liver. To investigate whether the integrated human angiotensinogen gene is prominently expressed in the kidney of transgenic mice, we have analyzed a production region of the transgene mRNA by in situ hybridization technique. This analysis clearly demonstrated that human angiotensinogen mRNA is localized specifically to the cortex region of transgenic mouse kidney. The present finding indicates a possible involvement of the renal renin-angiotensin system in the pathogenesis of high blood pressure in transgenic mice.

Angiotensinogen↗
PubMed1994

Dynamic changes in mRNA expression of neutrophils during the course of acute inflammation in rabbits.

To examine RNA/protein synthesis of neutrophils and related dynamic changes during the inflammatory process, we investigated mRNA expressions in neutrophils, by RNA blot hybridization analyses using 12 different rabbit gene probes. We first selected five candidate genes encoding inflammation-related proteins, i.e. tumor necrosis factor (TNF-alpha) IL-1 alpha, IL-1 beta, neutrophil activating peptide-1/IL-8 (NAP-1/IL-8) and monocyte chemoattractant protein (MCP)-1. We further selected several genes on basis of the results from gene subtraction between cDNA libraries from neutrophils at an early (5 h) and at a late (24 h) stage of casein-induced acute peritonitis in rabbits, i.e. immune activation gene-2 (Act-2), migration inhibitory factor-related protein-8 (MRP-8), MRP-14, gamma-actin, and formyl-methionyl-leucyl-phenylalanine receptor (fMLP-R), and ferritin light (L) chain. In addition to these genes we used ferritin heavy (H) chain gene, another component of the ferritin molecule. We examined mRNA expressions by cytoplasmic slot blot analysis of the above 12 genes in neutrophils obtained from blood and from various stages of casein-induced inflammation in rabbits. The observed patterns of mRNA expression kinetics were classified into three. Pattern 1: mRNAs of MRP-8, MRP-14, and gamma-actin were constitutively expressed in blood neutrophils, and increased rapidly after emigration into inflammatory sites. Pattern 2: mRNAs of IL-1 beta, NAP-1/IL-8, Act-2, and fMLP-R were undetectable in blood neutrophils, and were induced rapidly after the onset of inflammation. Pattern 3 mRNAs of ferritin L and H chain were induced slowly, and increased with progression of the inflammatory process.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Expression of recombinant mouse/human chimeric antibody specific to human GMP-140/P-selectin.

To construct a mouse/human chimeric antibody, we cloned the genomic DNAs for Ig from a murine hybridoma that produces PL7-6 monoclonal antibody specific to human P-selectin and expressed them in SP2/0 myelomas using a series of pSV2 vectors. Transfected cells that produce the mouse/human chimeric anti-human P-selectin antibody were geneticin-selected and screened by an immunoassay using immobilized antigen. The chimeric antibody, cPL-2R1, expressed by the resultant clone has the murine Ig variable region and the human Ig constant region. The native antibody PL7-6 and the chimeric antibody cPL-2R1 react equally with purified P-selectin and thrombin-stimulated platelets. Competitive inhibition tests demonstrated that the native antibody PL7-6 and the chimeric antibody cPL-2R1 had identical affinity for purified human P-selectin. Thus, although human IgG1 constant region was substituted for the murine counterpart in this chimeric antibody, its specificity and binding affinity for P-selectin was not altered. This chimeric antibody may prove useful when employed in combination with imaging reagents or therapeutic drugs for targeting activated platelets or endothelium in patients with thrombosis or intravascular inflammation.

Amino Acid Sequence↗
PubMed1994

The transmembrane region of microsomal cytochrome P450 identified as the endoplasmic reticulum retention signal.

Microsomal-type cytochrome P450s are integral membrane proteins bound to the membrane through their N-terminal transmembrane hydrophobic segment, the signal anchor sequence. To elucidate the determinants that enable the P450s to be located in the ER, we constructed cDNAs encoding chimeric proteins in which a secretory form of carboxyesterase, carboxyesterase Sec, was connected to the N-terminus of the full-length or truncated forms of a microsomal-type P450, P450(M1), and the constructed plasmids were expressed in COS cells. Since carboxyesterase Sec is an N-glycosylated secretory protein, endo H treatment could be used to determine whether these chimeric proteins were located in the ER or not. Carboxyesterase Sec with the N-terminal 20 amino acids, containing the transmembrane region, of P450(M1), was located in the ER, as determined from the endo H sensitivity of the expressed protein and immunofluorescence staining of the cells. As the expressed protein exhibited carboxyesterase activity, it was not retained in the ER through the BiP-dependent quality control system recognizing unfolded proteins. Another chimeric protein construct in which carboxyesterase Sec was connected to the C-terminal region of rat UDP-glucuronosyltransferase (UDP-GT), that contained a double-lysin ER retention motif, was also located in the ER, as determined from the endo H sensitivity and immunofluorescence staining. On the other hand, the sugar moiety of the carboxyesterase Sec connected to the transmembrane segment of UDP-GT, Sec/GTd, was partially resistant to the endo H treatment. From the results of immunofluorescent staining and cell fractionation, it was concluded that the Sec/GTd product was located in the Golgi apparatus. These observations indicated that the N-terminal hydrophobic segment of P450(M1) is sufficient for the ER membrane retention, whereas the transmembrane segment of UDP-GT is not. To determine whether microsomal P450s are recycled between the ER and Golgi compartments or not, a DNA construct encoding cathepsin D connected to the N-terminus of P450(M1) was prepared and expressed in COS cells. The fusion protein was phosphorylated, but the phosphorylation was sensitive to alkaline phosphatase. As a control, authentic cathepsin D was subjected to phosphorylation of its oligosaccharide chain that was resistant to the alkaline phosphatase treatment. Since GlcNAc-P-transferase, which forms the alkaline phosphatase-resistant phosphodiester in the sugar chains of lysosome-targeting proteins, is located in the Golgi apparatus, it was concluded that the oligosaccharide chain of the cathepsin D portion of the fusion protein was not phosphorylated, and that the chimeric protein did not go to the Golgi apparatus.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Sequence requirements for endoproteolytic processing of precursor proteins by furin: transfection and in vitro experiments.

We have recently presented some sequence rules that govern the endoproteolytic processing of precursor proteins within the constitutive secretory pathway [Watanabe, T. et al. (1992) J. Biol. Chem. 267, 8270-8274; Watanabe, T. et al. (1993) FEBS Lett. 320, 215-218]. This cleavage seems to be catalyzed by furin, a mammalian homologue of the yeast processing endoprotease Kex2. In this study, not only by coexpression of furin and substrate precursors in a furin-deficient cell line but also by in vitro experiments using purified recombinant furin and substrate precursors, we determined the substrate specificity of furin. The sequence requirements of furin determined by the coexpression and in vitro experiments were essentially the same, and were consistent with the sequence rules for precursor cleavage within the constitutive secretory pathway.

Amino Acid Sequence↗
PubMed1994

Histological and immunohistochemical studies on the articular cartilage after experimental discectomy of the temporomandibular joint in rabbits.

Total discectomy was performed experimentally in the rabbit temporomandibular joint (TMJ). In order to evaluate the injury and repair of the articular cartilage of the mandibular condyle after discectomy, both histologic and immunohistochemical observations were made. Immunohistochemical observation by using bromodeoxyuridine (BrdU) and its monoclonal antibody in vivo labelling method was used. The present study demonstrated that the cartilage on the articular surface of the condyle disappeared at 1 week post-operatively. This defect in the injured articular cartilage was not repaired by the cartilage, but was covered by a fibrous connective tissue at 6 weeks post-operatively, where the condylar recontouring by the adjacent articular cartilage, and flattening and hyperplasia of the condyle were observed. Thus, the cartilaginous repair following discectomy was characterized by the proliferation of the adjacent cartilage to the injured site and osteoblastic formation in the bone marrow at the exposed subchondral bone.

Animals↗
PubMed1994

Cloning and characterization of a gene affecting the methicillin resistance level and the autolysis rate in Staphylococcus aureus.

Tn918 mutagenesis of a high-level methicillin-resistant Staphylococcus aureus (methicillin MIC, 800 micrograms/ml) led to the isolation of a low-resistance mutant. The Tn918 insert was transferred back to the parent to produce strain SRM563 (methicillin MIC, 12.5 micrograms/ml), which showed heterogeneous resistance. Twenty-two clinical isolates of methicillin-resistant S. aureus were transformed with DNA of SRM563. In the transformants of most strains, instances of reduced resistance were observed with concomitant increases of autolysis rate induced by Triton X-100 and were generally more profound in high-resistance strains. Two transformants exhibited a decrease of the autolysis rate and little reduction of resistance. In the transformant of methicillin-susceptible strain RN2677, an increase of the autolysis rate and little reduction of resistance were observed. The production of low-affinity penicillin-binding protein (PBP2') did not significantly decrease in the mutants. Insertion of Tn918 occurred within the 3'-terminal region of a novel gene designated llm, which was cloned and sequenced. RNA blot analysis demonstrated that the gene was transcribed. The encoded protein was composed of 351 amino acid residues with a molecular weight of 38,512 and was hydrophobic, suggesting its location on the membrane. The gene was detected by PCR in all S. aureus strains tested but not in the other 26 staphylococcal species. Comparison of the 3'-terminal sequences of the gene among several S. aureus strains showed that, whereas nucleotide substitutions occurred at the third position in seven of eight 3'-terminal codons, only C-terminal amino acid variation of glutamate or aspartate was observed.

Amino Acid Sequence↗
PubMed1994

A Mucor pusillus mutant defective in asparagine-linked glycosylation.

A Mucor pusillus mutant defective in asparagine-linked glycosylation was found in our stock cultures. This mutant, designated 1116, secreted aspartic proteinase (MPP) in a less-glycosylated form than that secreted by the wild-type strain. Analysis of enzyme susceptibility, lectin binding, and carbohydrate composition indicated that this mutant secreted three glycoforms of MPPs, one of which contained no carbohydrate; the other two had truncated asparagine-linked oligosaccharide chains such as Man0-1GlcNAc2. Further analysis using oligosaccharide processing inhibitors, such as castanospermine, 1-deoxynojirimycin and N-methyldeoxynojirimycin, suggested that MPPs in the mutant were glycosylated through a transfer of the truncated lipid-linked oligosaccharides, Man0-1GlcNAc2, to the MPP protein but not through an aberrant processing. In addition, genetic studies with forced primary heterokaryons indicated that the mutation in strain 1116 was recessive.

1-Deoxynojirimycin↗
PubMed1994

Molecular mechanism of adipogenic activation of the angiotensinogen gene.

Angiotensinogen gene expression is controlled in a tissue- and development-specific manner. Interestingly, the angiotensinogen gene is abundantly expressed in adipose tissues other than the liver, where it is mainly produced. We investigated the molecular mechanism of angiotensinogen gene expression in a 3T3-L1 preadipocyte-adipocyte system. Although angiotensinogen mRNA was barely detectable in preadipocytes, its levels increased significantly during differentiation. As a whole, the pattern of the change in transcriptional activity of the angiotensinogen promoter was similar to that of the angiotensinogen mRNA levels during adipogenic differentiation, indicating that the activation of the angiotensinogen promoter might be involved in the adipogenic differentiation-coupled gene expression. The proximal promoter region, from -96 to +22 of the transcriptional start site, was sufficient to confer adipogenic activation, and the proximal element from -96 to -52 of the transcriptional start site was necessary for this promoter stimulation. DNA-protein binding experiments showed that this proximal element specifically bound to a nuclear factor induced by adipogenic differentiation. These results suggest that the proximal promoter element from -96 to -52 plays a role in adipogenic activation of the angiotensinogen promoter.

3T3 Cells↗
PubMed1994

Molecular mechanism of transcriptional activation of angiotensinogen gene by proximal promoter.

Angiotensinogen is shown to be produced by the liver and the hepatoma cell line HepG2. As a first step for understanding the molecular relationship between the transcriptional regulation of the angiotensinogen gene and the pathogenesis of hypertension, we have analyzed the basal promoter of the angiotensinogen gene. Chloramphenicol acetyltransferase (CAT) assays with 5'-deleted constructs showed that the proximal promoter region from -96 to +22 of the transcriptional start site was enough to express HepG2-specific CAT activity. Electrophoretic mobility shift assay and DNase I footprinting demonstrated that the liver- and HepG2-specific nuclear factor (angiotensinogen gene-activating factor [AGF2]) and ubiquitous nuclear factor (AGF3) bound to the proximal promoter element from -96 to -52 (angiotensinogen gene-activating element [AGE2]) and to the core promoter element from -6 to +22 (AGE3), respectively. The site-directed disruption of either AGE2 or AGE3 decreased CAT expression, and the sequential titration of AGF3 binding by in vivo competition remarkably suppressed HepG2-specific CAT activity. Finally, the heterologous thymidine kinase promoter assay showed that AGE2 and AGE3 synergistically conferred HepG2-specific CAT expression. These results suggest that the synergistic interplay between AGF2 and AGF3 is important for the angiotensinogen promoter activation.

Angiotensinogen↗
PubMed1994

Mechanism of cAMP regulation of renin gene transcription by proximal promoter.

Renin is produced mainly by the kidney, and cAMP is a main positive regulator of its synthesis. This study was undertaken to analyze the molecular mechanism of cAMP-mediated regulation of Ren-1C gene transcription by the proximal promoter. We first showed that the promoter region from -365 to +16 of the mouse renin gene (Ren-1C) mediated the cAMP-induced chloramphenicol acetyltransferase gene expression in embryonic kidney-derived 293 cells. Deletion analysis and heterologous promoter assay disclosed that the proximal promoter region from -75 to +16 was able to activate chloramphenicol acetyltransferase expression by cAMP, and indicated that the proximal promoter element from -75 to -47 (RP-2 element) overlapping the TATA-like region was able to confer cAMP responsiveness. Electrophoretic mobility shift assay and DNase I footprinting analysis demonstrated that novel nuclear factors in 293 cells interacted with the RP-2 element, and that cAMP increased the binding activity of these nuclear factors to the RP-2 element. Furthermore, we demonstrated that cAMP enhanced the binding of nuclear factors derived from juxtaglomerular cells, the main production site of renin in the kidney, to the RP-2 element in vivo. These results suggest that the RP-2 element plays an important role in the cAMP-mediated regulation of Ren-1C gene transcription through the proximal promoter.

Base Sequence↗
PubMed1994

[Studies on attenuation of post-ischemic brain injury by kampo medicines-inhibitory effects of free radical production. I].

In view of the recent investigation, highly reactive oxygen-derived free radicals may play a role in cerebral ischemia and brain damage, free radical scavenging activity of kampo formulations (Oren-gedoku-to, Keishi-bukuryogan, Saiko-ka-ryukostu-borei-to and Daio-botampi-to) and Kampo components (Ityou, Shini, Menamomi, Arinosudama and Denshiti), used for the attenuation of brain damage, was investigated in vitro. The used experimental system was a radical scavenging action against 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide anion radical (O2-.) and hydroxyl radical (OH.) by the electron spin resonance technique at room temperature. All kampo formulations and kampo components showed radical scavenging activity in a concentration-dependent manner. This result indicates that kampo formulations, treated for brain damage, were good radical scavenger that could possibly prevent ischemic complications such as formation of brain damage. The intensity of scavening activity of kampo formulations for three types of radicals were as follows; 1. DPPH: Daio-botampi-to > Saiko-ka-ryukotsu-borei-to = Keishi-bukuryo-gan > Oren-gedokuto, 2. O2-.: Daio-botampi-to = Keishi-bukuryo-gan > Saiko-ka-ryukotsu-borei-to > Oren-gedoku-to, 3. OH.: Daio-botampi-to = Saiko-ka-ryukotsu-borei-to > Keishi-bukuryo-gan = Oren-gedoku-to. Daio-botampi-to indicated the strongest scavenging activity against three types of radicals. Ischemic brain damage is thought to be an "oketsu" syndrome, which is correlated with abnormalities of the microcirculation. Therefore, Daio-botampi-to, which has been used for "oketsu" syndrome, showed superior free-radical scavenging activity.

Brain Ischemia↗