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Biomedical subjects

S Okuno

Publications and source records attributed to S Okuno.

At least 127 records · Page 7Linked to original sources
PubMed1997

Studies on the substrate specificity of Ca2+/calmodulin-dependent protein kinase kinase alpha.

Ca2+/calmodulin-dependent protein kinase (CaM-kinase) kinase a, which is known to activate CaM-kinases IV and I by phosphorylation of Thr196 and Thr177, respectively, can only phosphorylate Thr196 among many phosphorylation sites of CaM-kinase IV [Kitani, T., Okuno, S., and Fujisawa, H. (1997) J. Biochem. 121, 804-810], indicating its high degree of substrate specificity. In the present study, the substrate specificity of CaM-kinase kinase a was examined using various proteins and synthetic peptides as substrates as a means to address its physiological function. Among a number of proteins and synthetic peptides, including several known as good substrates for various protein kinases, only CaM-kinases IV and I and peptides containing the sequence surrounding Thr196 of CaM-kinase IV or Thr177 of CaM-kinase I were significantly phosphorylated by CaM-kinase kinase alpha, while the heat-denatured (at 60 degrees C for 5 min) CaM-kinases IV and I were not phosphorylated. Peptides containing the phosphorylation site of CaM-kinase IV or I were far less active as substrates for CaM-kinase kinase a than were native CaM-kinase IV or I. Thus, CaM-kinase kinase a showed a high degree of substrate specificity, recognizing not only specific amino acid sequences but also the native conformation of CaM-kinases IV and I.

Adenosine Triphosphate↗
PubMed1997

Regulation of Ca2+/calmodulin-dependent protein kinase IV (CaM-kinase IV) by changing its susceptibility to phosphorylation by CaM-kinase kinases.

Calmodulin-dependent protein kinase IV (CaM-kinase IV) is markedly activated on the phosphorylation of Thr196 by an upstream protein kinase, CaM-kinase kinase. The phosphorylation of CaM-kinase IV by CaM-kinase kinase is strongly suppressed on incubation with calmodulin in the presence of Ca2+, which results in a decrease in the enzyme activity, and completely restored on subsequent incubation with Mg2+ in the absence of Ca2+, which results in an increase in the activity. That a downstream protein kinase regulates its activity through reversible changes in its susceptibility to phosphorylation by an upstream protein kinase is a new regulatory mechanism.

Animals↗
PubMed1997

Isolation and mapping of karyopherin alpha 3 (KPNA3), a human gene that is highly homologous to genes encoding Xenopus importin, yeast SRP1 and human RCH1.

From a human fetal-brain cDNA library, we isolated and characterized a novel gene (KPNA3) encoding a protein highly homologous to certain nuclear transport proteins of Xenopus and human. The complete cDNA clone, designated karyopherin alpha 3, contained an open reading frame of 1,563 nucleotides encoding 521 amino acids. The predicted amino acid sequence showed 48%, 45% and 48% identity with Xenopus importin, yeast SRP1 and human RCH1, respectively. The similarities among these proteins suggest that karyopherin alpha 3 may be involved in the nuclear transport system. Eight repeats of the arm motif were well conserved among these proteins. The N-terminal region of the predicted karyopherin alpha 3 product was highly basic and the C-terminal region was strongly acidic. A 4.3-kb transcript was expressed in all adult human tissues examined by Northern blotting. The cDNA clone was assigned to chromosome band 13q14.3 by fluorescence in situ hybridization.

Amino Acid Sequence↗
PubMed1997

Cloning and chromosome assignment to 1q32 of a human cDNA (RAB7L1) encoding a small GTP-binding protein, a member of the RAS superfamily.

A full-length cDNA homologous to RAB7, a member of the RAB-related GTP-binding protein subfamily, was isolated from a human placenta cDNA library. This cDNA, designated RAB7L1, has an open reading frame of 609 nucleotides encoding 203 amino acids. Northern analysis showed that the mRNA is ubiquitously expressed in human tissues, although signal intensities were different among the various organs examined. This gene was located on chromosome band 1q32 by fluorescence in situ hybridization.

Amino Acid Sequence↗
PubMed1997

Antitumor effects and toxicities of carboxymethylpullulan-peptide-doxorubicin conjugates.

In vivo antitumor effects of the conjugates of doxorubicin (DXR) with carboxymethylpullulan (CMPul) through tetrapeptide spacers were compared with those of DXR against tumor-bearing rats. CMPul-DXR conjugates bound through Gly-Gly-Phe-Gly and Gly-Phe-Gly-Gly spacers were found to be more potent than DXR after a single intravenous injection in rats bearing Walker 256 carcinosarcoma. These conjugates were also more effective than DXR in rats bearing Yoshida sarcoma. However, CMPul-DXR conjugate bound through Gly-Gly-Gly-Gly was less effective against Walker 256-bearing rats than DXR. Body weight loss of CMPul-DXR conjugates in rats, on the other hand, was less than that of DXR at a DXR dose of 10 mg/kg. Lethal doses of CMPul-DXR conjugates in CDF1 mice were about 3-times higher than that of DXR. These data suggest that the therapeutic index of CMPul-DXR conjugates bound through appropriate peptide spacers was increased more than that of DXR. However, CMPul-DXR conjugates tested were all less effective than DXR against Walker 256 cells in vitro. Also, 125I-labeled CMPul-DXR conjugate accumulated much less in the cells than 14C-DXR.

Animals↗
PubMed1996

Inactivation of calmodulin-dependent protein kinase IV by autophosphorylation of serine 332 within the putative calmodulin-binding domain.

When brain calmodulin-dependent protein kinase IV is incubated with calmodulin-dependent protein kinase IV kinase under the phosphorylation conditions in the presence of Ca2+/calmodulin, rapid initial incorporation of 1 mol of phosphate into 1 mol of the enzyme by the action of the kinase kinase occurs, resulting in marked activation of the enzyme, and the subsequent incorporation of more than 3 mol of phosphate by autophosphorylation occurs, resulting in no significant change in the activity (Okuno, S., Kitani, T., and Fujisawa, H. (1994) J. Biochem. (Tokyo) 116, 923-930; Okuno, S., Kitani, T., and Fujisawa, H. (1995) J. Biochem. (Tokyo) 117, 686-690). After the maximal phosphorylation, the continued incubation in the presence of excess EGTA resulted in additional autophosphorylation of the enzyme, leading to a complete loss of the Ca2+/calmodulin-dependent activity, while causing no significant change in the Ca2+/calmodulin-independent activity. The amino acid sequence analysis revealed that the autophosphorylation after removal of Ca2+ occurred on Ser332, Ser333, Ser337, and Ser341. Analysis by site-directed mutagenesis clearly showed that the autophosphorylation site responsible for the inactivation is Ser332. Thus, calmodulin-dependent protein kinase IV activated by the kinase kinase may lose its Ca2+/calmodulin-dependent activity by autophosphorylation on Ser332 located within the putative calmodulin-binding domain in the absence of Ca2+.

Amino Acid Sequence↗
PubMed1996

Distribution of Ca2+/calmodulin-dependent protein kinase kinase alpha in the rat central nervous system: an immunohistochemical study.

Ca2+/calmodulin-dependent protein kinase IV (CaM-kinase IV) is activated by CaM-kinase IV kinase. We provided a rabbit antiserum against 20 amino acid residues at the carboxyl-terminal end of CaM-kinase IV kinase, and examined regional and intracellular distribution of CaM-kinase IV kinase immunohistochemically in the central nervous system of the rat by light and electron microscopy. The immunoreactivity was found in cellular nuclei of virtually all neurons. However, the immunoreactivity was weak in the nuclei of the granule cells in the cerebellar cortex, although the nuclei of the granule cells were reported to contain high CaM-kinase IV activity. Thus, it was suggested that other types of CaM-kinase IV kinase might exist in the cerebellum, and the present CaM-kinase IV kinase was named as CaM-kinase kinase alpha.

Amino Acid Sequence↗
PubMed1996

Dissecting aneurysm of the anterior cerebral artery causing hemorrhagic infarction.

BACKGROUND: Dissecting aneurysms of the cerebral arteries are infrequent, and they occur preferentially in the middle cerebral, internal carotid, and vertebrobasilar arteries. The anterior cerebral artery (ACA) is usually involved in association with dissection in other locations, but a lesion confined to the ACA is an extremely rare event. No previous reports have well documented the clinical and pathological features of the ACA dissection. Moreover, surgical management for this lesion has not been reported. CASE REPORT: A case of dissecting aneurysm confined to the ACA causing hemorrhagic infarction is presented. To prevent subsequent rupture, we employed a trapping procedure 12 days after the onset. A dark purplish discoloration of the right A2 portion was encountered, with an intact anterior communicating complex. The involved vessel was partly sectioned for further pathological examination. RESULTS: Pathologically, subintimal clots dissected the vascular lumen in the inner layer of the media. Other abnormalities such as deficiency of the internal elastic lamina and medial defects were not found. The postoperative clinical course was not eventful. CONCLUSIONS: This is the first case to document an ACA dissecting aneurysm treated by a direct surgical approach. Spontaneous resolution is not infrequent in cerebral dissection, but subsequent rupture has commonly resulted in poor outcome. Surgical management is thought to be the most effective method to prevent further hemorrhagic event, even for an ACA dissection. Revascularization distal to the compromised artery should be considered whenever necessary.

Cerebral Angiography↗
PubMed1996

Echogenic fetal bowel in the third trimester associated with trisomy 18.

We present the first case of trisomy 18 in which echogenic fetal bowel was detected in the third trimester after normal echogenicity was documented during the second trimester. Fetal karyotyping should be considered in cases of increased echogenicity of the fetal lower abdomen in the second as well as the third trimester.

Abnormalities, Multiple↗
PubMed1996

Phosphorus intake regulates intestinal function and polyamine metabolism in uremia.

This study found that 5/6-nephrectomized uremic rats showed secondary hyperparathyroidism as reflected by an increase in their serum parathyroid hormone (PTH) level in association with a decrease in serum 1,25-dihydroxyvitamin D [1,25-(OH)2D]. These changes recovered partially upon phosphorus restriction. Calcium absorption and gene expression of calbindin-D9k were decreased in uremia and were also improved by phosphorus restriction. In uremia, intestinal spermidine/spermine N1-acetyltransferase activity was decreased, while ornithine decarboxylase (ODC) activity and its gene expression were potentiated. Enhancement of c-fos and c-jun gene expressions was also observed in uremia. These phenomena suggest that the intestinal villus may proliferate in uremia. Phosphorus restriction prevented increases in the expression of ODC, c-fos and c-jun observed in uremia. Since phosphorus restriction caused a rise in the serum 1,25-(OH)2D level, the role of 1,25-(OH)2D in uremia-induced intestinal dysfunction was examined. A single injection of 1,25-(OH)2D3 to uremic rats caused an increase in the steady-state calbindin-D9k mRNA level, and decreases in steady state c-fos and ODC mRNA levels, suggesting that the deficiency of 1,25-(OH)2D3 is responsible for intestinal dysfunction in uremia. In conclusion, altered polyamine metabolism caused by 1,25-(OH)2D deficiency is intimately involved in intestinal dysfunction and the development of the proliferative state of the intestinal villus in uremia.

Absorption↗
PubMed1996

Evidence for the existence of Ca2+/calmodulin-dependent protein kinase IV kinase isoforms in rat brain.

Calmodulin-dependent protein kinase IV (CaM-kinase IV), which plays crucial roles in the functioning of Ca2+ in the central nervous system and immune system, is markedly activated upon phosphorylation by the action of CaM-kinase IV kinase. Northern and Western blot analyses of CaM-kinase IV kinase showed relatively weak reactions in the rat cerebellum, where the activity of CaM-kinase IV kinase has been demonstrated to exist, indicating that CaM-kinase IV kinase isoforms distinct from the enzyme cloned from the cerebral cortex may exist in the cerebellum. When the crude extracts of rat cerebral cortex, brain stem, and cerebellum were immunotitrated with antibody against the cloned enzyme, only approximately 46, 56, and 25% of the enzyme activity of the respective extracts were immunoprecipitated. Thus, at least two distinct isoforms of CaM-kinase IV kinase appear to exist in the brain.

Amino Acid Sequence↗
PubMed1996

Molecular cloning of a novel protein containing the association domain of calmodulin-dependent protein kinase II.

The cDNA of a novel protein, which contains the association domain of alpha isoform of calmodulin-dependent protein kinase II (CaM-kinase II alpha), was cloned from rat skeletal muscle. This protein, called alpha KAP, consisted of 200 amino acid residues with a molecular weight of 22,583. alpha KAP has a highly hydrophobic amino-terminal stretch of 25 amino acids which is absent from CaM-kinase II alpha, suggesting that this protein is either a secretory protein or an integral membrane protein. Northern blot analysis with a probe specific for alpha KAP detected three distinct mRNA species of 4.0, 2.4, and 1.5 kb in rat skeletal muscle. The 4.0- and 2.4-kb RNAs were also detected in heart, and at much lower levels in lung, kidney, and testis. Western blot analysis, using antibody raised against a synthetic peptide corresponding to the carboxyl-terminal 15 amino acids, revealed a single band corresponding in mobility to a molecular weight of 21,000 in crude extracts of both rat skeletal muscle and bacteria transformed with the cDNA, suggesting that no significant post-translational modification, such as excision of the amino-terminal hydrophobic segment, occurred. This, together with the fact that alpha KAP was recovered in the high-speed pellet in skeletal muscle, indicated that this protein may be an integral membrane protein.

Amino Acid Sequence↗
PubMed1996

Cloning, expression, and mapping of UBE2I, a novel gene encoding a human homologue of yeast ubiquitin-conjugating enzymes which are critical for regulating the cell cycle.

From a human fetal-brain cDNA library we isolated a novel gene sharing significant homology with two yeast genes, UBC9 and hus5, which encode ubiquitin-conjugating enzyme 9 (UBC9). In yeast this protein is critical for normal mitosis, and seems to be closely involved in progression of G2 to M phase of the cell cycle. The human UBC9 (h-UBC9) cDNA, (gene symbol UBE2I), contained an open reading frame of 474 nucleotides encoding 158 amino acids. Its predicted peptide showed respectively 56% and 66% identity (75% and 82% similarity) with the products of UBC9 and hus5. Northern-blot analysis revealed expression of three transcripts, 6.4 kb, 3.3 kb, and 1.35 kb, in all human tissues examined. This gene, UBE2I, was mapped to chromosome band 16p13.3 by FISH.

Amino Acid Sequence↗
PubMed1996

Cloning, expression and chromosomal mapping of a novel cyclophilin-related gene (PPIL1) from human fetal brain.

We isolated a human cDNA clone encoding a novel protein homologous to cyclophilins, specific cellular targets of cyclosporin A, which are conserved in species ranging from human to prokaryotes. This cDNA, designated hCyPX, contained an open reading frame of 498 nucleotides encoding 166 amino acids. Computer analysis indicated that its predicted amino acid sequence had 41.6%, 40.4%, and 39.2% homology to those of human, bovine, and Drosophila cyclophilins, respectively. Northern blot analysis indicated ubiquitous expression in adult human tissues, but most abundant expression in heart. Fluorescence in situ hybridization to human metaphase chromosomes localized this gene (PPIL1, peptidylprolyl isomerase [cyclophilin]-like 1) to chromosome bands 2p23.3-->p23.1.

Amino Acid Isomerases↗
PubMed1996

Cloning, expression, and mapping of TCTEL1, a putative human homologue of murine Tcte1, to 6q.

From a human fetal-brain cDNA library we isolated a putative human homologue of the murine Tcte1 gene. The cDNA, designated TCTEL1, contained an open reading frame of 339 nucleotides encoding 113 amino acids. The predicted peptides of TCTEL1 showed 94% and 55% identity (100% and 94% similarity) with those of murine Tcte1 and human RP3. Northern-blot analysis revealed a 0.9-kb transcript in all tissues examined. This gene was mapped by FISH to chromosome bands 6q25.2 --> q25.3, the syntenic region of the murine t-complex locus of chromosome 17.

Animals↗
PubMed1996

Cloning, expression and chromosome mapping of adducin-like 70 (ADDL), a human cDNA highly homologous to human erythrocyte adducin.

From a human fetal-brain cDNA library we isolated a novel human cDNA, termed human adducin-like 70 (gene symbol ADDL), whose predicted amino acid sequence showed a high degree of homology to adducins. This cDNA clone (ADDL), which contained an open reading frame of 2,022 nucleotides encoding 674 amino acids, revealed 54%, 53%, and 59% identity in predicted amino acid sequence with alpha and beta components of human adducin and rat adducin 63, respectively. Human adducin-like 70 is likely to play an important role in the skeletal organization of the cell membrane. Northern blot analysis indicated ubiquitous expression of this gene in adult human tissues. We localized the gene to chromosome bands 10q24.2-->q24.3 by fluorescence in situ hybridization (FISH).

Adult↗