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Biomedical subjects

S Okuno

Publications and source records attributed to S Okuno.

At least 109 records · Page 6Linked to original sources
PubMed1998

Critical amino acid residues of AIP, a highly specific inhibitory peptide of calmodulin-dependent protein kinase II.

The importance of the individual amino acid residues of AIP (KKALRRQEAVDAL), a highly specific inhibitor of calmodulin-dependent protein kinase II (CaMKII), was studied. Replacement of Arg6, Gln7, or Ala9 by other amino acid residues produced a marked increase in the IC50 value. Leu4 and Val10 were also sensitive to replacement, but some hydrophobic amino acids could substitute for these residues. Although replacement of Ala3, Glu8, Ala12, and Leu13 by other residues produced no significant increase in the IC50, the substitution of Lys for Ala3 decreased the IC50. An AIP analog (KKKLRRQEAFDAY), in which Ala3 and Val10 were replaced with Lys and Phe, respectively, showed an IC50 value as low as 4 nM, suggesting that it is a useful tool for studying the physiological roles of CaMKII.

Animals↗
PubMed1998

Localization of the mRNAs for two isoforms of Ca2+/calmodulin-dependent protein kinase kinases in the adult rat brain.

Ca2+/calmodulin-dependent protein kinase (CaM kinase) I and IV are thought to be activated by CaM kinase kinases (CaMKK). We examined the distribution of mRNAs for two isoforms (alpha and beta) of CaMKKs in the brain by in situ hybridization histochemistry. In the adult rat brain, CaMKK alpha mRNAs are widely distributed throughout the brain, whereas CaMKK beta mRNAs are restricted to some neuronal populations, particularly the cerebellar granule cells.

Animals↗
PubMed1998

Prevention of hypoxic liver cell necrosis by in vivo human bcl-2 gene transfection.

Prevention of hypoxic cell death is a key to successful liver transplantation. We developed a new method for preventing liver hypoxic cell death by introducing an anti-cell death gene directly into rat livers. When the human bcl-2 gene (hbcl-2) was directly transfected into rat livers together with non-histone chromosomal protein high mobility group 1 (HMG1) by the hemagglutinating virus of Japan (Sendai virus; HVJ)-liposome method, human Bcl-2 protein (hBcl-2) was efficiently expressed. Electron microscopy and fluorescence microscopy revealed that hepatocytes expressing exogenous hBcl-2 were almost completely protected the hypoxic cell necrosis. The expression of the hBcl-2 also inhibited activation of caspase-3 (-like) proteases and liver dysfunction. Thus, we conclude that transfection of the hbcl-2 gene through HVJ-liposome method is useful to prevent liver cell necrosis induced by hypoxia. This finding could lead to new strategies to avoid the hypoxic cell death, the major problem in liver transplantation.

Animals↗
PubMed1998

Clinical, pathological, and genetic features of limb-girdle muscular dystrophy type 2A with new calpain 3 gene mutations in seven patients from three Japanese families.

We report on the clinical, pathological, and genetic features of 7 patients with limb-girdle muscular dystrophy type 2A (LGMD2A) from three Japanese families. The mean age of onset was 9.7+/-3.1 years (mean+/-SD), and loss of ambulance occurred at 38.5+/-2.1 years. Muscle atrophy was predominant in the pelvic and shoulder girdles, and proximal limb muscles. Muscle pathology revealed dystrophic changes. In two families, an identical G to C mutation at position 1080 the in calpain 3 gene was identified, and a frameshift mutation (1796insA) was found in the third family. The former mutation results in a W360R substitution in the proteolytic site of calpain 3, and the latter in a deletion of the Ca2+-binding domain.

Age of Onset↗
PubMed1998

Genetic dissection of "OLETF", a rat model for non-insulin-dependent diabetes mellitus.

To elucidate the genetic factors underlying non-insulin-dependent diabetes mellitus (NIDDM), we performed genome-wide quantitative trait locus (QTL) analysis, using the Otsuka Long-Evans Tokushima Fatty (OLETF) rat. The OLETF rat is an excellent animal model of NIDDM because the features of the disease closely resemble human NIDDM. Genetic dissection with two kinds of F2 intercross progeny, from matings between the OLETF rat and non-diabetic control rats F344 or BN, allowed us to identify on Chromosome (Chr) 1 a major QTL associated with features of NIDDM that was common to both crosses. We also mapped two additional significant loci, on Chrs 7 and 14, in the (OLETF x F344)F2 cross alone, and designated these three loci as Diabetes mellitus, OLETF type Dmo 1, Dmo2 and Dmo3 respectively. With regard to suggestive QTLs, we found loci on Chrs 10, 11, and 16 that were common to both crosses, as well as loci on Chrs 5 and 12 in the (OLETF x F344)F2 cross and on Chrs 4 and 13 in the (OLETF x BN)F2 cross. Our results showed that NIDDM in the OLETF rat is polygenic and demonstrated that different genetic backgrounds could affect "fitness" for QTLs and produce different phenotypic effects from the same locus.

Animals↗
PubMed1998

Cervical infarction associated with vertebral artery occlusion due to spondylotic degeneration: case report.

Cases of cervical infarction with clearly documented evidence of the underlying aetiology and associated neuroradiological abnormalities have not been frequently reported. A rare case of cervical infarction caused by midvertebral artery occlusion due to spondylotic degeneration of the spine is described. The most probable aetiological factor affecting this disease entity, and the usefulness of magnetic resonance imaging in the detection of this rare lesion, are briefly discussed.

Aged↗
PubMed1998

Cloning and chromosomal mapping of a novel ABC transporter gene (hABC7), a candidate for X-linked sideroblastic anemia with spinocerebellar ataxia.

We isolated a novel human ATP-binding cassette (ABC) transporter cDNA, determined its nucleotide sequence, and designated it human ABC7 (hABC7). The nucleotide sequence was highly homologous to the ATM1 gene in yeast, which encodes an ABC transporter (yAtm1p) located in the mitochondrial inner membrane. The deduced human product, a putative half-type transporter, consists of 752 amino acids that are 48.9% identical to those of yAtm1p. A computer-assisted protein structural and localization analysis revealed that the mitochondrial targeting signal of yAtm1p is conserved in the N-terminal region of the primary sequence of the hABC7 protein, and therefore this product is also likely to be located in the mitochondrial inner membrane. The evidence strongly suggests that the hABC7 gene is a counterpart of ATM1 and that its product is probably involved in heme transport. We mapped the hABC7 gene to chromosome Xq13.1-q13.3 by fluorescence in-situ hybridization. As band Xq13 has been implicated in X-linked sideroblastic anemia with spinocerebellar ataxia, hABC7 becomes a candidate gene for this heritable disorder.

5-Aminolevulinate Synthetase↗
PubMed1998

Comparison of umbilical artery Doppler velocimetry between maternal supine position and complete left lateral position in predicting obstetric complications.

The aim of this study was to compare the predictive value of umbilical artery Doppler velocimetry in women in the supine position with that in women in the complete left lateral position as a screening test for abnormal obstetric outcomes. Umbilical artery resistance index (RI) was measured at 27-29 weeks and 35-37 weeks in 202 pregnant women. The measurements were performed with the mother in the supine position in 100 cases (supine group), and in the complete left lateral position in 102 cases (lateral group). Predictive values of the tests for abnormal outcomes (small for gestational age, fetal distress, pregnancy-induced hypertension) were compared between both groups. When abnormal RIs were defined as being greater than the 90th centile in the supine group, the sensitivities for any of the abnormal outcomes at 27-29 weeks were 18% in the supine group and 6% in the lateral group; the positive predictive values were 30% and 25%, respectively. For measurement at 35-37 weeks, the sensitivity and positive predictive value were 29% and 45%, respectively in the supine group, and 0% in both cases in the lateral group. When abnormal RIs were defined as being greater than the 90th centile in the lateral group, the sensitivities at 27-29 weeks were 41% in the supine group and 6% in the lateral group; the positive predictive values were 44% and 8%, respectively. At 35-37 weeks, the sensitivity and positive predictive value were 53% and 43% in the supine group, and 6% and 8% in the lateral group. Umbilical artery Doppler velocimetry when the mother was in the complete left lateral position was of little value as a screening test. However, when the mother is in the supine position, it may serve as a kind of stress test and disclose latent obstetric abnormalities in certain cases.

Adult↗
PubMed1997

Isolation of a novel human cDNA (rhoHP1) homologous to rho genes.

A novel full-length cDNA showing homology with rho genes was isolated from a human placenta cDNA library. Sequencing of a total of 1086 nucleotides of this clone revealed an open reading frame of 630 nucleotides (210 amino acids). In view of its degree of homology to members of the Rho family of molecules (50-54% identical amino acids, 60-63% identical nucleotides within the coding region), the predicted product was designated RhoHP1(Rho-related protein HP1). Northern analysis indicated that a message about 1.2-kb long is expressed in human heart, placenta, liver, skeletal muscle, and pancreas and, with weaker intensity, in several other tissues.

Amino Acid Sequence↗
PubMed1997

Detection of protein phosphatase activities in sodium dodecyl sulfate-polyacrylamide gel using peptide substrates.

A method for detection of protein phosphatase activity toward phosphorylated oligopeptides in SDS-polyacrylamide gel was developed. A synthetic peptide (MHRQETVDC) corresponding to the autophosphorylation site of calmodulin-dependent protein kinase II (residue 281-289) was conjugated to poly-L-lysine and phosphorylated with [gamma-32P]ATP by the action of calmodulin-dependent protein kinase II, and the [32P]-phosphopeptide-polymer conjugate was included as a substrate for protein phosphatases in gels. When a crude extract from rat brain was electrophoresed on polyacrylamide gel containing the [32P]phosphopeptide conjugate, followed by treatment for in situ renaturation and autoradiography, three transparent bands corresponding to apparent molecular weights of 52,000, 58,000 and 74,000, resulting from the removal of the [32P]phosphate from the phosphopeptide conjugate included in the gel were observed, indicating the existence of at least three different phosphoprotein phosphatases catalyzing dephosphorylation of the phosphopeptide in the brain. Among the three, two bands corresponding to molecular weights of 52,000 and 58,000 were not clearly observed when other phosphopeptide-polymer conjugates such as C-syntide-2 and CAMKAKS peptide were included in gels, suggesting that site-specific protein phosphatases can be detected in crude tissue extracts by this in-gel protein phosphatase assay.

Amino Acid Sequence↗
PubMed1997

Reticalmin: a novel calcium/calmodulin-dependent protein kinase IV-like protein in rat retina.

Western blot analysis of 100,000 g supernatant of rat retina using a polyclonal anti-Ca2+/ calmodulin-dependent protein kinase IV (CaM-kinase IV) antibody revealed an immunoreactive mass of 35 kDa, termed reticalmin. Lower amount of a isoform of CaM-kinase IV was also expressed in rat retina. Reticalmin did not react with anti-CaM-kinase IV C-terminal peptide antibody which recognized alpha and beta isoforms of CaM-kinase IV and calspermin. Immunohistochemically reticalmin was shown to be localized mainly in the outer segment of photo-receptor cells, and in dendrites of inner plexiform layers and may be in nuclei of ganglion cells and some inner nuclear layer cells.

Animals↗
PubMed1997

Purification and characterization of Ca2+/calmodulin-dependent protein kinase kinase beta from rat cerebellum.

The existence of isoforms of calmodulin-dependent protein kinase kinase (CaM-kinase kinase) in the rat brain was recently suggested by Northern and Western blot analyses and immunotitration [Okuno, S., Kitani, T., and Fujisawa, H. (1996) J. Biochem. 119, 1176-1181]. In the present study, CaM-kinase kinase beta, distinct from Cam-kinase kinase alpha which had been purified and cloned from rat cerebral cortex, was purified approximately 5,000-fold from rat cerebellum and its properties were examined. The purified CaM-kinase kinase beta gave a doublet at positions corresponding to molecular weights of 66,000 to 67,000 on SDS-PAGE, and neither protein band reacted with antibody against CaM-kinase kinase alpha. Both CaM-kinase kinase alpha and beta markedly activated both CaM-kinase I and IV, but CaM-kinase kinase beta activated CaM-kinase IV more strongly than did CaM-kinase kinase alpha. The maximal extents of the activation of CaM-kinase I and IV by CaM-kinase kinase beta were almost the same as those by CaM-kinase kinase alpha, suggesting that the two CaM-kinase kinases activated CaM-kinase I and IV by the same mechanisms.

Amino Acid Sequence↗
PubMed1997

Studies on the site of phosphorylation of Ca2+/calmodulin-dependent protein kinase (CaM-kinase) IV by CaM-kinase kinase.

The phosphorylation site(s) involved in the activation of CaM-kinase IV by CaM-kinase kinase alpha was studied using a mutant CaM-kinase IV (K71R) in which Lys71 (ATP-binding site) was replaced with Arg, because the autophosphorylation of CaM-kinase IV occurring at multiple sites made it difficult to study phosphorylation of the enzyme by CaM-kinase kinase. Sequence analysis of the phosphopeptide from the trypsin digest of CaM-kinase IV (K71R) phosphorylated by CaM-kinase kinase alpha suggested that the phosphorylation of CaM-kinase IV by CaM-kinase kinase only occurred at Thr196. The recombinant mutant CaM-kinase IV in which Thr196 or Thr200 was replaced with nonphosphorylatable alanine showed little activity in the presence and absence of the kinase kinase. The mutant enzyme in which Thr196 was replaced with negatively charged aspartic acid showed almost 25 times as high activity as the wild-type enzyme in the absence of the kinase kinase, and no more activation was observed in its presence. In contrast, the enzyme in which Thr200 was replaced with aspartic acid showed little enzyme activity. Thus, it may be concluded that the phosphorylation of Thr196 in CaM-kinase IV by CaM-kinase kinase is necessary for the subsequent autophosphorylation and activation of CaM-kinase IV.

Amino Acid Sequence↗
PubMed1997

Molecular cloning of Ca2+/calmodulin-dependent protein kinase kinase beta.

Calmodulin-dependent protein kinase IV (CaM-kinase IV), which plays crucial roles in the functioning of Ca2+ in the central nervous and immune systems, is markedly activated upon phosphorylation through the action of CaM-kinase kinase. Our previous immunotitration analysis suggested the existence of an isoform different from CaM-kinase kinase alpha, the beta isoform, in rat brain [Okuno, S., Kitani, T., and Fujisawa, H. (1996) J. Biochem. 119, 1176-1181]. In the present study, cDNA for CaM-kinase kinase beta was cloned from a rat cerebellar cDNA library. The coded protein consisted of 587 amino acids with a molecular weight of 64,445. Western blot analysis revealed that CaM-kinase kinase beta significantly existed only in the brain. The enzyme was not significantly detected in the retina where CaM-kinase kinase alpha exists.

Amino Acid Sequence↗