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Biomedical subjects

H Hidaka

Publications and source records attributed to H Hidaka.

At least 343 records · Page 19Linked to original sources
PubMed1991

Molecular cloning of the gene for indolepyruvate decarboxylase from Enterobacter cloacae.

Although indole-3-acetic acid (IAA) is a well-known plant hormone, the main IAA biosynthetic pathway from L-tryptophan (Trp) via indole-3-pyruvic acid (IPyA) has yet to be elucidated. Previous studies have suggested that IAA is produced by Enterobacter cloacae isolated from the rhizosphere of cucumbers and its biosynthetic pathway may possibly be the same as that in plants. To elucidate this pathway, the IAA biosynthetic gene was isolated from a genomic library of E. cloacae by assaying for the ability to convert Trp to IAA. DNA sequence analysis showed that this gene codes for only one enzyme and its predicted protein sequence has extensive homology with pyruvate decarboxylase in yeast and Zymomonas mobilis. Cell-free extracts prepared from Escherichia coli harboring this gene could convert IPyA to indole-3-acetaldehyde (IAAld). These results clearly show that this pathway is mediated only by indolepyruvate decarboxylase, which catalyzes the conversion of IPyA to IAAld.

Amino Acid Sequence↗
PubMed1991

The effects of HA1077 on the cerebral circulation after subarachnoid haemorrhage in dogs.

We examined the effects of the recently developed calcium antagonist HA1077 on cerebral haemodynamics during the chronic stage of the two-haemorrhage canine model system of vasospasm. Regional cerebral blood flow (rCBF), regional cerebral blood velocity and regional cerebral blood volume in the canine parietal cortex were measured by Laser-doppler flowmeter. On the 7th day after the initial injection of autogenous blood, subarachnoid haemorrhage (SAH) produced a significant decrease in rCBF (59% of control, p less than 0.05) and blood velocity (48% of control, p less than 0.05) with no remarkable change in blood volume (108% of control). Bolus intravenous administration of HA1077 (0.1-0.3 mg/kg) dose-dependently increased the rCBF and blood velocity, without significantly changing the blood volume on Day 7 after SAH. HA1077 improves haemodynamic function manifested by an increase in rCBF and velocity in this SAH model, and may be suitable for the treatment of vasospasm in patients with SAH.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Phosphorylation of bovine brain 81-kDa acidic calmodulin binding protein (ACAMP-81) in vitro.

We found a novel 81-kDa acidic protein (ACAMP-81) in the bovine brain membrane fraction, which bound to calmodulin in a Ca(2+)-dependent manner. The present study reveals physicochemical properties and phosphorylation of this protein with various protein kinases in vitro. The Stokes radius and sedimentation coefficient were calculated to be 52 A and 2.05 S, respectively, suggesting that the structure of ACAMP-81 is highly elongated. Purified Ca2+/phospholipid-dependent protein kinase (protein kinase C), cAMP-dependent protein kinase, and Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) catalyzed the incorporation of 1.46, 0.72, and 0.44 mol of phosphate/mol of ACAMP-81, respectively. The amino acid residues of ACAMP-81 phosphorylated by either protein kinase C or cAMP-dependent protein kinase were almost exclusively on serine. Sequential phosphorylation of ACAMP-81 by cAMP-dependent protein kinase and protein kinase C resulted in the additional incorporation of 1.15 mol of [32P]phosphate into ACAMP-81. Comparison of phosphopeptide maps of ACAMP-81 phosphorylated by each kinase revealed that there are two classes of phosphorylatable polypeptide, one is phosphorylatable by both protein kinases which contained two polypeptides and the others are specific sites for protein kinase C.

Amino Acids↗
PubMed1991

Thrombin-induced translocation of protein kinase C in human megakaryoblastic leukemia cells (MEG-01).

Our previous immunocytochemical study showed that Ca2+ ionophore-induced translocation of protein kinase C (PKC) in human megakaryoblastic leukemia cells (MEG-01) was potentiated by a synthetic diacylglycerol (T. Ito, T. Tanaka, T. Yoshida, K. Onoda, H. Ohta, M. Hagiwara, Y. Itoh, M. Ogura, H. Saito, and H. Hidaka, 1988, J. Cell Biol. 107, 929). In the present study, we analyzed the roles of the intracellular Ca2+ levels ([Ca2+]i) and diacylglycerol (DG) levels in thrombin-induced translocation of PKC using MEG-01 cells. When the cells were treated with thrombin (0.5 U/ml), PKC was translocated from the cytosol to the plasma membrane after 15 s, and the maximal membrane association was observed after 90 s. The [Ca2+]i of the cells rapidly increased (15 s) and reached a maximum level at 60 s which was sustained for a total of 600 s after thrombin addition. The increase in DG was biphasic with the first phase occurring in the first 15 s and the increase during the second phase lasting less than 600 s. The experiments without extracellular Ca2+ indicated that Ca2+ efflux accompanied by DG in the first phase was sufficient to initiate the membrane association of the PKC and that the large Ca2+ influx enhanced the binding. PKC returned to the cytosol within 600 s despite high levels of both [Ca2+]i and DG. We found that a relatively selective PKC inhibitor, H-7, enhanced thrombin-induced translocation of PKC without modulating [Ca2+]i or DG levels. These results indicate that certain protein phosphorylation events, potentially those mediated by PKC, may be responsible for, at least in part, inhibiting membrane association and further activation of the enzyme.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

A calcium-binding protein from rabbit lung cytosol identified as the product of growth-regulated gene (2A9) and its binding proteins.

Using Ca(2+)-dependent affinity chromatography on a synthetic compound (W-77)-coupled Sepharose 4B column, we purified two different Ca(2+)-binding proteins from rabbit lung extracts. The molecular weights of these proteins were estimated to be 17 kDa (calmodulin) and 10 kDa, respectively. The partial amino acid sequence of the 10-kDa protein revealed that it has two EF-hand structures. In addition, the 10-kDa protein was highly homologous (91%) to the product of growth-regulated gene, 2A9 (calcyclin). The Ca(2+)-binding property of the 10-kDa protein was observed by a change in the uv difference spectrum. Equilibrium dialysis showed that 1 mol of the 10-kDa protein bound to 2.04 +/- 0.05 mol of Ca2+ in the presence of 10(-4) M Ca2+. However, the protein failed to activate calmodulin-dependent enzymes such as Ca2+/CaM kinase II, myosin light chain kinase, and phosphodiesterase. We found that a 50-kDa cytosolic protein of the rabbit lung, intestine, and spleen bound to the 10-kDa protein, in a Ca(2+)-dependent manner. The distribution of calcyclin and calcyclin binding proteins was unique and seems to differ from that of calmodulin and calmodulin-binding proteins. Thus, calcyclin probably plays a physiological role through its binding proteins for the Ca(2+)-dependent cellular response.

Amino Acid Sequence↗
PubMed1991

Identification and characterization of protein kinase C-related enzymes in frog retina.

We isolated two types of protein kinase C-related enzymes (fPKC) in frog (Rana catesbeiana) retina. Three sequential steps of column chromatography of DEAE-cellulose, butyl-Toyopearl, and hydroxylapatite were carried out and resulted in the separation of two types of distinct Ca2+/phospholipid-dependent protein kinase activities. These proteins reacted with antibodies raised against synthetic peptides based on the predicted amino acid sequences of rat protein kinase C delta or zeta subspecies, and showed molecular masses of 90 and 75 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. Immunocytochemical analysis of 90 and 75K fPKC in frog retina revealed that the 75K fPKC was localized in the outer segment of cone cells and in the microvillar projections of Müller cells. These results suggested that fPKCs play differential roles on the photosignal transduction in frog retina.

Amino Acid Sequence↗
PubMed1991

Low level hyperlipidemia impairs endothelium-dependent relaxation of porcine coronary arteries by two mechanisms. Functional change in endothelium and impairment of endothelium-dependent relaxation by two mediators.

We evaluated the effect of a low level of hyperlipidemia and the effects of in vitro exposure to atherogenic lipoproteins (LDL, VLDL) on the vascular responsiveness of isolated porcine coronary arteries. Firstly we studied the change in vascular responsiveness induced by feeding a cholesterol-rich diet to pigs for 4 and 9 weeks (C4 and C9 pigs). The serum cholesterol level in pigs fed a cholesterol-rich diet reached 218.5 +/- 32.9 mg/dl compared with 85.5 +/- 8.4 mg/dl in the controls. Segments of the left descending coronary artery were examined. The contraction induced by KCl or prostaglandin F2 alpha was not altered significantly by hypercholesterolemia nor was the relaxation induced by the Ca2+ ionophore, A23187, or by nitroglycerin. Endothelium-dependent relaxation (EDR) evoked by high, but not low, concentrations of bradykinin was reduced in the C4 pigs as compared with those in normal animals. EDRs evoked by bradykinin, substance P, and serotonin were significantly reduced in C9 pigs. Histologically, as observed by light and electron microscopy, fatty changes or intimal thickenings were not seen in the coronary arteries of the C4 pigs. Minimal changes (intimal thickening and fragmentation of internal elastic lamina) were observed only in parts of arteries of the C9 pigs. Secondly, the direct effects of LDL and VLDL on vascular responsiveness were studied. Although preincubation with LDL inhibited the EDR caused by exposure to bradykinin and A23187 in the coronary arteries of normal and cholesterol-fed pigs, preincubation with LDL inhibited the arterial relaxation induced by exposure to substance P or serotonin in both the C4 and the C9 pigs, but not in the control animals. The degree of inhibition was especially marked in the C9 pigs. The inhibitory effect of VLDL on EDR was weaker than that of LDL. Indomethacin (5 microM) did not alter this inhibitory effect of lipoproteins. Neither LDL nor VLDL had any effect on the vascular relaxation induced by nitroglycerin. These results are consistent with the idea that endothelium-dependent arterial relaxation is attenuated even at the very early stage of cholesterol-induced atherosclerosis. Atherogenic lipoproteins may further impair the decreased EDR in the arteries of hyperlipidemic pigs by two factors: one released on stimulation with bradykinin and the calcium ionophore A23187, the other released on stimulation with substance P and serotonin.

Animals↗
PubMed1991

In vitro stimulation of glucose utilization by insulin in primary cultures of rat hepatocytes.

The effect of glucose concentration and insulin on glucose incorporation was studied in primary cultures of rat hepatocytes. The rate of glucose incorporation into hepatocytes was proportional to the medium glucose concentration from 100 to 800 mg/dl. At 800 mg/dl glucose the rate reached a plateau. Of the glucose taken up by hepatocytes, 16 and 18% was incorporated into glycogen and lipid, respectively, and 58% into the nucleotide fraction after incubation for 4 h. In the medium, lactate was the major product found. Insulin stimulates glucose incorporation by 20-112% into all the above pathways at glucose concentrations between 100 and 800 mg/dl. The insulin effect was noted as early as 2-4 h (early effect) and up to 24 h (delayed effect). This effect of insulin was observed to be dose dependent from 5 to 200 ng/ml insulin. While the delayed insulin effect was abolished by cycloheximide, the early effect of insulin was not affected. With respect to the key enzyme activities of glucose utilization, activation of glycogen synthase (increase of I-activity/total activity) and pyruvate kinase (activation at 0.2 mM phosphoenolpyruvate) was noted 4 h after insulin addition, and these effects were not abolished by cycloheximide. These two enzymes increased in total activity after 24 h. Both glucokinase and glucose-6-phosphate dehydrogenase activities increased by 30-35% and 65-93% at 4 and 24 h, respectively. The results indicate that hepatocytes directly utilize glucose in a dose-dependent manner with respect to glucose and insulin. A major early and delayed effect of insulin appeared due to the activation and induction of the key hepatic enzymes of glucose utilization, respectively.

Animals↗
PubMed1991

Obviation of drug resistance and affinity purification of P-glycoprotein by isoquinolinesulfonamides.

A newly synthesized isoquinolinesulfonamide named H-85; N-[2-[N-formyl-N-[[3-(4-chlorophenyl)-2-propenyl] amino] ethyl]-5-isoquinolinesulfonamide was found to reverse drug resistance in multidrug resistant P388 murine leukemic cells (P388/ADR). The energy-dependent extrusion of [3H]vinblastine from P388/ADR-cells was significantly suppressed by 10 microM H-85 but not so the efflux from the sensitive P388 cells. A 140-kDa protein overexpressed in P388/ADR cells was photoaffinity labeled with a vinblastine analog; N-(P-azid-[3-125I]salicyl-N'-(beta-aminoethyl) vindesine and H-85 selectively inhibited photolabeling of the 140-kDa protein. This 140-kDa protein was purified to apparent homogeneity by succeeding steps of phosphocellulose, DEAE-cellulose, and W-66 (a derivative of H-85)-coupled sepharose chromatography. The purified 140-kDa protein proved to be immunopositive with the P-glycoprotein-specific monoclonal antibody, C219.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Differential localization of protein kinase C isozymes in retinal neurons.

We report the immunohistochemical localization of protein kinase C isozymes (types I, II, and III) in the rabbit retina using the monospecific monoclonal antibodies MC-1a, MC-2a, and MC-3a. Using immunoblot analysis of partially purified protein kinase C preparations of rabbit retina, types II and III isozymes alone were detected. The activity of type III was the stronger. By light microscopic immunohistochemical analysis, retinal neurons were negative for type I and positive for type II and type III isozymes. Type II was more diffusely distributed through the retinal layers, but was distinctive in ganglion cells, bipolar cells, and outer segments. The immunoreactivity was stronger for type III isozyme, and it was observed in mop (rod) bipolar cells and amacrine cells. By using immunoelectron microscopy, the cytoplasm of the cell body, the axon, and dendrites of the mop bipolar cells were strongly immunoreactive for type III. The so-called rod bipolar cells were for the first time seen to form synapses with rod photoreceptor cells. These differential localizations of respective isozymes in retinal neurons suggest that each isozyme has a different site of function in each neuron.

Animals↗
PubMed1991

Ca2+/calmodulin-dependent protein phosphorylation associated with the cytoskeleton of quiescent rat fibroblast (3Y1) cells.

Endogenous phosphorylation of the crude membrane fraction of cultured 3Y1 fibroblast cells was enhanced by the addition of Ca2+/calmodulin. Both Ca2+/calmodulin-dependent protein kinase activity and its substrate were present in a cytoskeletal fraction, obtained as a pellet after washing of the membrane fraction with 2 mM EGTA, 0.6 M NaCl, and 1% Triton X-100. The phosphorylatable protein in the Triton X-insoluble fraction was identified by immunoblotting as vimentin. This endogenous phosphorylation induced by calmodulin was inhibited by the addition of KN-62, a specific Ca2+/calmodulin-dependent protein kinase II inhibitor, in a dose-dependent manner. However, phosphorylation of the 59 kDa protein (vimentin) in this fraction was not stimulated by adding both phosphatidyl serine and cAMP, thereby suggesting the absence of protein kinase C or of cAMP-dependent protein kinase in this fraction. The protein kinase associated with the Triton X-insoluble fraction phosphorylated the Ca2+/calmodulin-dependent protein kinase II-specific site of synapsin I from the bovine cortex. Two-dimensional phosphopeptide maps of vimentin indicated that a major phosphopeptide phosphorylated by the endogenous calmodulin-dependent kinase also appears to be the same as a major phosphopeptide phosphorylated by the exogenous Ca2+/calmodulin-dependent protein kinase II. Our results suggest that cytoskeleton-associated Ca2+/calmodulin-dependent protein kinase II regulates dynamic cellular functions through the phosphorylation of cytoskeletal elements in non-neural cells.

Animals↗
PubMed1991

Calcium ion regulates the release of lipase of Fusarium oxysporum.

The lipase production of a plant pathogenic fungus, Fusarium oxysporum f. sp. lini SUF 402, was induced by fat as the carbon source, and its release was stimulated by the infusion of intracellular free calcium ion with a calcium ionophore, A23187. N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7, a calmodulin inhibitor) and 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl- L-tyrosyl]-4-phenylpiperazine (KN-62, a Ca2+/calmodulin dependent protein kinase II inhibitor) reduced the extracellular release of lipase in vivo. 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7, a protein kinase C inhibitor) did not have this ability. After K2H32PO4 had been incorporated into the cells, they were treated with W-7 or KN-62 and stimulated by Ca2+ ionophore. On SDS-PAGE of intracellular proteins followed by autoradiography, W-7- and KN-62-treated cells showed inhibition of the incorporation of 32Pi into the 20 kDa protein resulting from Ca2+ stimulation. F. oxysporum had calmodulin (CaM)-dependent protein kinase activity in the cytoplasmic fraction and had the ability to phosphorylate of syntide 2, a specific substrate of CaM kinase II. The partially purified CaM-dependent protein kinase was inhibited by 10 microM KN-62 in vitro. Increase of the intracellular Ca2+ concentration of F. oxysporum activated CaM and CaM-dependent protein kinase, resulting in the extracellular lipase release. These results suggest the existence of a Ca2+ signalling system in F. oxysporum like those observed in higher eucaryotes.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Inhibition of cyclic GMP phosphodiesterase by xanthine derivatives relaxes guinea-pig trachealis smooth muscle.

1. For the purpose of clarifying the mechanism of the airways smooth muscle relaxant action of xanthines, cyclic guanosine monophosphate (GMP) phosphodiesterase (PDE) from guinea-pig trachealis muscle was purified with diethylaminoethyl ether (DEAE) cellulose column chromatography. 2. Five 3-alkylxanthines (3-methylxanthine, 3-ethylxanthine, 3-n-propylxanthine (enprofylline), 3-n-butylxanthine, and 3-iso-butylxanthine), and five 1-methyl-3-alkylxanthines (1-methyl-3-methyl-xanthine (theophylline), 1-methyl-3-ethylxanthine, 1-methyl-3-n-propylxanthine, 1-methyl-3-n-butylxanthine, and 1-methyl-3-iso-butylxanthine (IBMX] were compared in terms of purified cyclic GMP PDE inhibition. The relationship between the structure and inhibition of cyclic GMP PDE was studied. 3. The -log EC50 values for relaxation of spontaneous tone of isolated guinea-pig trachealis preparations by the 3-alkylxanthines and 1-methyl-3-alkylxanthines were determined. 4. The five 1-methyl-3-alkylxanthines were each more potent in relaxing isolated trachealis smooth muscle than the corresponding 3-alkylxanthines. The 1-methyl-3-alkylxanthines were also more potent than the corresponding 3-alkylxanthines in their cyclic GMP PDE inhibitory effect. There was a strong positive correlation between the concentration of inhibitor which inhibited hydrolysis by 50% (IC50) values for cyclic GMP PDE inhibition by the xanthine derivatives and their EC50 values for trachealis muscle relaxation. 5. It is suggested that the mechanism by which xanthine derivatives relax trachealis smooth muscle involves inhibition of cyclic GMP PDE in addition to inhibition of cyclic adenosine monophosphate PDE.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1991

Blockade of intracellular actions of calcium may protect against ischaemic damage to the gerbil brain.

1. The brain cytoprotective effects of a putative calcium-associated protein kinase inhibitor, HA1077, as well as a calcium entry blocker nicardipine were evaluated in models of cerebral ischaemia in Mongolian gerbils. Morphological changes characterizing delayed neuronal death of selectively vulnerable CA1 pyramidal neurones in the hippocampus of the Mongolian gerbil brain occurred 7 days after transient bilateral occlusion of the common carotid arteries. 2. A single injection of HA1077 (1 and 3 mg kg-1, i.p.) 5 min after the occlusion led to a dose-dependent protection of the CA1 neurones. Repeated administrations of HA1077 (1 and 3 mg kg-1, i.p., twice daily for 7 days post-ischaemia) revealed an increase in the number of normal cells, compared to findings with a single administration. 3. In contrast to HA1077, nicardipine (0.3 and 1 mg kg-1, i.p.) did not reduce neuronal degeneration. 4. HA1077 did not interact with the ion channel within which MK-801 binds, as determined by receptor binding. 5. The calcium ionophore, A23187, caused a tonic contraction in canine cerebral arterial strips. HA1077, but not nicardipine, relaxed the A23187-induced contraction, concentration-dependently. 6. These results suggest that blockade of the intracellular actions of calcium may provide protection against ischaemic damage in the brain.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Neuropeptide regulation of interleukin-6 production from the pituitary: stimulation by pituitary adenylate cyclase activating polypeptide and calcitonin gene-related peptide.

Interleukin 6 (IL-6) production was shown to be stimulated by vasoactive intestinal peptide via cAMP dependent signal transduction pathway in the pituitary. We were interested in whether other hypothalamic neuropeptides, which activate adenylate cyclase in the pituitary, also stimulate pituitary IL-6 production. Whereas vasoactive intestinal peptide was effective in stimulating pituitary IL-6 production only at concentrations of 10(-6) M or higher, pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38) and calcitonin gene-related peptide (CGRP) at concentrations from 10(-10) to 10(-9) M significantly stimulated IL-6 production. Similar effective concentrations of each peptide were required for activating adenylate cyclase, as measured by extracellular cAMP accumulation. H89, a specific inhibitor of cAMP dependent protein kinase (protein kinase A), inhibited IL-6 production stimulated by PACAP38, CGRP, and (Bu)2cAMP. However, H89 failed to inhibit the IL-6 production stimulated by lipopolysaccharide, a ligand which enhanced IL-6 production in the absence of cAMP accumulation. Two other peptides which are known to activate pituitary adenylate cyclase, corticotropin-releasing factor and GRF failed to stimulate IL-6 production in pituitary cells. Using discontinuous Percoll gradients to fractionate the pituitary cells, the greatest PACAP38-stimulated IL-6 secretion was observed in the low density fraction 1 (F1). This fraction also contained the highest percentage of folliculo-stellate (FS) cells, one of the nonhormone secreting pituitary cells. However, the largest PACAP38-induced accumulation of cAMP was observed in F4. These results suggest that the production of IL-6 stimulated by PACAP and CGRP is mediated by the adenylate cyclase/protein kinase A signal transduction system. FS cells appear to be the most likely target cell type for PACAP-induced IL-6 production. However, IL-6 producing FS cells may not be an exclusive target for PACAP in the pituitary.

Animals↗