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K Otsu

Publications and source records attributed to K Otsu.

At least 55 records · Page 3Linked to original sources
PubMed1993

[Calcium release channel of cardiac muscle sarcoplasmic reticulum].

Cardiac muscle contractility is controlled by myoplasmic calcium (Ca) concentration. Sarcoplasmic reticulum plays an essential role in the regulation of [Ca]. Depolarization of the sarcolemma induces Ca release from the sarcoplasmic reticulum, leading to the muscle contraction. On the other hand, Ca uptake by the sarcoplasmic reticulum into the lumen results in the muscle relaxation. The Ca release from the sarcoplasmic reticulum is mediated by Ca release channel. Using ryanodine as a molecular probe, the calcium release channel has been isolated, purified and characterized. Morphological studies have confirmed its identity with the feet structure which spans between the transverse tubule and the junctional face of the sarcoplasmic reticulum. The cardiac Ca release channel cDNA encodes 4969 amino acids with a molecular weight of 564,711. The analysis of the sequence indicates that 10 potential transmembrane sequences in the COOH-terminal fifth of the molecule and two additional nearer to the center of the molecule could contribute to the formation of the Ca conducting pore. The remainder of the molecule is hydrophilic and constitutes the cytoplasmic domain which corresponds to the feet structure. Northern blot analysis has shown that the cardiac Ca release channel is expressed in heart and brain. The channel is modulated by Ca, ATP, calmodulin, and phosphorylation. A potential modulator binding domain has been identified in the molecule by searching consensus sequences and investigation of a causal mutation for malignant hyperthermia, the primary defect of which exists in the skeletal Ca release channel.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Alteration of rat liver proteoglycans during regeneration.

Sepharose CL-6B column chromatography of crude extracts from the slices of regenerating rat livers after partial hepatectomy and sham-operated controls labeled with [35S]sulfuric acid revealed an enhancement of [35S]sulfate incorporation into proteoglycan fractions during regeneration. The 35S-labeled proteoglycans contained heparan sulfate (more than 80% of the total) and chondroitin/dermatan sulfate. The 35S-incorporation into both glycosaminoglycans increased to maxima 3-5 days after partial hepatectomy and decreased thereafter toward the respective control levels. When [35S]sulfuric acid was replaced by [3H]glucosamine, similar results were obtained. These results suggest that the maximal stimulation of proteoglycan synthesis in regenerating rat liver follows the maximal mitosis of hepatic cells 1-2 days after partial hepatectomy. The 35S-labeled proteoglycans from regenerating liver 3 days after partial hepatectomy and control were analyzed further. They were similar in chromatographic behavior on a gel filtration or an anion-exchange column and in glycosaminoglycan composition. Their glycosaminoglycans were indistinguishable in electrophoretic mobility. However, these proteoglycans were slightly but significantly different in their affinity to octyl-Sepharose and in the molecular-weight distribution of their glycosaminoglycans.

Animals↗
PubMed1992

Proton dependence of the partial reactions of the sodium-proton exchanger in renal brush border membranes.

The pre-steady state time dependence of Na+ accumulation by the Na(+)-H+ exchanger in renal brush border membrane vesicles was investigated at 0 degree C by a manual mixing technique using amiloride to quench the reaction. Dilution of acid-loaded (pHi 5.7) vesicles into an alkaline medium (pHo 7.7) containing 1 mM 22Na+ produced a time course of amiloride-sensitive Na+ uptake that consisted of three distinct phases: 1) a lag, 2) a monoexponential "burst," and 3) a linear or steady state phase. Experiments testing for the presence of 22Na+ backflux, residual Na+ binding to the membrane, and hysteresis were negative, lending support to the hypothesis that the burst phase corresponds to Na+ translocation during the initial turnover of Na(+)-H+ exchanger. Lowering the internal pH increased the amount of na+ uptake in each of the phases without affecting the apparent burst rate, whereas lowering the external pH inhibited Na+ uptake while increasing the duration of the lag phase. The pattern of inhibition produced by external H+ was of the simple competitive type, indicating that Na+ and H+ share a common binding site. Steady state Na+ uptake showed a sigmoidal dependence on internal pH (Hill coefficient = 1.67), consistent with the presence of an internal allosteric H+ activation site. Alkaline loading conditions (pHi 7.7), which favor desaturation of the internal H+ binding sites, completely abolished Na+ uptake in the steady state. In contrast, Na+ accumulation during the burst phase was reduced to 25% of an acid-loaded (pHi 5.7) control. The persistence of the burst phase and the disappearance of steady state Na+ uptake under alkaline loading conditions suggest that recycling of the H(+)-loaded exchanger is a late event in the transport cycle that follows Na+ translocation (ping-pong mechanism) and controls the steady state rate of Na+ accumulation. Activation of the recycling step involves sequential binding of H+ to the allosteric and transport sites, thus accounting for the cooperative dependence of steady state Na+ uptake on the internal [H+].

Allosteric Site↗
PubMed1992

Concurrent changes in sinusoidal expression of laminin and affinity of hepatocytes to laminin during rat liver regeneration.

Distribution of fibronectin, laminin, and collagens type I, III, IV, and V in the lobular regions of regenerating rat liver was studied by indirect immunofluorescence. Little or no laminin was detected in sham-operated controls throughout the experimental period, while it was detected in sinusoids of regenerating liver as early as 6 h after partial hepatectomy (PH). After reaching a maximum at 24 h, it decreased and was barely detectable 6 days after PH. Changes in the other extracellular matrix (ECM) proteins were evident 3 days after PH, but not earlier than 24 h. Hepatocytes isolated from regenerating rat livers were tested in a short term assay for attachment to the substrates coated with the ECM proteins. The attachment of hepatocytes to laminin substrates increased 12 h after PH, reached a maximum at 24 h, and decreased to the control level 6 days after PH, while that of the control remained constant. The attachment to fibronectin substrates was not different between regenerating livers and controls at any time point. The attachment to collagen did not change earlier than 24 h after PH, but increased slightly 3 days after PH. Primary rat hepatocytes cultured on the substrates coated with the ECM proteins were determined for replicative DNA synthesis in response to epidermal growth factor. Both in normal liver and in regenerating liver 24 h after PH, laminin was one of the most effective substrates in supporting the responsiveness of hepatocytes to the growth stimulus. Taken together, these results suggest the importance of hepatocyte-laminin interaction during the early stage of liver regeneration possibly in growth stimulation of hepatocytes and/or maintenance of hepatocyte-specific functions.

Animals↗
PubMed1992

Polymorphisms and deduced amino acid substitutions in the coding sequence of the ryanodine receptor (RYR1) gene in individuals with malignant hyperthermia.

Twenty-one polymorphic sequence variants of the RYR1 gene, including 13 restriction fragment length polymorphisms (RFLPs), were identified by sequence analysis of human ryanodine receptor (RYR1) cDNAs from three individuals predisposed to malignant hyperthermia (MH). All RFLPs were detectable in PCR-amplified products, and their segregation was consistent with our initial finding of linkage to MH in the nine families previously informative for one or more intragenic markers (MacLennan et al., 1990, Nature 343:559-561). Four amino acid substitutions were identified in the study: Arg for Gly248, Cys for Arg470, Leu for Pro1785, and Cys for Gly2059. Of 45 families tested, a single family presented the Arg for Gly248 substitution where it segregated with malignant hyperthermia, making it a candidate mutation for predisposition to MH in man. The other three polymorphic substitutions failed to segregate with malignant hyperthermia in those families in which they occurred, implying that they represent polymorphisms with little or no effect on the function of the RYR1 gene.

Amino Acids↗
PubMed1992

Refinement of diagnostic assays for a probable causal mutation for porcine and human malignant hyperthermia.

The substitutions of T for C1843 in the porcine ryanodine receptor (RYR1) gene, which deletes a HinPI restriction endonuclease site and creates a HgiAI site, and of T for C1840 in human RYR1, which deletes a RsaI site, lead to Cys for Arg substitutions in the ryanodine receptors and are probable causal mutations for malignant hyperthermia (MH). To improve the restriction endonuclease assay of these sites, thereby providing an accurate, reliable diagnosis for MH, introns flanking the exon containing the mutation were sequenced, permitting identification and PCR amplification of a 659-bp porcine gene sequence that contains both constant and variant HgiAI sites and a 922-bp human gene sequence that contains both constant and variant RsaI sites. As a result, these PCR-amplified sequences contain constant internal controls for the reliable differentiation by restriction endonuclease digestion of normal, heterozygous, and MH genotypes.

Animals↗
PubMed1992

Recombinant Leishmania Hsp90 and Hsp70 are recognized by sera from visceral leishmaniasis patients but not Chagas' disease patients.

Approximately 70% of the cDNA clones identified by immunoscreening Leishmania donovani expression libraries with serum from a patient with visceral leishmaniasis (kala-azar) were found to encode the highly conserved Hsp90 and Hsp70 members of the heat shock protein family. Recombinant fusion proteins containing the C-terminal portions of L. donovani Hsp90 and Hsp70 were used as target antigens in enzyme-linked immunosorbent assays of various sera. Sera from four patients with visceral leishmaniasis recognized recombinant Leishmania Hsp90 and Hsp70, while sera from seven patients with Chagas' disease did not, despite the fact that Trypanosoma cruzi Hsp90 and Hsp70 share more than 80% amino acid identity with their counterparts in Leishmania spp. Thus, Leishmania Hsp90 and Hsp70 elicit strong humoral responses and are potential candidates for specific serodiagnostic assays capable of distinguishing between L. donovani and T. cruzi infections.

Amino Acid Sequence↗
PubMed1992

Regulation of sarcoplasmic reticulum gene expression during cardiac and skeletal muscle development.

The expression of major sarcoplasmic reticulum proteins during cardiac and fast-twitch skeletal muscle development was examined using gene-specific probes. Through the use of S1 nuclease mapping, Northern blot, and RNA slot-blot analysis, sarcoplasmic reticulum proteins were shown to exhibit both narrow tissue specificity and plasticity in their expression during muscle development. In fast-twitch skeletal muscle, the cardiac/slow-twitch isoforms of Ca(2+)-ATPase and calsequestrin were detected at high levels in fetal stages but were gradually replaced by fast-twitch isoforms in adult muscle. In contrast, cardiac muscle expressed exclusively cardiac/slow-twitch isoforms of Ca(2+)-ATPase and calsequestrin at all stages. Both fast-twitch and slow-twitch skeletal muscle expressed the same skeletal muscle ryanodine receptor isoform, whereas cardiac muscle expressed a cardiac isoform. Phospholamban expression was restricted to cardiac and slow-twitch skeletal muscle and did not appear in developing fast-twitch skeletal muscle. During in vitro myogenesis of C2C12 cells, the mRNA transcripts encoding sarcoplasmic reticulum proteins were found to be coordinately induced in synchrony with that of contractile protein mRNA. The myogenic factor "myogenin" induced sarcoplasmic reticulum gene transcripts along with contractile protein mRNAs in nonmyogenic cells. These data suggest that the induction of both sarcoplasmic reticulum and contractile protein gene families is under the control of a common myogenic differentiation program.

Actins↗
PubMed1992

The role of the skeletal muscle ryanodine receptor gene in malignant hyperthermia.

Malignant hyperthermia (MH) is an inherited, potentially lethal condition in which sustained muscle contracture with attendant hypermetabolism and hyperthermia is triggered in humans, heterozygous for the gene defect, by inhalational anaesthetics and skeletal muscle relaxants, and in pigs, homozygous for the defect, by stress. Because muscle contracture could result from a defective Ca2+ release channel, we have focussed our attention on the linkage of MH to defects in the gene (RYR1) encoding the skeletal muscle Ca2+ release channel. We have cloned and sequenced human RYR1 cDNA and found restriction fragment length polymorphisms (RFLPs) in the human gene. We also localized RYR1 to human chromosome 19q13.1. Studies of the cosegregation of MH with these RFLPs established RYR1/MH linkage on human chromosome 19q13.1 (lod score of 4.2; recombinant fraction 0.0). We then sequenced MH and normal porcine RYR1 cDNAs. Mutation of C1843 to T, leading to substitution of Cys for Arg615, was the sole amino acid change noted between MH and normal animals. Linkage of this mutation to MH was established in a study of 338 informative meioses (lod score of 102; recombinant fraction 0.0). We identified the corresponding mutation in 1 of 35 human MH families studied and found cosegregation of the mutation and MH. The combination of a high lod score with crossing of a species barrier supports the causal nature of this mutation. Future studies are aimed at finding the major human MH mutations and establishing assays for their accurate diagnosis.

Amino Acid Sequence↗
PubMed1991

Identification of a mutation in porcine ryanodine receptor associated with malignant hyperthermia.

Malignant hyperthermia (MH) causes neurological, liver, and kidney damage and death in humans and major economic losses in the swine industry. A single point mutation in the porcine gene for the skeletal muscle ryanodine receptor (ryr1) was found to be correlated with MH in five major breeds of lean, heavily muscled swine. Haplotyping suggests that the mutation in all five breeds has a common origin. Assuming that this is the causal mutation for MH, the development of a noninvasive diagnostic test will provide the basis for elimination of the MH gene or its controlled inclusion in swine breeding programs.

Amino Acid Sequence↗
PubMed1991

Cosegregation of porcine malignant hyperthermia and a probable causal mutation in the skeletal muscle ryanodine receptor gene in backcross families.

A study of the inheritance of malignant hyperthermia (MH) in the British Landrace breed revealed the same substitution of T for C at nucleotide 1843 in the ryanodine receptor (RYR1) gene that was previously shown to be correlated with MG in five Canadian swine breeds. Cosegregation of the mutation with MH in 338 informative meioses led to a lod score of 101.75 for linkage at Omax = 0.0. The substitution was also associated with a HinPI- BanII+ RsaI- haplotype in this breed, as in the five breeds tested earlier, suggesting its origin in a common founder animal. DNA-based detection of the MH status in 376 MH-susceptible heterozygous (N/n) and homozygous (n/n) pigs was shown to be accurate, eliminating the 5% diagnostic error that is associated with the halothane challenge test and flanking marker haplotyping procedures in current diagnostic use. These results strongly support the view that the substitution of T for C at nucleotide 1843 is the causative mutation in porcine MH and demonstrate the feasibility of rapid, accurate, noninvasive, large-scale testing for porcine MH status using DNA-based tests for the mutation.

Animals↗
PubMed1991

A substitution of cysteine for arginine 614 in the ryanodine receptor is potentially causative of human malignant hyperthermia.

Malignant hyperthermia (MH) is a devastating, potentially lethal response to anesthetics that occurs in genetically predisposed individuals. The skeletal muscle ryanodine receptor (RYR1) gene has been linked to porcine and human MH. Furthermore, a Cys for Arg substitution tightly linked to, and potentially causative of, porcine MH has been identified in the ryanodine receptor. Analysis of 35 human families predisposed to malignant hyperthermia has revealed the presence, and cosegregation with phenotype, of the corresponding substitution in a single family. This substitution, by analogy to the findings in pig, may be causal for predisposition to MH in this family.

Adolescent↗
PubMed1991

Effect of thyroid hormone on the expression of mRNA encoding sarcoplasmic reticulum proteins.

The purpose of this study was to determine the expression of genes encoding various sarcoplasmic reticulum components that are functionally coupled with calcium release, uptake, and storage function during cardiac hypertrophy induced by thyroid hormone. Hyperthyroidism was induced in two groups of rabbits by the injection of 200 micrograms/kg L-thyroxine (T4) daily for 4 days (T4-4-day group) and 8 days (T4-8-day group). Hypothyroidism was induced in another group of rabbits by adding 0.8 mg/ml propylthiouracil to the drinking water for 4 weeks. The relative expression level of mRNA encoding different sarcoplasmic reticulum proteins was determined by RNA slot blot and Northern blot analysis. In hyperthyroid hearts, the steady-state level of cardiac ryanodine receptor mRNA and sarcoplasmic reticulum cardiac/slow-twitch Ca(2+)-ATPase mRNA were both increased to 147% (T4-4-day group) and 186% (T4-8-day group) of control, respectively, but decreased to 71% and 75%, respectively, in hypothyroid ventricles. The mRNA level for phospholamban was decreased in both hyperthyroidism (T4-8-day group, 72%) and hypothyroidism (77%) in these hearts. On the other hand, calsequestrin mRNA levels did not change in hyperthyroid and hypothyroid ventricles. In accord with the changes in Ca(2+)-ATPase mRNA levels, the Ca(2+)-ATPase protein was increased to 199% (T4-8-day group) in hyperthyroid ventricles and decreased to 86% of control in hypothyroid ventricles. The expression levels of ryanodine receptor, Ca(2+)-ATPase, phospholamban, and calsequestrin mRNAs were similarly altered in skeletal muscle tissues from hyperthyroid and hypothyroid rabbits. These results indicate that the mRNA levels of sarcoplasmic reticulum proteins responsible for calcium release and calcium uptake are coordinately regulated in response to changes in thyroid hormone level in both heart and skeletal muscle. These changes in mRNA level should lead to changes in protein levels and thus to altered calcium release and uptake in the chronic stages of hyperthyroidism and hypothyroidism.

Animals↗
PubMed1990

Molecular cloning of cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum.

We have cloned and sequenced cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum. The cDNA, 16,532 base pairs in length, encodes a protein of 4,969 amino acids with a Mr of 564,711. The deduced amino acid sequence is 66% identical with that of the skeletal muscle ryanodine receptor, but analysis of predicted secondary structures and hydropathy plots suggests that the two isoforms exhibit the same topology in both transmembrane and cytoplasmic domains. A potential ATP binding domain was identified at residues 2619-2652, a potential phosphorylation site at residue 2809, and potential calmodulin binding sites at residues 2775-2807, 2877-2898, and 2998-3016. We suggest that a modulator binding domain in the protein lies between residues 2619 and 3016. Northern blot analysis of mRNA from a variety of tissues demonstrated that the cardiac isoform is expressed in heart and brain, while the skeletal muscle isoform is expressed in both fast- and slow-twitch muscle. No ryanodine receptor mRNA was detected in extracts from smooth muscle or any other non-muscle tissue examined. The two receptors are clearly the products of separate genes, and the gene encoding the cardiac muscle ryanodine receptor was localized to chromosome 1.

Amino Acid Sequence↗
PubMed1990

Molecular cloning of cDNA encoding human and rabbit forms of the Ca2+ release channel (ryanodine receptor) of skeletal muscle sarcoplasmic reticulum.

We have cloned cDNAs encoding the rabbit and human forms of the Ca2+ release channel of sarcoplasmic reticulum. The human cDNA encodes a protein of 5032 amino acids, with a molecular weight of 563,584, which is made without an NH2-terminal signal sequence. Amino acid substitutions between rabbit and human sequences were noted in 163 positions and deletions or insertions in eight regions accounted for additional sequence differences between the two proteins. Analysis of the sequence indicates that 10 potential transmembrane sequences in the COOH-terminal fifth of the molecule and two additional, potential transmembrane sequences nearer to the center of the molecule could contribute to the formation of the Ca2+ conducting pore. The remainder of the molecule is hydrophilic and presumably constitutes the cytoplasmic domain of the protein. A 114-120 amino acid motif is repeated four times in the protein, in residues 841-954, 955-1068, 2725-2844, and 2845-2958 and a 16 amino acid part of the motif is repeated twice more in residues 1344-1359 and 1371-1386. Although the channel is modulated by Ca2+, ATP, and calmodulin, no clear high affinity Ca2(+)-binding domain of the EF hand type and no clear high affinity ATP-binding domain were detected in the primary sequence. An acidic sequence in residues 1872-1923 contains 79% glutamate or aspartate residues and this sequence is a potential low affinity Ca2(+)-binding site. Several potential calmodulin-binding sites were observed in the sequence, in the region 2800 to 3050.

Amino Acid Sequence↗
PubMed1989

Transient state kinetic evidence for an oligomer in the mechanism of Na+-H+ exchange.

Pre-steady-state kinetic measurements of 22Na+ uptake by the amiloride-sensitive Na+-H+ exchanger in renal brush border membrane vesicles (BBMV) were performed at 0 degrees C to characterize the intermediate reactions of the exchange cycle. At 1 mM Na+, the initial time course of Na+ uptake was resolved into three separate components: (i) a lag phase, (ii) an exponential or "burst" phase, and (iii) a constant velocity or steady-state phase. Pulse-chase experiments using partially loaded BBMV showed no evidence for 22Na+ back-flux, suggesting that the decline in the rate of Na+ uptake rate following the burst represents completion of the first turnover of the exchanger. Gramicidin completely abolished Na+ uptake, indicating that the burst phase results from the translocation of Na+ rather than from residual Na+ binding to external sites. Raising the [Na+] from 1 to 10 mM at constant pH (internal pH 5.7; external pH 7.7) produced a sigmoidal increase in the amplitude of the burst phase without affecting the lag duration or the apparent burst rate. In contrast, Na+ uptake in the steady state obeyed Michaelis-Menten kinetics. These results suggest that a minimum of two Na+ transport sites must be occupied to activate Na+ uptake in the pre-steady state. The transition to Michaelis-Menten kinetics in the steady state can be explained by a "flip-flop" or alternating site mechanism in which the functional transport unit is an oligomer and only one promoter per cycle is allowed to form a translocation complex with Na+ after the first turnover.

Amiloride↗
PubMed1989

Trypanosoma cruzi expresses diverse repetitive protein antigens.

We screened a Trypanosoma cruzi cDNA expression library with human and rabbit anti-T. cruzi sera and identified cDNA clones that encode polypeptides containing tandemly arranged repeats which are 6 to 34 amino acids in length. The peptide repeats encoded by these cDNAs varied markedly in sequence, copy number, and location relative to the polyadenylation site of the mRNAs from which they were derived. The repeats were specific for T. cruzi, but in each case the sizes of the corresponding mRNAs and the total number of repeat copies encoded varied considerably among different isolates of the parasite. Expression of the peptide repeats was not stage specific. One of the peptide repeats occurred in a protein with an Mr of greater than 200,000 and one was in a protein of Mr 75,000 to 105,000. The frequent occurrence and diversity of these peptide repeats suggested that they may play a role in the ability of the parasite to evade immune destruction in its invertebrate and mammalian hosts, but the primary roles of these macromolecules may be unrelated to the host-parasite relationship.

Amino Acid Sequence↗