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K Takimoto

Publications and source records attributed to K Takimoto.

At least 55 records · Page 3Linked to original sources
PubMed1996

Altered K+ channel subunit composition following hormone induction of Kv1.5 gene expression.

Diverse voltage-gated K+ channels are produced by tetramerization of channel alpha subunits that can be encoded by multiple genes. In GH3 pituitary cells, the Kv1.4 and Kv1.5 subunit genes are constitutively expressed, and dexamethasone selectively up-regulates expression of the latter gene. Here we use subunit-specific antibodies to determine the effect of Kv1.5 gene induction on expression of homomeric and heteromeric channels. In control cells, almost all detectable Kv1.4 proteins are associated with Kv1.5 protein, whereas only approximately 30% Kv1.5 protein is present as Kv1.4/Kv1.5 heteromeric channels. Dexamethasone treatment doubled expression of Kv1.5 homomeric channels but only modestly up-regulated Kv1.4/Kv1.5 heteromeric complexes. The steroid treatment similarly increased total cellular and cell surface Kv1.5 protein without affecting Kv1.4 proteins in either fraction. Thus, hormone induction of one K+ channel subunit gene differentially influences expression of cell surface homomeric and heteromeric channels leading to specific alteration of excitability.

Animals↗
PubMed1996

Characterization of the rat Class 3 aldehyde dehydrogenase gene promoter.

The Class 3 aldehyde dehydrogenase gene (ALDH-3) is differentially expressed. Expression is either constitutive or xenobiotic inducible via an aromatic hydrocarbon (Ah) receptor-mediated pathway, depending upon the tissue. A series of studies were performed to examine the regulation of rat ALDH-3 basal expression. DNase I footprint analysis identified four DNA regions within the proximal 1 kb of the 5' flanking region of rat ALDH-3 which interact with regulatory proteins. Reporter gene and gel mobility shift assays indicate that Sp1-like proteins interact with two proximal DNase I footprinted sites to confer strong promoter activity. Two distal DNase I footprinted sites are found within a region that inhibits rat ALDH-3 promoter activity. This negative region is bound by NF1-like proteins and/or unique proteins. This 1 kb 5' flanking region of rat ALDH-3 may act constitutively in many cell types. In contrast with other Ah receptor regulated genes, no DNA elements or transcription factors acting within this region appear to be involved in regulating xenobiotic-inducible expression of rat ALDH-3.

Aldehyde Dehydrogenase↗
PubMed1996

Cryopreservation of isolated rat islets of Langerhans in the presence of ethylene glycol or dimethyl sulfoxide: evaluation of toxicity and the dynamic pattern of subsequent insulin release in vitro.

The toxic effect of ethylene glycol (EG) on the pattern of dynamic insulin release from rat pancreatic islets with or without freezing was investigated in comparison with that of dimethyl sulfoxide (Me2SO). Sixty islets were perifused (1 ml/min) consecutively with D-glucose (1.67 mM for 30 min followed by 16.7 mM for 60 min and 1.67 mM for 60 min) after exposure to 2.0 M EG or Me2SO for 1 h at either 22 or 0 degrees C. During the second period of perifusion, the insulin output from islets exposed to Me2SO or EG at 22 degrees C decreased to 53 and 51% of that from nontreated control islets, respectively. On the other hand, the islets exposed to EG at 0 degrees C exhibited 86% of the control insulin output under the same perifusion conditions, and this appeared to be higher than that of islets exposed to Me2SO (60%) at 0 degrees C. Frozen islets, after exposure to 2.0 M EG or Me2SO for 1 h at 0 degrees C, responded positively to 16.7 mM D-glucose, and the typical biphasic pattern of insulin secretion was observed. The insulin output from these islets during the second period of perifusion was not comparable to that from unfrozen control islets. In particular, the mean insulin output of EG-cryopreserved islets during the second period accounted for 99% of that from unfrozen control islets. The present findings suggest the possible use of EG as an alternative cryoprotectant to Me2SO.

Animals↗
PubMed1996

Dexamethasone rapidly increases calcium channel subunit messenger RNA expression and high voltage-activated calcium current in clonal pituitary cells.

Glucocorticoid hormones increase voltage-gated Ca(2)+ current density in clonal pituitary cells. To test whether these steroids might stimulate expression of Ca(2)+ channel genes, messenger RNase protection assays were used to measure alpha IC and alpha ID RNAs that encode pore-forming subunits of L-type Ca2+ channels. We show here that dexamethasone rapidly increases alpha IC messenger RNA expression without affecting alpha ID messenger RNA level. This up-regulation of channel messenger RNA is also produced by natural glucocorticoids and is blocked by the glucocorticoid antagonist Ru48386. The up-regulation of the channel subunit messenger RNA expression is associated with an increase in high voltage-activated Ca(2)+ current density. Thus, glucocorticoids may produce a long-term effect on Ca(2)+ homeostasis in clonal pituitary cells by differentially regulating expression of Ca(2)+ channel subunit genes.

Animals↗
PubMed1996

The cDNA sequence encoding bovine SP-22, a new defence system against reactive oxygen species in mitochondria.

We have isolated cDNA clones coding SP-22, an antioxidant protein in mitochondria, from a bovine adrenal medulla cDNA library constructed with (lambda)gt11. The largest clone contained the entire coding sequence for mature SP-22. Since the isolated cDNA clones lacked 5'- and 3'-ends, we determined the sequences of both ends by the "Rapid Amplification of cDNA Ends (RACE)" tecnique. The deduced amino acid sequence of the mature protein region was the same as that determined by protein sequencing. Since SP-22 had a mitochondrial targetting signal, its mitochondrial localization was confirmed.

Adrenal Medulla↗
PubMed1996

Lysosomal glycolipid storage in the renal tubular epithelium in mastomys (Praomys coucha).

The renal proximal tubular epithelium of MCC strain of mastomys (Praomys coucha) exhibited a number of cytoplasmic vacuoles after conventional paraffin-embedding procedures. These vacuoles were strongly PAS-positive in cryostat sections. Ultra-structurally, they were double membrane-bound structures filled with myelin figures and acid phosphatase-positive electron-dense matrix. Immunofluorescent microscopy revealed that these structures contained GM2 ganglioside. Other tissues or organs were histologically normal. Mating experiments indicated that the ganglioside storage in MCC mastomys is inherited as an autosomal recessive trait.

Animals↗
PubMed1996

Dexamethasone and stress upregulate Kv1.5 K+ channel gene expression in rat ventricular myocytes.

Hormones may produce long-term effects on excitability by regulating K+ channel gene expression. Previous studies demonstrated that administration of dexamethasone, a glucocorticoid receptor agonist, to adrenalectomized rats, rapidly induces Kv1.5 K+ channel expression in the ventricle of the hear. Here, RNase protection assays and Northern blots are used to examine the cell type specificity of dexamethasone action and to test whether Kv1.5 gene expression can be regulated by a physiological stimulus. We show that Kv1.5 mRNA expression in the central nervous system is highest in the hypothalamus. However, dexamethasone treatment of adrenalectomized rats fails to affect Kv1.5 mRNA levels in hypothalamus or lung. In contrast, dramatic upregulation of Kv1.5 mRNA is seen in skeletal muscle and pituitary. Increased Kv1.5 message also found in isolated ventricular cardiomyocytes following in vivo treatment with dexamethasone. Finally, it is shown that cold stress of intact rats significantly increases cardiac Kv1.5 mRNA expression. We conclude that dexamethasone induction of Kv1.5 gene is tissue-specific. Furthermore, our results suggest that stress may act via glucocorticoids to increase Kv1.5 gene expression in ventricular cardiomyocytes. Hence, K+ channel gene expression can be influenced by physiological and pharmacological stimuli.

Adrenalectomy↗
PubMed1995

Possible function of SP-22, a substrate of mitochondrial ATP-dependent protease, as a radical scavenger.

SP-22 was found to be a substrate protein of a mitochondrial ATP-dependent protease in bovine adrenal cortex. Its amino acid sequence was homologous to that of some prokaryotic and eukaryotic proteins such as thioredoxin peroxidase (formerly called thiol-specific antioxidant) in yeast and mammalian brains and the C22 component of alkyl hydroperoxide reductase in Salmonella typhimurium. In the present study, we found SP-22 to have the ability to scavenge reactive oxygen species, thus protecting radical-sensitive proteins such as tryptophan hydroxylase, glutamine synthetase and hemoglobin from oxidation. The protecting activity was enhanced by the addition of horse serum. The "serum factor(s)" seemed to be protein(s), since the physiological roles of SP-22 in adrenocortical mitochondria are discussed.

ATP-Dependent Proteases↗
PubMed1995

Membrane depolarization inhibits Kv1.5 voltage-gated K+ channel gene transcription and protein expression in pituitary cells.

Voltage-gated K+ channels play an essential role in the production of action potential activity by excitable cells. Recent studies have suggested that expression of K+ channel genes may be regulated by stimuli that affect electrical activity. Elevating the concentration of extracellular KCl causes membrane depolarization and, thus, is widely used for studying electrical activity-dependent changes in neurons, muscle, and endocrine cells. Here we show that elevated KCl decreases Kv1.5 K+ channel mRNA expression in clonal pituitary cells without affecting Kv1.4 and Kv2.1 mRNA levels. K+ channel blockers, which cause depolarization, also produce down-regulation of Kv1.5 mRNA, while NaCl addition had no effect. Thus, the effect of KCl is mediated by K(+)-induced membrane depolarization. Unlike many known effects of K+, down-regulation of Kv1.5 mRNA does not require Ca2+ or Na+ influx, or Na(+)-H+ exchange. Furthermore, the decrease in Kv1.5 mRNA expression is due to inhibition of channel gene transcription and persists after inhibition of protein synthesis, excluding a role for induction of intermediary regulatory proteins. Finally, immunoblots with antibody specific for the Kv1.5 polypeptide show that depolarization for 8 h reduces the expression of Kv1.5 channel protein. The decrease in K+ channel protein expression caused by depolarization-induced Ca(2+)-independent inhibition of Kv1.5 gene transcription may produce a long-term enhancement of pituitary cell excitability and secretory activity.

Amiloride↗
PubMed1995

Multiple protein kinases are required for basal Kv1.5 K+ channel gene expression in GH3 clonal pituitary cells.

The role of protein kinases in maintaining basal expression of voltage-gated K+ channel mRNA was examined in GH3 clonal pituitary cells. Nonspecific inhibition of protein kinases with H7 or staurosporine markedly decreases Kv1.5 K+ channel gene transcription and mRNA without producing a substantial change in Kv1.4 mRNA. Selective inhibitors for protein kinase C, Ca(2+)-calmodulin kinases, and tyrosine kinases do not affect Kv1.5 mRNA expression. In contrast, the Rp-diastereomer of adenosine 3',5'-cyclic monophosphorothioate, a specific inhibitor of protein kinase A, partially inhibits Kv1.5 mRNA expression (approximately 40%), and this effect was antagonized by 8-bromo-adenosine 3',5'-cyclic monophosphate. Thus, protein kinase A and at least one other kinase are required for basal Kv1.5 mRNA expression in pituitary cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Differential freezing tolerance of rat pancreatic islets depending on their size variation.

We have investigated the freezing tolerance of rat pancreatic islets. Freshly isolated rat pancreatic islets were divided into three groups based on their longest diameter (small; 100 - 200 microns, medium; 201 - 300 microns, large; > 300 microns). They were then cryopreserved at a slow cooling rate (-0.3 degrees C/min) in the presence of dimethyl sulfoxide (Me2SO) or ethylene glycol (EG). After storage at -196 degrees C for 1 - 4 weeks, they were thawed and their ability to secrete insulin in response to fluctuations in glucose concentration was examined during three consecutive static incubations in vitro (1st; 2.8 mM, 2nd; 16.7 mM, 3rd; 2.8 mM). Morphological examination of the beta-granule population was determined by image analysis, and correlation with islets size was analyzed. The amount of insulin released from large-sized islets was significantly suppressed in EG (p < 0.05) and Me2SO (p < 0.01) groups compared to unfrozen islets. However, the mean volume of the large-sized islets isolated from one rat accounted for 43.0% of the total volume. On the other hand, the amount of insulin released from small- and medium-sized islets did not differ from those of unfrozen islets, and their mean volumes were 13.2 and 43.8% respectively. The percentage of cells with beta-granules was significantly correlated with size in both EG (r = -0.52) and Me2SO (r = -0.35) groups, but no significant correlation was observed in the unfrozen islets groups. These findings suggest that large-sized islets are more susceptible to freezing injury than small- or medium-sized islets. Moreover, the volume distribution of isolated islets indicated that it may be important to retain the ability of insulin secretion from the large-sized islets.

Animals↗
PubMed1995

Inhibition of voltage-gated K+ channel gene expression by the neuropeptide thyrotropin-releasing hormone.

Many neurotransmitters regulate action potential activity in neuronal, endocrine, and cardiac cells by rapidly modulating the gating of K+ channels. Neurotransmitters might also produce prolonged effects on excitability by regulating the expression of K+ channel genes. Here we show that the neuropeptide thyrotropin-releasing hormone (TRH) down-regulates Kv1.5 and Kv2.1 K+ channel mRNAs in clonal pituitary cells. The effect on Kv1.5 mRNA expression does not require protein synthesis and is due to decreased transcription. Immunoblots demonstrate that Kv1.5 and Kv2.1 immunoreactivities are significantly reduced by TRH within 12 hr. The change in channel protein expression is associated with a decrease in voltage-gated K+ currents. Thus, TRH enhances excitability by inhibiting K+ channel gene expression. Neuropeptide regulation of K+ channel gene expression may produce long-term changes in neuronal action potential activity and synaptic transmission.

Calcium Channels↗
PubMed1994

Structure of the 5' flanking region of class 3 aldehyde dehydrogenase in the rat.

Class 3 aldehyde dehydrogenase (ALDH-3) is induced by exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and during chemical carcinogenesis. These inductions as well as the basal expression of ALDH-3 vary significantly in different organs. In order to identify DNA elements controlling ALDH-3 expression, we have cloned and analyzed approximately 5.5 kb of the 5' flanking region of the ALDH-3 gene. Deletion analysis showed that the 5' flanking region contains at least three functional domains: a strong promoter proximal to the transcription start site, inhibitory regions upstream of the promoter, and TCDD-responsive enhancers. The TCDD-responsive enhancers in the ALDH-3 gene were functionally similar to xenobiotic responsive elements in the cytochrome P450IA1 gene. These results indicate that transcription of the ALDH-3 gene is controlled by cooperation of at least three functional domains.

Aldehyde Dehydrogenase↗
PubMed1994

Spectrum of proton-induced mutagenesis of the Escherichia coli crp gene.

Mutation of the adenosine 3',5'-cyclic monophosphate receptor protein gene (crp) of Escherichia coli induced by protons, ionizing radiation of charged particles, was analyzed to determine the specificity of the mutational spectrum. The majority, 44 of 49 mutations detected, were base substitutions, and three frameshifts and two gross structural changes were also found. Base substitutions included 35 transversions and nine transitions. G:C to T:A transversions were the dominant type of base substitution, followed by G:C to C:G and A:T to T:A transversions. Almost all transitions were eight G:C to A:T changes. The spectrum of proton mutagenesis was quite different from that of X-ray mutagenesis of the crp gene, in which G:C to A:T transitions dominated.

Base Sequence↗
PubMed1994

Molecular cloning and sequencing of cDNA that encodes cysteine proteinase in the eggs of the silkmoth, Bombyx mori.

We have isolated and sequenced a 1,486-base-pair near full-length cDNA coding for Bombyx egg cysteine proteinase. The cDNA encodes 344 amino acid residues containing a typical signal peptide sequence (16 residues), pro-peptide (104 residues), and the sequence for mature enzyme (224 residues). Sequence alignments show that the egg cysteine proteinase is similar to lobster cysteine proteinase (61% identity), barley cysteine proteinase, Aleurain (52%), rice cysteine proteinase, Oryzain (54%), and rat cathepsin L (59%). The amino-terminal sequencing of the egg cysteine proteinase indicates that the enzyme purified as an inactive form from eggs is a pro-enzyme. Pro-egg cysteine proteinase was detected in other silkmoth tissues such as ovary, fat body, hemocyte, and hemolymph by immunoblotting.

Amino Acid Sequence↗
PubMed1994

AP-811, a novel ANP-C receptor selective agonist.

AP-811 is a derivative of the Phe8-Ile15 region of atrial natriuretic peptide (ANP) and is one of the smallest linear ligands for ANP receptors. The binding and agonist activities of AP-811 have been compared with those of other ANP analogs for the ANP-A and ANP-C receptors. AP-811 binds with a high binding affinity to and is a strong agonist for the ANP-C receptor, indicating that the binding and agonist sites for this receptor are the same or near each other in the ANP sequence. In contrast, AP-811 showed no agonistic effect for the ANP-A receptor, although it could bind to this receptor. Comparing the biological activities of AP-811 with those of other ANP analogs, we propose that the binding and agonist sites for the ANP-A receptor may consist of separate regions of ANP. In conclusion, AP-811 is the smallest C-receptor-selective agonist.

Adenylyl Cyclase Inhibitors↗
PubMed1994

Glucocorticoid induction of Kv1.5 K+ channel gene expression in ventricle of rat heart.

Multiple voltage-gated K+ channels contribute to the repolarization phases of the cardiac action potential and are targets of several antiarrhythmic drugs. The Kv1.5 K+ channel gene is expressed in the heart, and heterologous expression of this gene generates a slowly inactivating K+ current. Previously, we found that glucocorticoids specifically upregulate pituitary Kv1.5 gene expression. To test whether these steroids might also induce Kv1.5 gene expression in the heart, cardiac channel mRNA and protein were measured by RNase protection assay and by immunoblotting with antibody specific for the extracellular domain of Kv1.5 polypeptide. Kv1.5 mRNA and immunoreactive protein appeared to be more abundant in rat ventricle than atrium. Reduction of endogenous glucocorticoids by adrenalectomy decreased ventricular Kv1.5 mRNA approximately 8-fold, which was estimated by using cyclophilin mRNA as an internal control. Kv1.5 immunoreactive protein also decreased approximately 6-fold. Injection of dexamethasone into adrenalectomized rats acted within a day to increase ventricular Kv1.5 mRNA and immunoreactive protein approximately 50-fold and approximately 20-fold, respectively. In contrast, atrial Kv1.5 mRNA expression was unaffected by either adrenalectomy or injection of the glucocorticoid agonist. Furthermore, dexamethasone-induced upregulation was specific for Kv1.5, since whole-heart Kv1.4 and Kv2.1 mRNA levels, as well as ventricular Kv2.1 mRNA expression, were unchanged. Thus, dexamethasone specifically upregulates Kv1.5 K+ channel gene expression in rat ventricle but not atrium. Glucocorticoids may affect excitability of ventricular myocytes and the efficacy of clinically useful drugs by changing the expression of the Kv1.5 K+ channel.

Adrenalectomy↗