Biomedical subjects

H Hidaka

Publications and source records attributed to H Hidaka.

At least 289 records · Page 16Linked to original sources

PubMed1992

Increased plasma post-heparin diamine oxidase activity and plant sterol levels in streptozotocin diabetic rat.

Plasma post-heparin diamine oxidase (DAO) activity and plasma levels of plant sterols were examined in streptozotocin diabetic rats fed with chow containing plant sterols, to investigate the enzyme activity in relation to the morphological changes of small intestine as well as sterol absorption in the diabetic rats. Diabetic rats showed increased small intestinal mass and surface area compared with control rats. Plasma post-heparin DAO activity and plant sterol level were also increased more than 2.5-fold in the diabetic rats. Insulin treatment improved these abnormalities. Plasma DAO activity correlated to both the small intestinal hyperplastic change and plasma plant sterol levels. These results indicate that plasma post-heparin DAO activity may be used as a marker of intestinal hypertrophy as well as ability to absorb dietary sterols.

Amine Oxidase (Copper-Containing)↗

PubMed1992

Specific binding of CAP-50 to calcyclin.

CAP-50, a calcyclin-associated protein with an apparent molecular mass of 50 kDa, was purified and proved to be a novel annexin [Tokumitsu, H. et al. (1992) J. Biol. Chem. 267, 8919-8924]. We examined the binding of CAP-50 to other Ca(2+)-binding proteins which have two of four EF-hand structures, by a co-precipitation assay with phospholipid (phosphatidylserine). Among nine Ca(2+)-binding proteins (calcyclin, S-100 proteins, p11, calgizzarin, calvasculin, calmodulin and troponin C) examined, only calcyclin interacted with CAP-50. These results clearly show that the interaction of CAP-50 to calcyclin is specific, i.e. other Ca(2+)-binding proteins with the EF-hand structure could not substitute for calcyclin, thereby suggesting the possible role in specific regulation of the function of CAP-50 by Ca2+/calcyclin.

Animals↗

PubMed1992

CAP-50, a newly identified annexin, localizes in nuclei of cultured fibroblast 3Y1 cells.

A 50-kDa protein, which binds to the growth-regulated gene (2A9) product, calcyclin in a calcium-dependent manner, was purified from bovine lung. Partial amino acid sequencing of the protein revealed it to be the bovine equivalent of rabbit lung CAP-50 (calcyclin-associated protein, 50 kDa), which is a member of the annexin family and binds to calcyclin in a calcium-dependent manner. Specific polyclonal antibodies to bovine lung CAP-50 were prepared. Comparative studies between CAP-50 and synexin (annexin VII) on the immunoreactivity against anti-CAP-50 antibodies and the ability of binding to calcyclin revealed that CAP-50 was a distinct molecule from synexin. Using specific polyclonal antibodies to bovine lung CAP-50, tissue distribution and subcellular distribution of CAP-50 were investigated. In most rat tissues, except those in the central nervous systems and kidney, CAP-50 is expressed at a high or moderate level. Both studies by subcellular fractionation and by indirect immunofluorescence staining of the rat embryonic fibroblast cell line, 3Y1, revealed that CAP-50 mainly localized in nuclei. Moreover, between the cells at interphase and at mitotic phase, different distributions of CAP-50 were observed. That is, in the cells at interphase, CAP-50 seemed to localize throughout the nucleoplasm. On the other hand, in the cells during mitosis, CAP-50 was concentrated at the loop-like structure around the mitotic apparatus. CAP-50 was found in isolated 3Y1 nuclei lacking outer nuclear membranes, and approximately 50% of CAP-50 was extracted from the nuclei by chelating calcium. Thus, CAP-50, a unique annexin, localizes in nuclei.

Amino Acid Sequence↗

PubMed1992

Identification of Ca2+/calmodulin-dependent protein kinase and endogenous substrate of Fusarium oxysporum.

Ca2+/calmodulin-dependent phosphorylation and cross-reactivity between anti-rat brain Ca2+/calmodulin-dependent protein kinase II (CaMK) antibody and partially purified CaMK from Fusarium oxysporum were detected in the component of high-molecular mass (M(r) greater than 100,000). In vitro, Ca2+/CaM-dependent phosphorylation of only a 16-kDa protein was detected. The 16-kDa protein was localized in the membrane fraction. Amino acid sequence of one of the peptides derived from partial hydrolysis of the 16-kDa protein had a high homology (65.5%) with the bovine transducin beta chain. It is assumed that the 16-kDa protein is an endogenous substrate of F. oxysporum CaMK.

Amino Acid Sequence↗

PubMed1992

Phosphorylation of myosin light chain kinase by the multifunctional calmodulin-dependent protein kinase II in smooth muscle cells.

Stimulation of tracheal smooth muscle cells in culture with ionomycin resulted in a rapid increase in cytosolic free Ca2+ concentration ([Ca2+]i) and an increase in both myosin light chain kinase and myosin light chain phosphorylation. These responses were markedly inhibited in the absence of extracellular Ca2+. Pretreatment of cells with 1-[N-O-bis(5-isoquinolinesulfonyl)-N- methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a specific inhibitor of the multifunctional calmodulin-dependent protein kinase II (CaM kinase II), did not affect the increase in [Ca2+]i but inhibited ionomycin-induced phosphorylation of myosin light chain kinase at the regulatory site near the calmodulin-binding domain. KN-62 inhibited CaM kinase II activity toward purified myosin light chain kinase. Phosphorylation of myosin light chain kinase decreased its sensitivity to activation by Ca2+ in cell lysates. Pretreatment of cells with KN-62 prevented this desensitization to Ca2+ and potentiated myosin light chain phosphorylation. We propose that the Ca(2+)-dependent phosphorylation of myosin light chain kinase by CaM kinase II decreases the Ca2+ sensitivity of myosin light chain phosphorylation in smooth muscle.

Animals↗

PubMed1992

Full sequence of neurocalcin, a novel calcium-binding protein abundant in central nervous system.

We determined the cDNA sequence for neurocalcin, a novel calcium-binding protein in bovine brain. This clone (pCalN) has 582 nucleotides in the open reading frame including the termination codon TGA, 11 nucleotides of the 5' leader and 1251 nucleotides of the 3' noncoding region. The deduced amino acid sequence revealed that neurocalcin is composed of 193 amino acids, has a molecular mass of 22,284 daltons, and contains three putative calcium-binding sites (EF-hand motifs). By Northern blot analysis, 3.8kbp mRNA was detected in brain. The deduced amino acid sequence had a strong homology to visinin (46.5%) and recoverin (51.6%) in retina, suggesting that neurocalcin may play a visinin- or recoverin-like role in brain.

Amino Acid Sequence↗

PubMed1992

Affinity purification of the hepatic high-density lipoprotein receptor identifies two acidic glycoproteins and enables further characterization of their binding properties.

Several high-density lipoprotein (HDL)-binding proteins, candidates for the putative HDL receptor, have recently been identified, including two membrane proteins: HB1 of 120 kDa and HB2 of 100 kDa, present in rat and human liver plasma membranes respectively. Further insights into their function however, have been hampered by poor recoveries of these hydrophobic peptides, and the present work was undertaken to improve yields and enable a more detailed investigation of their properties. A significant improvement has been achieved using two affinity chromatographic procedures, one exploiting the glycoprotein nature of the proteins and the other exploiting their ligand properties, which in combination resulted in considerable enrichment of HB1 and HB2. Thus DEAE-Sephacel fractionation (0.05-0.2 M-NaCl) of CHAPS-solubilized plasma membranes yielded active HDL-binding proteins which bound to concanavalin A-Sepharose or wheat-germ-lectin-Sepharose columns and retained their binding activity after eluting with methyl-alpha-D-mannoside or N-acetylglucosamine respectively. These glycoproteins were further purified by affinity chromatography using apo-HDL-Sepharose columns. Final purification required preparative SDS/PAGE. Investigation of the carbohydrate moieties of the proteins using glycosidases and two-dimensional gel electrophoresis revealed pI values ranging from 4.6 to 4.9 and from 4.5 to 4.7 for HB1 and HB2 respectively, which after treatment with neuraminidase shifted towards basic pH (5.4-5.7 and 5.3-5.5 respectively). The molecular masses were decreased to 115 kDa and 95 kDa respectively, demonstrating that sialic acid residues contributed significantly to the negative charge of the glycosylated peptides. Treatment with the enzyme peptide N-glycosidase F (N-glycanase) resulted in a decrease in molecular mass of HB1 and HB2 to 105 kDa and 80 kDa respectively, but endo-alpha-N-acetylgalactosaminidase (O-glycanase) treatment was not effective. Neither neuraminidase nor N-glycanase treatment destroyed activity, suggesting that sialic acids or N-linked oligosaccharides are not important determinants of HDL binding. Digestion of plasma membranes with trypsin or Pronase resulted in a loss of activity of both HB1 and HB2 that was not influenced by prior treatment with neuraminidase, suggesting that sialic acid residues play no protective role against proteolytic cleavage of HDL receptor proteins.

Animals↗

PubMed1992

A calcyclin-associated protein is a newly identified member of the Ca2+/phospholipid-binding proteins, annexin family.

A calcyclin-associated protein with an apparent molecular weight of 50,000 (CAP-50) was purified from rabbit lung. The procedure included ammonium sulfate precipitation, anion and cation ion-exchange, and calcyclin affinity chromatographies. Interestingly, partial amino acid sequences of lysyl-endpeptidase-digested fragments indicated that CAP-50 was a member of the Ca2+/phospholipid-binding proteins, the annexin family. The sequence of a proteolytic peptide with Staphylococcus aureus V8 protease on NH2-terminal region is not homologous with any other annexin family proteins. Phospholipid binding studies showed that CAP-50 bound to phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid-containing vesicles, in a Ca(2+)-dependent manner. In the presence of Ca2+/calcyclin, CAP-50 formed a complex with calcyclin and bound to the PS-containing vesicles. The apparent Kd value of calcyclin for CAP-50 was calculated to be 1.61 x 10(-6) M. Zero-length cross-linking studies indicated that 1 mol of CAP-50 bound to an equimolar unit of calcyclin. CAP-50 inhibited the phospholipase A2 activity, dose-dependently (IC50 = 0.2 microM), however, calcyclin did not alter the inhibitory effect. With the 125I-calcyclin gel overlay method, calcyclin bound tightly to CAP-50 in a Ca(2+)-dependent manner after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that rabbit lung CAP-50 is a newly identified member of the annexin family. Ca2+/calcyclin apparently regulates the function of CAP-50 on cytosolic face of the plasma membrane.

Amino Acid Sequence↗

PubMed1992

Proteinkinase C beta-isoform triggers the formation of prostanoids and superoxide in liver macrophages.

The zymosan- and phorbolester-induced formation of prostanoids in cultured rat liver macrophages has been shown recently to be controlled by proteinkinase C (1). Using specific antibodies raised against the alpha-, beta-, gamma- and epsilon-isoforms of proteinkinase C, we show that proteinkinase-beta is the predominant isoform in rat liver macrophages. Northern blot analysis with a beta-isoform-specific c-DNA probe revealed the expression of m-RNA for proteinkinase-beta. In resting cells the beta-isoform of proteinkinase C is nearly equally distributed between the cytosolic and membrane fractions. Zymosan and phorbolester led to a translocation of proteinkinase-beta from the cytosol to the membranes, whereas exogenously added arachidonic acid and the calcium ionophore A23187 had no effect. These data indicate that the beta-isoform of proteinkinase C takes part in the prostaglandin and superoxide formation following PMA and zymosan treatment of rat liver macrophages.

Animals↗

PubMed1992

Protein kinase C and calmodulin kinase are required for endothelin-stimulated atrial natriuretic factor secretion from primary atrial myocytes.

Endothelin (ET), a potent stimulator of atrial natriuretic factor (ANF) secretion in atrial myocyte cultures, has been hypothesized to act via the stimulation of protein kinase C (PKC). This study was carried out in order to determine if ET activates PKC in atrial cultures and whether this activation fully accounts for the effects of ET on ANF secretion. By monitoring the phosphorylation of p80 upon exposure to phorbol ester or ET, it was shown that ET activated PKC in atrial cultures, but to a lesser extent than phorbol ester. In contrast, ET stimulated ANF secretion to a level five times greater than phorbol ester, indicating that PKC activation alone does not fully account for the effects of ET on ANF secretion. Down-regulation of PKC or exposure to the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) resulted in a 50% decrease in ET-stimulated ANF secretion. Interestingly, increasing calcium influx with BAY K 8644 stimulated ANF secretion but did not effect the phosphorylation of p80, indicating a PKC-independent pathway of ANF secretion. Similarly, a component of ET-stimulated secretion that required calcium influx was independent of PKC activation but was sensitive to the Ca2+/calmodulin kinase (CaMK) inhibitor KN-62. Complete inhibition of ET-mediated ANF secretion was obtained only in the presence of both H7 and KN-62. These results demonstrate that ET activates PKC in atrial myocyte cultures and that the full effects of ET on ANF secretion require both PKC and Ca2+/calmodulin kinase activities.

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

PubMed1992

Purification and characterization of a new member of the S-100 protein family from human placenta.

A novel Ca(2+)-binding protein which is termed S-100P was purified from human placenta with a hydrophobic column followed by an anion exchange column and reverse phase high performance liquid chromatography (HPLC). Molecular mass of the protein was 10 kDa according to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Using immunoblotting technique, anti-human calcyclin antibodies did not bind to the S-100P. Isoelectric point of S-100P was pI = 4.6. S-100P did not formed disulfide-linked dimer. Calcium binding ability was proved by UV difference spectrometry, urea/alkaline gel electrophoresis, and 45Ca overlay technique. A ninety amino acid sequence of S-100P was determined. It is 49% identical with human S-100 beta, 38% with human calcyclin, and 37% with human cystic fibrosis antigen.

Amino Acid Sequence↗

PubMed1992

Acidic calmodulin binding protein, ACAMP-81, is MARCKS protein interacting with synapsin I.

ACAMP-81 is an acidic calmodulin binding protein with molecular mass of 81 kDa. We report partial amino acid analysis of ACAMP-81 and its interaction with synapsin I. 123 amino acids of ACAMP-81 were determined and the sequence was completely identical with that of MARCKS protein which was thought to be a substrate for calcium/phospholipid dependent protein kinase (PKC). We found ACAMP-81 bound to synapsin I with 125I-labeled ACAMP-81 overlay method. ACAMP-81 bound to the cysteine specific cleaved 51 kDa fragment derived from middle/tail region of synapsin I.

Amino Acid Sequence↗

PubMed1992

Isolation and characterization of a calcium-binding protein derived from mRNA termed p9Ka, pEL-98, 18A2, or 42A by the newly synthesized vasorelaxant W-66 affinity chromatography.

W-66 (N-(2-aminoethyl)-N-[2-(4-chlorocinnamylamino) ethyl]-5-isoquinolinesulfonamide), a newly synthesized isoquinolinesulfonamide, was shown to have a potent vasodilatory action and calmodulin (CaM)-antagonizing action. Using the W-66 affinity chromatographic technique, we purified two Ca(2+)-binding proteins from the EGTA-soluble fraction of bovine aorta. One was CaM and the other was an acidic protein with a molecular mass of 11 kDa. It was tentatively named "calvasculin." Calvasculin was a dimeric protein. Equilibrium dialysis showed that 1 mol of calvasculin (dimer) bound to 1.98 +/- 0.30 mol of Ca2+ in the presence of 10(-3) M Ca2+. Calvasculin failed to activate Ca2+/CaM-dependent enzymes such as myosin light chain kinase, Ca2+/CaM-dependent phosphodiesterase, or Ca2+/CaM-dependent protein kinase II and to inhibit the CaM stimulation of these enzymes. The partial amino acid sequence of calvasculin revealed a high homology to the predicted protein derived from mRNA, named pEL-98, 18A2, 42A, or p9Ka. We also examined the physicochemical and biochemical properties of calvasculin. Using the antibody specific for calvasculin, we obtained evidence that calvasculin is present in abundance in bovine aorta but not in brain, lung, heart, or testis.

Amino Acid Sequence↗

PubMed1992

Structure of the mouse tyrosine hydroxylase gene.

The mouse tyrosine hydroxylase (TH) gene was isolated from a genomic library by cross-hybridization with human TH cDNA probe. Nucleotide sequence analysis of two overlapping genomic clones showed that this gene is split into 13 exons distributed about 7.5 kb in length. The transcription initiation site was determined by primer extension analysis with mouse adrenal gland poly(A)+RNA. The structure of the mouse TH gene was similar to that of the human TH gene, but it contained neither the alternative splice donor site around the 3'-end of the first exon nor an independent exon corresponding to the second exon of the human TH gene. There were the canonical TATA and GC boxes, cyclic AMP responsive element (CRE), and AP1 binding site in the 5'-flanking region of the mouse TH gene.

Amino Acid Sequence↗

PubMed1992

Identification of a 80 kDa calmodulin-binding protein as a new Ca2+/calmodulin-dependent kinase by renaturation blotting assay (RBA).

We surveyed rabbit brain cytosol for a new Ca2+/calmodulin (CaM)-dependent kinase. The renaturation blotting assay (RBA) exploits the ability of blotted SDS-denatured proteins to regain enzymic activity after guanidine treatment. Using RBA, we found that the eluate of rabbit brain cytosol from a CaM affinity column contains at least four electrophoretically distinct protein kinase bands which were autophosphorylated in a Ca2+/CaM-dependent manner. The 49 kDa band and the 60 kDa band were alpha and beta subunit of CaM kinase II, and the 42 kDa band was presumed to be CaM kinase I, but the 80 kDa band could not be attributed to any reported Ca2+/CaM-dependent protein kinases. The 80 kDa protein kinase was isolated by three-step chromatography. We examined the phosphorylation of exogenous substrates by 80 kDa protein kinase, and histone IIIs and myosin light chain were phosphorylated in a Ca2+/CaM-dependent manner. W-7, a specific inhibitor for calmodulin, inhibited this kinase activity, but KN-62, a specific inhibitor for CaM kinase II, had no effect on this protein kinase activity. Autoradiography using boiled rabbit brain homogenate as substrate showed three intrinsic substrates (80 kDa, 60 kDa and 42 kDa), which were phosphorylated in a Ca2+/CaM-dependent manner. These findings suggest that a new Ca2+/CaM-dependent protein kinase could be identified by the RBA.

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

PubMed1992

MAPKAP kinase-2; a novel protein kinase activated by mitogen-activated protein kinase.

A novel protein kinase, which was only active when phosphorylated by the mitogen-activated protein kinase (MAP kinase), has been purified 85,000-fold to homogeneity from rabbit skeletal muscle. This MAP kinase activated protein kinase, termed MAPKAP kinase-2, was distinguished from S6 kinase-II (MAPKAP kinase-1) by its response to inhibitors, lack of phosphorylation of S6 peptides and amino acid sequence. MAPKAP kinase-2 phosphorylated glycogen synthase at Ser7 and the equivalent serine (*) in the peptide KKPLNRTLS*VASLPGLamide whose sequence is similar to the N terminus of glycogen synthase. MAPKAP kinase-2 was resolved into two monomeric species of apparent molecular mass 60 and 53 kDa that had similar specific activities and substrate specificities. Peptide sequences of the 60 and 53 kDa species were identical, indicating that they are either closely related isoforms or derived from the same gene. MAP kinase activated the 60 and 53 kDa forms of MAPKAP kinase-2 by phosphorylating the first threonine residue in the sequence VPQTPLHTSR. Furthermore, Mono Q chromatography of extracts from rat phaeochromocytoma and skeletal muscle demonstrated that two MAP kinase isoforms (p42mapk and p44mapk) were the only enzymes in these cells that were capable of reactivating MAPKAP kinase-2. These results indicate that MAP kinase activates at least two distinct protein kinases, suggesting that it represents a point at which the growth factor-stimulated protein kinase cascade bifurcates.

3T3 Cells↗

PubMed1992

Immunohistochemical analysis of spinal intradural xanthomatosis developed in a patient with phytosterolemia.

Multiple intradural xanthomatous tumors developed in 48-year-old female with familial phytosterolemia. These tumors were restricted to the spinal denticulate ligaments. Histological and immunohistochemical findings were fundamentally similar to those of tendinous xanthomas. The major cellular component of these tumors were identified as of mono-histiocytic origin because they possessed myeloid histiocytic antigen (Mac 387), CD11c and lysozyme but not CD15. Sitosterols, campesterols and cholestanols were recovered from the extract of the tumors and the lesions were confirmed to be phytosterolemic xanthomas. Schwann cells stained with anti-S100 protein were confined to the perivascular small nerve bundles and did not show xanthomatous change. Although immunohistochemical preparation of epithelial membrane antigen and desmoplakin I+II revealed the presence of non-neoplastic meningothelial cells in the superficial portion of the tumors, they were too few to play a significant role in the development of these xanthomas.

Chromatography, Liquid↗

PubMed1992

Hyperlipidemia and atherosclerosis in experimental insulinopenic diabetic monkeys.

Chronic insulinopenic diabetes was induced by i.v. streptozotocin in the non-human primate Macaca fuscata. Five diabetic monkeys were kept for 8-19 months and nine for 24-48 months without any insulin treatment. Hyperglycemia (241 +/- 22 mg/dl, M +/- SE less than or equal to 1 year) progressed to 376 +/- 34 mg/dl (greater than 2 years) and ketosis to 3.5 mM (greater than 2 years) during the course of diabetes; this was roughly inversely proportional to hypoinsulinemia (3.4 microU/ml, 2 years). Serum cholesterol increased from 184 +/- 11 (less than or equal to 1 year) to 328 +/- 66 mg/dl (greater than 2 years) with the major increase in LDL-cholesterol (2.7-fold over control, greater than 2 years). HDL-cholesterol did not change at all throughout the experimental period. TG increased from 144 +/- 25 (less than or equal to 1 year) to 676 +/- 116 (greater than 2 years) with a major increase in the VLDL fraction (15-fold over control, greater than 2 years). Serum levels of apo B increased to 141 +/- 16 (less than or equal to 2 years) and 223 +/- 8 mg/dl (greater than 2 years) in contrast to control, 73 +/- 2. Morphologically, lipid deposition in the intima and fatty streaks have been observed in the abdominal aorta of all the diabetic monkeys with duration of more than 2 years. In six of the diabetic monkeys atheromatous changes such as intimal and medial thickening with smooth muscle cell proliferation were observed with foam cell formation. Similar atherosclerotic lesions were observed in renal and coronary arteries in at least six of these monkeys. In diabetic monkeys with duration of less than 2 years, mild atherosclerotic lesions were observed in two out of five. The results indicate that long standing insulinopenia leads to metabolic derangements characterized by hyperglycemia, ketonemia and hyperlipidemia. Elevation of LDL-cholesterol and VLDL TG with an increase of apo B is a characteristic of lipoprotein disorder. Morphologically, early to moderately advanced lesions of atherosclerosis were observed in aorta, renal and coronary arteries as a result of metabolic derangement due to insulin deficiency.

Animals↗