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T Takumi

Publications and source records attributed to T Takumi.

At least 37 records · Page 2Linked to original sources
PubMed1995

A novel ATP-dependent inward rectifier potassium channel expressed predominantly in glial cells.

We have isolated a novel inward rectifier K+ channel predominantly expressed in glial cells of the central nervous system. Its amino acid sequence exhibited 53% identity with ROMK1 and approximately 40% identity with other inward rectifier K+ channels. Xenopus oocytes injected with cRNA derived from this clone expressed a K+ current, which showed classical inward rectifier K+ channel characteristics. Intracellular Mg.ATP was required to sustain channel activity in excised membrane patches, which is consistent with a Walker type-A ATP-binding domain on this clone. We designate this new clone as KAB-2 (the second type of inward rectifying K+ channel with an ATP-binding domain). In situ hybridization showed KAB-2 mRNA to be expressed predominantly in glial cells of the cerebellum and forebrain. This is the first description of the cloning of a glial cell inward rectifier potassium channel, which may be responsible for K+ buffering action of glial cells in the brain.

Adenosine Triphosphate↗
PubMed1995

Molecular basis of IsK protein regulation by oxidation or chelation.

Slowly activating IsK channels were expressed in Xenopus oocytes and exposed to oxidative agents. Oxidative treatment reduced the resulting current IsK, while no inhibition was observed for IsK protein mutants carrying a Ser mutation instead of a highly conserved Cys residue in the intracellular domain. In contrast, Hg2+, which may not only oxidize thiol groups but also form chelates with dibasic amino acids, caused a use-dependent, positive regulation of IsK. This effect was reversed in an IsK protein mutant with a deletion in the extracellular domain. These data suggest opposite effects of peroxides and Hg2+ on IsK, a peroxide-mediated IsK inhibition by intracellular oxidation and a Hg(2+)-mediated IsK increase, caused by extracellular Hg2+ chelation of the IsK protein.

Animals↗
PubMed1995

GH3 pituitary tumor cells contain heteromeric type I and type II receptor complexes for transforming growth factor beta and activin-A.

Transforming growth factors beta (TGF-beta s) and activins induce and inhibins block secretion of follicle-stimulating hormone by rat GH3 pituitary tumor cells. Cheifetz et al. (Cheifetz, S., Ling, N., Guillemin, R., and Massagué, J. (1988) J. Biol. Chem. 263, 17225-17228) reported that GH3 cells express a approximately 50-kDa surface protein, termed the type IV TGF-beta receptor, that directly binds all of these peptide hormones. Here we show that GH3 cells express the previously identified type I and type II receptors for TGF-beta and activin-A. Immunoprecipitation of affinity-labeled surface binding proteins with antisera specific to known receptors demonstrated independent heteromeric complexes of TGF-beta types I and II receptors and of activin types I and II receptors. As judged by ligand-binding and cross-linking analysis, TGF-beta binding to the TGF-beta receptors is not inhibited by activin-A and activin-A binding to its receptors is not inhibited by TGF-beta. Screening of a cDNA library from GH3 cells for potential receptor serine-threonine kinases yielded the known types I and II TGF-beta and activin receptors. The presumed common intracellular signaling pathway for TGF-beta and activin in GH3 cells appears to be mediated by distinct cell-surface receptors.

Activin Receptors↗
PubMed1995

Molecular characterization of a type I serine-threonine kinase receptor for TGF-beta and activin in the rat pituitary tumor cell line GH3.

GH3 pituitary tumor cells have surface receptors for transforming growth factors-beta (TGF-beta s) and activins/inhibins. GH3 cell mRNA was screened by a novel reverse transcriptase-polymerase chain reaction technique with primers for receptor serine-threonine kinases. We isolated rat homologs of previously identified clones for type I (ALK-2 and ALK-5) and type II (ActRII, TGF-beta RII) activin and TGF-beta receptors, together with a novel clone, whose full-length version was isolated from a GH3 cell cDNA library. Named B1, it encodes a 505-amino-acid protein belonging to the family of type I receptor serine/threonine kinases. The kinase domain of B1 exhibits 90% identity to that of the TGF-beta type I receptor. B1 mRNA is expressed not only in pituitary cells but also in all other cells and tissues examined. B1 protein can be expressed on the cell surface, but cannot bind ligand unless a type II receptor is also present. When coexpressed with the type II receptors specific for TGF-beta or activin, B1 can be efficiently cross-linked to either ligand, suggesting that it can form heteromeric complexes with both type II receptor subunits.

Activin Receptors↗
PubMed1994

Blockade of human IsK channels expressed in Xenopus oocytes by the novel class III antiarrhythmic NE-10064.

cRNA encoding the human IsK protein was injected into Xenopus oocytes and the expressed channels were investigated using the two-microelectrode voltage-clamp method. The novel class III antiarrhythmic NE-10064 (1-[[[5-(4-chlorophenyl)-2-furanyl]methylene]-amino]-3- [4-(4-methyl-1-piperazinyl)-butyl]-2,4-imidazolidinedione dihydrochloride) was tested for its ability to block these channels. The compound displayed potent inhibitory effects with an EC50 of 5.4 microM. The block caused by NE-10064 was use-dependent, i.e. channels had to be activated for the inhibition to occur. Further, the reversal of the inhibition during the wash-out period was use-dependent. Finally, the blockade of human IsK channels by NE-10064 appeared to be voltage-dependent, being more pronounced at depolarized potentials. We conclude that this novel class III antiarrhythmic is a potent inhibitor of human IsK channels and suggest that such effects could be involved in its antiarrhythmic action.

Animals↗
PubMed1992

Amplification of ten deletion-rich exons of the dystrophin gene by polymerase chain reaction shows deletions in 36 of 90 Japanese families with Duchenne muscular dystrophy.

We analyzed DNA samples taken from 95 Duchenne muscular dystrophy (DMD) patients belonging to 90 different families in Japan using the polymerase chain reaction. Ten different regions at the 5' end or in the central region of the dystrophin cDNA gene that were previously shown to be prone to deletion were selected for amplification and analysis. Patients in 36 of the 90 families (40%) had deletions in at least one of these segments of the gene. Identical deletions were detected in the dystrophin gene of patients from the same family. The deletions were heterogeneous in size and location. One patient had deletions in 7 of the 10 amplified regions, while 19 patients from 18 families had a deletion in only one of the regions studied. Deletions at the 5' end were generally larger and more heterogeneous than those in the central region of the gene. One third of deletions had their proximal end breakpoints between exons 44 and 45. This region seems to be particularly vulnerable to gene breakage in DMD patients.

Asian People↗
PubMed1991

Alteration of channel activities and gating by mutations of slow ISK potassium channel.

ISK is a small membrane protein consisting of 129-130 amino acid residues with a single putative transmembrane domain and induces a very slow voltage-dependent K+ channel activity in the Xenopus oocyte expression system. We investigated the nature and structure-function relation of ISK by examining the effects of various mutations of ISK on the K+ channel activities measured in Xenopus oocytes. Deletion and truncation of the ISK protein indicated that the 63-amino acid sequence covering a transmembrane domain is sufficient for eliciting a K+ channel activity characteristic of ISK. Amino acid substitutions at a total of 31 positions within and surrounding the transmembrane domain caused different effects on the channel activity. A channel activity was enhanced by substitution of leucine with isoleucine at position 52 within the transmembrane domain, and the kinetic analysis of this mutation indicated that the enhancement of the channel activity is due to an alteration of a gating property of the ISK protein and thus supported the view that ISK forms an integral part of the K+ channel itself. The substitutions at many positions of the membrane-following region produced drastic reduction of the channel activity, and this is in marked contrast to the lack of effects of amino acid substitutions at the membrane-preceding region. Thus, the cytoplasmic portion immediately following the transmembrane domain plays a crucial role in inducing the channel activity of ISK.

Amino Acid Sequence↗
PubMed1991

Screening of gene deletions by polymerase chain reaction in Japanese patients with Duchenne muscular dystrophy.

Gene deletions were screened in 49 Japanese Duchenne muscular dystrophy patients from 43 families, using the polymerase chain reaction. Enzymatic amplification was carried out on six regions prone to deletion. Fifteen of 43 families (33%) had gene deletions in at least one of the six regions. This frequency was almost the same as that previously reported in Caucasians. The mid-part of the dystrophin gene was the location most frequently deleted. The frequency of deletion of the region encompassing exon 45 was higher in Japanese families (18.4%) than in Caucasians.

Blotting, Southern↗
PubMed1991

Cellular localization of rat Isk protein in the stria vascularis by immunohistochemical observation.

A novel rat membrane protein, termed Isk protein, that exhibits a voltage-dependent potassium channel activity was first reported through molecular cloning combined with an electrophysiological assay (Takumi et al., 1988). In the present study, we made an attempt to identify the cellular localization of the rat Isk protein in the stria vascularis using two types of antibodies that specifically react with the distinct parts of the rat Isk protein. Immunohistochemical analysis showed that the rat Isk protein was present only on the endolymphatic surface of the marginal cell. The possibility that the Isk protein is involved in potassium permeation in the luminal membrane of the marginal cell will be also discussed.

Amino Acid Sequence↗
PubMed1991

Sideroblastic anemia showing unique response to pyridoxine.

We treated and followed up for 6 years a patient with pyridoxine-responsive sideroblastic anemia. The patient was a boy age 1 year and 9 months, who was diagnosed on the basis of peripheral red cell morphology and an increased number of sideroblasts in the bone marrow. Bone marrow erythroblasts showed a marked reduction of delta-aminolevulinic acid synthase (ALA-S) activity. The response of the patient to pyridoxine and its active form, pyridoxal phosphate, was unique. After the first course of pyridoxal phosphate therapy (300 to 500 mg/day i.v. for 4 days), all hematological data were restored to normal and remained normal for 29 months without the further administration of pyridoxal phosphate. The second course of pyridoxal phosphate therapy (500 mg/day i.v. for 2 days) was effective for 6 months. The third, fourth, and fifth courses of the therapy consisted of daily oral pyridoxine hydrochloride at a dose of 180 mg/day for 4 to 6 weeks, and the respective periods of hematological remission were 7, 12, and greater than 18 months. These observations suggest the presence of a complicated ALA-S activating or inactivating system, or both, in our patient.

5-Aminolevulinate Synthetase↗
PubMed1991

Exon skipping during splicing of dystrophin mRNA precursor due to an intraexon deletion in the dystrophin gene of Duchenne muscular dystrophy kobe.

Recent molecular studies have shown that in a patient with Duchenne muscular dystrophy (DMD) Kobe, the size of exon 19 of the dystrophin gene was reduced to 36 bp due to the deletion of 52 bp out of 88 bp of the exon. The consensus sequences at the 5' and 3' splice sites of exon 19 were unaltered (Matsuo, M., et al. 1990. Biochem. Biophys. Res. Commun. 170:963-967). To further elucidate the molecular nature of the defect, we examined the primary structure of cytoplasmic dystrophin mRNA of the DMD Kobe patient across the junctions of exons 18, 19, and 20 by gel electrophoresis and sequencing of polymerase chain reaction-amplified cDNA. The mRNA coding for dystrophin was reverse transcribed using random primers, and the cDNA was then enzymatically amplified in vitro. The targeted fragment was smaller than expected from the genomic DNA analysis. By sequencing of the amplified product, we found that exon 18 was joined directly to exon 20, so that exon 19 was completely absent, suggesting that this exon was skipped during processing of the dystrophin mRNA precursor. All other bases in the amplified product were unaltered. Therefore, the data strongly suggest that the internal exon deletion generates an abnormally spliced mRNA in which the sequence of exon 18 is joined to the sequence of exon 20. We propose that the deletion is responsible for abnormal processing of the DMD Kobe allele. This finding has important implications regarding the determinants of a functional splice site.

Base Sequence↗
PubMed1990

A very small frame-shifting deletion within exon 19 of the Duchenne muscular dystrophy gene.

We report the molecular characterization of a Japanese Duchenne muscular dystrophy (DMD) patient. The analysis of genomic gene by polymerase chain reaction indicates that the individuals have a limited deletion within an amplified region, which encompasses exon 19 of DMD gene. The amplified region was sequenced. Comparison of the deletion joint sequence with the normal amplified region sequence indicates that both 5' and 3' deletion end points are present within exon 19 and the deletion removes total 52 bp out of 88 bp of exon 19. Both his mother and sister are carriers of the deletion-containing allele. The mutation introduces a termination codon at residue 791 in exon 20, and is predicted to result in the production of a severely truncated protein. This sort of deletion (designated as DMD-Kobe) might be classified as a new type of DMD gene abnormality.

Adolescent↗
PubMed1990

Immunohistochemical study of a rat membrane protein which induces a selective potassium permeation: its localization in the apical membrane portion of epithelial cells.

We previously reported a novel rat membrane protein that exhibits a voltage-dependent potassium channel activity on the basis of molecular cloning combined with an electrophysiological assay. This protein, termed IsK protein, is small and different from the conventional potassium channel proteins but induces selective permeation of potassium ions on its expression in Xenopus oocytes. In this investigation, we examined cellular localization of rat IsK protein by preparing three different types of antibody that specifically reacts with a distinct part of rat IsK protein. Immunohistochemical analysis using these antibody preparations demonstrated that rat IsK protein is confined to the apical membrane portion of epithelial cells in the proximal tubule of the kidney, the submandibular duct and the uterine endometrium. The observed tissue distribution of rat IsK protein was consistent with that of the IsK protein mRNA determined by blot hybridization analysis. In epithelial cells, the sodium, potassium-ATPase pump in the basolateral membrane generates a sodium gradient across the epithelial cell and allows sodium ions to enter the cell through the apical membrane. Thus, taking into account the cellular localization of the IsK protein, together with its electrophysiological properties, we discussed a possible function of the IsK protein, namely that this protein is involved in potassium permeation in the apical membrane of epithelial cells through the depolarizing effect of sodium entry.

Amino Acid Sequence↗
PubMed1990

Generation of transgenic mice with elevated blood pressure by introduction of the rat renin and angiotensinogen genes.

The role of the renin-angiotensin system in blood pressure control and in the development of hypertension was investigated by generating transgenic mice carrying the rat renin or angiotensinogen gene or both genes under the control of the mouse metallothionein I promoter. The systolic blood pressure was significantly elevated in transgenic mice carrying both transgenes but was maintained normally in those bearing either of the transgenes. The transgene was effectively and properly transcribed to form the mature mRNA in the transgenic mice. The production of rat renin and angiotensinogen in the transgenic mice carrying the corresponding transgene was also verified by immunoanalyses of these proteins. Furthermore, the specific angiotensin-converting enzyme inhibitor captopril was effective in reducing the elevated blood pressure of the hypertensive transgenic mice. These results indicate that the combined action of the exogenous rat renin and angiotensinogen is responsible and necessary for elevation of blood pressure in the hypertensive transgenic mice.

Angiotensinogen↗
PubMed1989

Molecular cloning and sequence analysis of human genomic DNA encoding a novel membrane protein which exhibits a slowly activating potassium channel activity.

The amino acid sequence for a novel human membrane protein that induces selective potassium permeation by membrane depolarization was deduced by molecular cloning and sequence analysis of its genomic DNA. This protein consists of 129 amino acid residues and shares several structural characteristics with the rat counterpart. These include a single putative transmembrane domain surrounded by many charged amino acid residues, two potential N-glycosylation sites at the amino-terminal portion and a single cysteine residue at the carboxyl-terminal portion. The transmembrane domain and its flanking carboxyl-terminal sequence are highly conserved between the human and rat sequences. Because the slowly activating potassium current elicited by the human protein on its expression in Xenopus oocytes is indistinguishable from that induced by the rat protein, the sequence conserved at the transmembrane domain and its following sequence should play an essential role in the induction of selective K+ permeation.

Amino Acid Sequence↗