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K Otsu

Publications and source records attributed to K Otsu.

At least 37 records · Page 2Linked to original sources
PubMed1998

Involvement of NF-Y in transcriptional regulation of the phospholamban gene.

To understand the transcriptional regulation of the phospholamban gene, we analyzed a 5'-upstream region of the gene. Using a series of deletion constructs, we demonstrated that the region from -96 bp to -78 bp, containing the CCAAT sequence, is essential for transcription of this gene. This region specifically bound to nuclear proteins extracted from rat hearts, and gel-shift assays using competitive oligonucleotides, antibodies and recombinant proteins showed that this region binds to the NF-YA and NF-YB, members of the CCAAT-binding nuclear protein family. This region-dependent transcription in cardiac myocytes transfected with antisense cDNAs encoding NF-YA and NF-YB was decreased to approximately 50% of that seen in cells transfected with the same sense cDNAs. We, therefore, conclude that the region from -96 bp to -78 bp plays a critical role in expression of the phospholamban gene, which is regulated by binding of the nuclear protein NF-Y.

Animals↗
PubMed1998

Synergistic induction by collagen and fibronectin of liver-specific genes in rat primary cultured hepatocytes.

The extracellular matrix plays an important role for maintaining liver functions. We examined the effects of type I collagen and fibronectin on the expression of liver-specific genes in rat primary hepatocytes. When primary culture hepatocytes were overlaid with a type I collagen-gel, the expression of liver-specific genes (tyrosine aminotransferase, aldolase B, and albumin) increased by 4-5 times, compared with not overlaid hepatocytes. In contrast, the expression of non-liver-specific genes (GAPDH and beta-actin) was suppressed under the same conditions. The addition of fibronectin together with type I collagen-gel further enhanced the expression of liver-specific genes by 1.4-1.8 times. The addition of GRGDS peptide instead of fibronectin with the collagen-gel had a similar effect on hepatic gene expression to that of fibronectin. Addition of fibronectin alone exhibited had no effect on gene expression. These results suggest that type I collagen and fibronectin synergistically induce liver-specific genes.

Animals↗
PubMed1998

Direct association of the gap junction protein connexin-43 with ZO-1 in cardiac myocytes.

The gap junction protein connexin-43 is normally located at the intercalated discs of cardiac myocytes, and it plays a critical role in the synchronization of their contraction. The mechanism by which connexin-43 is localized within cardiac myocytes is unknown. However, localization of connexin-43 likely involves an interaction with the cytoskeleton; immunofluorescence microscopy showed that in cardiac myocytes, connexin-43 specifically colocalizes with the cytoskeletal proteins ZO-1 and alpha-spectrin. In transfected HEK293 cells, immunoprecipitation experiments using coexpressed epitope-tagged connexin-43 and ZO-1 indicated that ZO-1 links connexin-43 with alpha-spectrin. The domains responsible for the protein-protein interaction between connexin-43 and ZO-1 were identified using affinity binding assays with deleted ZO-1 and connexin-43 fusion proteins. Immunoblot analysis of associated proteins showed that the C-terminal domain of connexin-43 binds to the N-terminal domain of ZO-1. The role of this linkage in gap junction formation was examined by a dominant-negative assay using the N-terminal domain of ZO-1. Overexpression of the N-terminal domain of ZO-1 in connexin-43-expressing cells resulted in redistribution of connexin-43 from cell-cell interfaces to cytoplasmic structures; this intracellular redistribution of connexin-43 coincided with a loss of electrical coupling. We therefore conclude that the linkage between connexin-43 and alpha-spectrin, via ZO-1, may serve to localize connexin-43 at the intercalated discs, thereby generating functional gap junctions in cardiac myocytes.

Amino Acid Sequence↗
PubMed1998

Hormonal regulation of aldolase B gene expression in rat primary cultured hepatocytes.

Gene expression of aldolase B, an important enzyme for glucose and fructose metabolism, is regulated by hormones. We examined direct effects of major hormones on aldolase B gene expression in rat primary cultured hepatocytes, in comparison with those on the gene expression of phospho(enol)pyruvate carboxykinase (PEPCK), a key enzyme for gluconeogenesis. Insulin, dexamethasone, and high concentration of glucose increased aldolase B mRNA abundance in the hepatocytes. Glucagon strongly suppressed aldolase B gene expression, and this hormone canceled the stimulative effects of insulin, dexamethasone, and high concentration of glucose. Epinephrine and thyroxine slightly reduced aldolase B mRNA abundance, but these hormones did not cancel the stimulative effects of insulin and dexamethasone. To the contrary, expression of PEPCK gene was suppressed by insulin, dexamethasone, and high concentration of glucose, and remarkably induced by glucagon. Glucagon rapidly suppressed aldolase B gene expression at the transcriptional level. Forskolin and dibutyryl cAMP mimicked the suppressive effect of glucagon on aldolase B gene expression. These results suggest that glucagon may be a key regulator of aldolase B gene transcription through a cAMP/protein kinase A-signaling pathway.

Animals↗
PubMed1998

Intercellular calcium signaling via gap junction in connexin-43-transfected cells.

In excitable cells, intracellular Ca2+ is released via the ryanodine receptor from the intracellular Ca2+ storing structure, the sarcoplasmic reticulum. To determine whether this released Ca2+ propagates through gap junctions to neighboring cells and thereby constitutes a long range signaling network, we developed a cell system in which cells expressing both connexin-43 and ryanodine receptor are surrounded by cells expressing only connexin-43. When the ryanodine receptor in cells was activated by caffeine, propagation of Ca2+ from these caffeine-responsive cells to neighboring cells was observed with a Ca2+ imaging system using fura-2/AM. Inhibitors of gap junctional communication rapidly and reversibly abolished this propagation of Ca2+. Together with the electrophysiological analysis of transfected cells, the observed intercellular Ca2+ wave was revealed to be due to the reconstituted gap junction of transfected cells. We next evaluated the functional roles of cysteine residues in the extracellular loops of connexin-43 in gap junctional communication. Mutations of Cys54, Cys187, Cys192, and Cys198 to Ser showed the failure of Ca2+ propagation to neighboring cells in accordance with the electrical uncoupling between transfected cells, whereas mutations of Cys61 and Cys68 to Ser showed the same pattern as the wild type. [14C]Iodoacetamide labeling of free thiols of cysteine residues in mutant connexin-43s showed that two pairs of intramolecular disulfide bonds are formed between Cys54 and Cys192 and between Cys187 and Cys198. These results suggest that intercellular Ca2+ signaling takes place in cultured cells expressing connexin-43, leading to their own synchronization and that the extracellular disulfide bonds of connexin-43 are crucial for this process.

Calcium↗
PubMed1996

Cloning and characterization of the 5'-upstream regulatory region of the Ca(2+)-release channel gene of cardiac sarcoplasmic reticulum.

To elucidate the transcriptional regulation mechanism for the Ca(2+)-release channel gene of the cardiac sarcoplasmic reticulum (RYR2), we isolated and analyzed the 5'-upstream flanking region of the gene. Sequence analysis indicated that the core promoter region lacks canonical TATA and CAAT boxes, but contains three overlapping GC boxes. A gel shift assay indicated that Sp1 binds to the region containing the GC boxes. Different 5'-deletion constructs in the 5'-flanking region of the RYR2 gene were fused to the luciferase gene, and their promoter activity in rat neonatal cardiac myocytes was subsequently determined. The results revealed the presence of a region containing positive regulatory elements in the 5'-flanking region. Analyses of substitutional mutations introduced into the GC boxes and the regulatory region indicated that in addition to the GC box located at -56 to -51, two regulatory elements (RYR2P1 and RYR2P2) are essential for the promoter activity. These results indicated that Sp1 and transcription factors that bind to RYR2P1 and RYR2P2 cooperatively enhance the expression of the RYR2 gene. In a transient transfection experiment involving promoter-luciferase gene constructs in skeletal muscle cells, we identified a negative regulatory region between positions -209 and -90 that represses the expression of the RYR2 gene in skeletal muscle cells.

Animals↗
PubMed1996

Direct cardiotoxic effects of cocaine and cocaethylene on isolated cardiomyocytes.

We investigated the cardiotoxic effects of cocaine and cocaethylene on the Ca2+ flux responsible for excitation-contraction coupling in isolated ventricular rat myocytes. We simultaneously measured intracellular Ca2+ transients and cell length in isolated cardiac myocytes loaded with a fluorescent Ca2+ indicator, indo-1, during electrical field stimulation at 1 Hz. The cell length was estimated by video dimension analysis. We also measured the activities of Ca2+ ATPase and Ca2+ release channels of cardiac sarcoplasmic reticulum membrane vesicles. Both cocaine and cocaethylene produced significant decreases in both peak intracellular Ca2+ and the cell-contraction rate in a dose-dependent manner. The K0.5 for the reduction of peak intracellular Ca2+ was 157.5 microM for cocaine, but 90.0 microM for cocaethylene. Both cocaethylene and cocaine inhibited neither Ca2+ ATPase nor Ca2+ release channel activity. These results demonstrate that cocaethylene has a more potent direct negative inotropic action on cardiomyocytes, without preventing Ca2+ flux through the cardiac sarcoplasmic reticulum membrane.

Animals↗
PubMed1995

Interleukin-6 down-regulates expressions of the aldolase B and albumin genes through a pathway involving the activation of tyrosine kinase.

Interleukin-6 plays a key role in mediating acute-phase protein synthesis in hepatocytes. However, the mechanism of how interleukin-6 regulates aldolase B and albumin syntheses in hepatocytes is not completely understood. In this study, using primary cultured rat hepatocytes, we have shown that interleukin-6 down-regulates expressions of the aldolase B and albumin genes in a dose- and time-dependent manner. We examined whether the decrease in aldolase B and albumin mRNA expressions by interleukin-6 reflected transcriptional down-regulation or stability of the mRNA. Actinomycin D and cycloheximide did not affect the interleukin-6-mediated decrease in the expressions of both genes. These results suggest that the decreased expressions of both genes induced by interleukin-6 is controlled at the transcriptional level, and that it is due neither to increased degradation of mRNA nor to synthesis of new proteins. Protein kinases play a fundamental role in the intracellular signal transduction. To examine the interleukin-6 signal pathway(s) leading to the decrease of aldolase B and albumin mRNA expressions, we tested various kinds of protein kinase inhibitors in this system. Herbimycin A, an inhibitor of tyrosine kinase(s), prevented the decrease in the expression of aldolase B and albumin mRNAs by interleukin-6. H-7, an inhibitor of protein kinase C, prevented the decrease in the expression of albumin mRNA by interleukin-6, but did not induce recovery of that of aldolase B mRNA. These results suggest that a tyrosine kinase(s) or a herbimycin A-sensitive kinase(s) constitutes a common pathway for interleukin-6-mediated reduction of aldolase B and albumin mRNA expressions and that distinct pathways exist for the modes of expression of the two mRNAs.

Albumins↗
PubMed1995

Trypanosoma cruzi: interruption of both alleles of a gene encoding a protein containing 14-amino-acid repeats by targeted insertion of NEOr and HYGr.

In Trypanosoma cruzi approximately 90% of the 121- and 176-kDa cytoskeletal proteins encoded by the two alleles of the TCR27 gene is composed of 14-amino-acid repeats. To gain insight into the function of the TCR27 proteins we replaced the corresponding regions of 42-nucleotide repeats in the two alleles with the NEOr and HYGr genes. Analyses of DNAs and RNAs from four clones resistant to both G418, a neomycin analogue, and hygromycin showed that in both cases the repetitive regions had in fact been deleted. In addition, the absence of expression of the 14-amino-acid repeats was confirmed in Western blots. In axenic cultures growth rates of the morphologically unchanged, doubly resistant organisms were not different from those of wild-type parasites. However, the doubly resistant organisms proliferated more slowly in cultured mammalian cells than did wild-type parasites. These findings indicate that the absence of the TCR27 repetitive regions is detrimental, but not fatal, to the parasites.

Alleles↗
PubMed1994

Dietary and hormonal regulation of aldolase B gene transcription in rat liver.

In the liver of the fasted rat, the aldolase B (AldB) mRNA level decreased to about half of that of the control rat. When the control rat was refed the glucose-rich diet, the AldB mRNA level increased about six to seven times more than in the fasted rat. This increase was shown as the activation of the AldB gene transcription by a nuclear run-on assay. To understand the causal factor(s) for this activation, the relationship between the AldB mRNA level in the liver and the plasma concentrations of hormones, which are known as major regulators of carbohydrate metabolism during fasting and refeeding, was investigated. The plasma insulin level in the rat which was refed the glucose-rich diet increased in parallel to AldB mRNA level, while the plasma glucagon level decreased reciprocally to it. The relationship of the plasma corticosterone level to the AldB mRNA level was not obvious. To directly confirm the effects of these hormones on AldB gene transcription in the liver, the responses of AldB gene in the primary cultured hepatocytes to these hormones were examined. Insulin and dexamethasone were effective to activate AldB gene, while glucagon and thyroxine were suppressive. Thyroxine did not extinguish the effects of insulin and dexamethasone, but glucagon canceled them. Thus, it is probable that in vivo these hormones synergistically regulate the AldB gene transcription. In vitro transcription analysis of two AldB promoter constructs suggested that the proximal half of the AldB promoter (up to -92 bp from the transcription start site) is, at least in part, involved for this induction, and the distal half which contains liver-specific elements (-93 to -202 bp) is not involved. The possible explanation for the dietary regulation of aldolase B gene transcription in the liver is discussed.

Animals↗
PubMed1994

A novel growth-inducible gene that encodes a protein with a conserved cold-shock domain.

We have isolated a cDNA that encodes a novel member of the Y-box binding protein family, termed as RYB-a (Rat Y-box Binding protein-a). RYB-a is a 31 kDa protein that contains a conserved cold-shock domain and an amino acid alignment similar to those of charge zipper proteins. Expression of RYB-a mRNA was highly abundant in the skeletal muscle, spleen, and fetal liver. The expression is very low in new-born and adult livers, suggesting its expression is under developmental regulation. In addition, the expression of RYB-a mRNA was induced in the liver during regeneration and by stimulation of quiescent fibroblast cells with serum. Induction in the fibroblasts was inhibited by treating the cell with a specific tyrosine kinase inhibitor, genistein or by detachment of cell-adhesion. Since both treatments are known to inhibit G1 cells to enter S phase, RYB-a gene is thought to be a member of growth-inducible genes.

3T3 Cells↗
PubMed1994

The point mutation Arg615-->Cys in the Ca2+ release channel of skeletal sarcoplasmic reticulum is responsible for hypersensitivity to caffeine and halothane in malignant hyperthermia.

Malignant hyperthermia (MH) is an autosomal dominant myopathy. Molecular genetic studies have shown that the alteration of Arg615 to Cys in the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (ryanodine receptor) is cosegregated with porcine MH (Fujii, J., Otsu, K., Zorzato, F., de Leon, S., Khanna, V. K., Weiler, J. E., O'Brien, P. J., and MacLennan, D. H. (1991) Science 253, 448-451; Otsu, K., Khanna, V. K., Archibald, A., and MacLennan, D. H. (1991) Genomics 11, 744-750). Here, using the fluorescence calcium indicator indo-1, we determined the concentration of ionized cytosolic calcium in myoblastic cells transfected with either the wild-type or mutated ryanodine receptor cDNA. The cells expressing the mutant ryanodine receptor showed higher sensitivity to caffeine, which induces Ca2+ release from the sarcoplasmic reticulum through the ryanodine receptor. Exposure to clinical doses of halothane resulted in a rapid increase of [Ca2+]i in cells expressing the mutated ryanodine receptor, whereas no [Ca2+] changes were observed in cells expressing the wild-type ryanodine receptor. These results provide definite evidence that a single amino acid mutation, Arg615-->Cys, in the ryanodine receptor is causative of MH.

Amino Acid Sequence↗
PubMed1994

Thyroid hormone enhances Ca2+ pumping activity of the cardiac sarcoplasmic reticulum by increasing Ca2+ ATPase and decreasing phospholamban expression.

Phospholamban is a putative suppressor of the Ca2+ ATPase of the cardiac sarcoplasmic reticulum. The level of mRNA encoding the Ca2+ ATPase has been shown to be increased, whereas the phospholamban mRNA level to be decreased in the ventricles obtained from hyperthyroid rabbits [Nagai R, Zarain-Herzberg A. Brandl CJ, Fujii J. Tada M. MacLennan DH, Alpert NR, Periasamy M. (1989) Proc Natl Acad Sci USA 86: 2966-2970]. The present study was designed to examine whether these effects of thyroid hormone on the expression of the Ca2+ ATPase and phospholamban are exerted directly on cardiac myocytes and whether the resultant incoordinate expression of these proteins alters Ca2+ pumping activity. We studied the levels of phospholamban and Ca2+ ATPase mRNA in primary isolated neonatal rat myocardial cells incubated with triiodothyronine (T3) for 3-48 h and the Ca2+ uptake activity of the microsomes prepared from the cells. Northern blot analysis showed that T3 decreased phospholamban mRNA levels to about a half of control in 24 h. On the other hand, Ca2+ ATPase mRNA gradually increased with time. EC50 for phospholamban mRNA expression was 2.5 x 10(-10) M which was approximately 10 times higher than that for the Ca2+ ATPase. T3 increased Vmax of Ca2+ uptake with the significant reduction of K0.5 for Ca2+ (0.40 +/- 0.02 microM for control v 0.31 +/- 0.02 microM for T3-treated vesicles), indicating that thyroid hormone stimulates Ca2+ pumping activity not only by increasing the Ca2+ ATPase but also decreasing phospholamban. These results suggested that phospholamban regulates the Ca2+ ATPase in dual modes; in short time range, by decreasing the affinity of the Ca2+ ATPase for Ca2+ by phosphorylation of phospholamban with cAMP-dependent protein kinase, and in long time range, by changing the molecular ratio between the two proteins through the regulation of gene expression.

Animals↗
PubMed1993

Sodium dependence of the Na(+)-H+ exchanger in the pre-steady state. Implications for the exchange mechanism.

The pre-steady state time course of amiloride-sensitive Na+o uptake by the Na(+)-H+ exchanger in renal brush border membrane vesicles (BBMV) exhibits a burst phase at 0 degrees C which corresponds to the initial turnover of the exchanger (Otsu, K., Kinsella, J. L., Sacktor, B. S., and Froehlich, J. P. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4818-4822). Investigation of the Na+o dependence of the Na(+)-H+ exchanger between 1 and 10 mM Na+ revealed that activation of the burst phase involves at least two Na+ transport sites interacting with positive cooperativity. In this study, characterization of the Na+ transport sites contributing to the burst phase was extended to include Na+ concentrations below 1 mM. Between 0.1 and 1 mM Na+ the amplitude of the burst phase in acid-loaded BBMV (pHi 5.7; pHo 7.7) exhibited a sigmoidal dependence on [Na+]o, consistent with the presence of a second class of high affinity Na+ transport sites with cooperative binding characteristics. In contrast, steady state Na+ uptake obeyed Michaelis-Menten kinetics, similar to the behavior observed previously at higher (1-10 mM) Na+o concentrations. Treatment of the vesicles with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, which induced the formation of an inside-negative membrane potential, increased the burst amplitude but had no effect on the steady state uptake velocity. Experiments performed with alkaline-loaded BBMV (pHi 7.7; pHo 7.7), which permit only a single turnover of the exchanger, gave a simple hyperbolic dependence of the burst amplitude on [Na+]o (0.5-5 mM). We propose that the change in multiplicity of Na+ transport sites and membrane potential sensitivity that occurs in the transition between the pre-steady state and the steady state of Na+ uptake in acid-loaded vesicles reflects the presence of an oligomer which operates according to a "flip-flop" mechanism. The minimum subunit composition inferred from the biphasic [Na+]o dependence of the burst amplitude is a dimer at low (< 1 mM) Na+o levels and a tetramer at high [Na+]o. Communication between the subunits producing the complex [Na+]o dependence is controlled by the intravesicular (cytoplasmic) H+ modifier site. Under alkaline conditions (pH 7.7), where this site is unoccupied, the subunits behave as independent units and cease operation after the first turnover. Occupation of the H+ modifier site activates a conformational interaction between the subunits that leads to cooperative Na+o binding, alternation of the transport sites, and repetitive cycling of the Na(+)-H+ exchanger.

Amiloride↗
PubMed1993

Chromosome mapping of five human cardiac and skeletal muscle sarcoplasmic reticulum protein genes.

Fluorescence in situ hybridization (FISH) experiments were performed using genomic and complementary DNA probes in order to determine the location on human chromosomes for five genes expressed in cardiac and skeletal muscle sarcoplasmic reticulum. The chromosome location of each gene was determined in terms of both cytogenetic bands and fractional chromosome length. The ATP2A2 gene, expressing the SERCA2 isoform of the Ca2+ pump, maps to bands 12q23-q24.1, the phospholamban gene (PLN) to 6q22.1, the human skeletal muscle calsequestrin gene (CASQ1) to band 1q21, the cardiac calsequestrin gene (CASQ2) to bands 1p11-p13.3, and the cardiac calcium release channel gene (RYR2) to the interval between band 1q42.1 (distal) and band 1q43 (proximal).

Adenosine Triphosphatases↗
PubMed1993

Interruption of a Trypanosoma cruzi gene encoding a protein containing 14-amino acid repeats by targeted insertion of the neomycin phosphotransferase gene.

In Trypanosoma cruzi, the cause of Chagas' disease in Latin America, a large proportion of the antigenic proteins described to date have repetitive domains. In earlier work we identified a partial length cDNA, designated TCR27, encoding approx. 26 copies of a 14-amino acid repeat and a unique 61-amino acid C-terminal region. The goal of the current project was to replace the repetitive region of a TCR27 gene with the neomycin phosphotransferase gene (NEOr). A pBluescript-based vector was constructed in which the 0.9-kb NEOr coding region replaced the 2.9-kb internal repetitive segment of a TCR27 gene and was in frame with its nonrepetitive 5' coding sequence (pTCR27-2::NEO). Epimastigotes were electroporated in the presence of linearized pTCR27-2::NEO and transfected clones were selected on solid medium containing G418. Southern and Northern analyses of DNAs and RNAs from four G418-resistant clones showed that in all cases the repetitive region in the smaller of the two TCR27 genes (TCR27-2) had been replaced by NEOr. The absence of the native TCR27-2 protein in the transfected clones was confirmed by Western blot. In axenic cultures growth rates of epimastigotes bearing an interrupted TCR27-2 gene were not different from those of wild-type parasites. In addition, there was no relative impairment of the four transfected clones' ability to proliferate in cultured mammalian cells. The fact that the clones having the interrupted TCR27-2 gene were not impaired biologically suggests that the length of the repetitive region of the TCR27 protein is not a critical factor for survival.

Amino Acid Sequence↗