Biomedical subjects

K Haga

Publications and source records attributed to K Haga.

At least 73 records · Page 4Linked to original sources

PubMed1994

[Regulation of G protein-coupled receptor kinase activity].

Recent progress on the activation of G protein-coupled receptor kinases is reviewed. beta-Adrenergic receptor kinase (beta ARK) is activated by G protein beta gamma -subunits, which interact with the carboxyl terminal portion of beta ARK. Muscarinic receptor m2-subtypes are phosphorylated by beta ARK1 in the central part of the third intracellular loop (I3). Phosphorylation of I3-GST fusion protein by beta ARK1 is synergistically stimulated by the beta gamma -subunits and mastoparan or a peptide corresponding to portions adjacent to the transmembrane segments of m2-receptors or by beta gamma -subunits and the agonist-bound I3-deleted m2 variant. These results indicate that agonist-bound receptors serve as both substrates and activators of beta ARK.

Animals↗

PubMed1994

[A case of examination of skeletal remains--how many bodies did they come from?].

We examined skeletal remains, with the main intention of estimating how many bodies they had come from. The samples were a skull with defect of its base, an upper jaw, two lower jaws (No. 1, No. 2) and two skeletal bodies (No. 1: with no skull, No. 2: consisting mainly of the lower extremities). In examining the mutual relationship among them, we utilized adjustability between the skull and the lower jaws at the temporomandibular joint, and between the lower jaws and the upper jaw in the biting manner of their teeth. We concluded that the most probable combination was of two bodies, i.e. [skull + lower jaw No. 1 + skeletal body No. 1] and [upper jaw + lower jaw No. 2 + skeletal body No. 2]. Beside the above presentation, we did comment on several problems in personal identification and estimation of postmortem interval.

Adult↗

PubMed1994

[Estimation of age from epiphyseal union degrees of the sternal end of the clavicle].

The sternal ends of right clavicles of Japanese aged 13-31 years (n = 54) autopsied during 1982-1992 were observed for determining degree of epiphyseal union to establish a criterion for age estimation. As the degree of union 5 stages (McKern and Stewart, 1957) were applied to samples: Stage 0; without any union between the trunk and the epiphyseal cap, stage 1; between stage 0 and stage 2, stage 2; half united, stage 3; between stage 2 and stage 4, and stage 4; completely united. As results, in females union appears to proceed faster than in males. There were cases in which union reached stage 4 in younger ages. As a criterion for age estimation it is proposed at present that in both sex, when union is in stage 0, age is certainly estimated to be less than 22 years old and in stage 4, more than 19 years old. Comparing the present data with the investigation about American males by McKern and Stewart (1957), in Japanese males union proceeds more fast than them. The authors realized that the correct criteria should be used in age estimation on a sample belonging to the same race and to the times. The clavicles in skeletal remains tend to be preserved outdoors and are suitable for estimating age from adolescence to about 30 years old. Therefore the present criterion would be very useful in districts such as Hokkaido where skeletal remains are commonly found.

Adolescent↗

PubMed1993

Three histidine residues in the active center of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011: effects of the replacement on pH dependence and transition-state stabilization.

Cyclodextrin glucanotransferase (CGTase) catalyzes the formation of cyclodextrins from amylose through an intramolecular transglycosylation reaction. On the basis of the three-dimensional structures of CGTases three histidine residues, which are conserved between CGTases and alpha-amylases, are located at the active center and are proposed to constitute the substrate binding sites. The three histidine residues (His-140, His-233, and His-327) of CGTase from alkalophilic Bacillus sp. 1011 were individually replaced by site-directed mutagenesis to probe their roles in catalysis. Asparagine-replaced CGTases (H140N-, H233N-, and H327N-CGTase) retained cyclization activity but had altered production ratios of alpha-, beta-, and gamma-cyclodextrin. Replacement of histidine by asparagine residues strongly affected the kcat for beta-cyclodextrin-forming, coupling, and hydrolyzing activities, whereas it barely affected the Km values. The activation energies for alpha-cyclodextrin hydrolysis were increased more than 12 kJ/mol by the replacement. Furthermore, the Ki values of acarbose, which is thought to be a transition-state analog of glycosidase catalysis, were 2-3 orders of magnitude larger in asparagine-replaced CGTases than that in wild-type CGTase. Therefore, the three histidine residues participate in the stabilization of the transition state, whereas they participate little in ground-state substrate binding. H327N-CGTase had decreased activity over an alkaline pH range, indicating that His-327 is important for catalysis over an alkaline pH range.

Acarbose↗

PubMed1993

Activation by G protein beta gamma subunits of beta-adrenergic and muscarinic receptor kinase.

We have shown previously that GTP-binding regulatory protein (G protein) beta gamma subunits stimulate the agonist- or light-dependent phosphorylation of muscarinic acetylcholine receptors (mAChRs) and rhodopsin by a protein kinase partially purified from porcine brain (mAChR kinase) but not the phosphorylation of rhodopsin by rhodopsin kinase (Haga, K., and Haga, T. (1992) J. Biol. Chem. 267, 2222-2227). We report here that the mAChR kinase phosphorylates beta-adrenergic receptors (beta-ARs) purified from bovine lung in an agonist-dependent manner, and the phosphorylation is also stimulated by G protein beta gamma subunits. We also report that recombinant beta-adrenergic receptor kinase 1 (beta-ARK1) expressed in COS-7 cells phosphorylates mAChRs (human m2 subtype) and rhodopsin in an agonist- or light-dependent manner, respectively, and that this phosphorylation is stimulated by G protein beta gamma subunits. By contrast, the beta gamma subunits do not stimulate the phosphorylation of mAChRs or rhodopsin by a beta-ARK1 mutant lacking a part of the carboxyl-terminal region which is present in beta-ARKs but not in rhodopsin kinase. These results indicate that the beta-ARK1 is the same as or very similar to the mAChR kinase but is distinguished from the rhodopsin kinase with respect to activation by the beta gamma subunits and that the extra carboxyl-terminal sequence in beta-ARKs is required for the stimulation by the beta gamma subunits.

Alprenolol↗

PubMed1993

Phosphorylation of muscarinic receptors: regulation by G proteins.

Effects of G proteins on the phosphorylation of muscarinic receptors (mAChRs) have been examined. Cerebral but not atrial mAChRs were phosphorylated by any one of three types of protein kinase C and 4-6 mol of phosphate were incorporated per mol of mAChR, mostly in the 12-14 kDa from the carboxyterminus. Atrial mAChRs were better substrates of cAMP-dependent protein kinase than cerebral mAChRs. Phosphorylation of mAChRs by protein kinase C or cAMP-dependent protein kinase was not dependent on the presence of agonists and G proteins except that a slight inhibition by G proteins was observed probably because G proteins were also substrates of the two kinases. Agonist-dependent phosphorylation of atrial mAChRs or recombinant human mAChRs (m2 subtype) by a kinase (mAChR kinase), which is the same or very similar to beta adrenergic receptor kinase (beta ARK), was found to be regulated by the G proteins in a dual manner; stimulation by G protein beta gamma subunits and inhibition by G protein alpha beta gamma trimer. The inhibition by the G protein trimer is restored by addition of guanine nucleotides and is considered to be due to the formation of a ternary complex of agonist, mAChR and guanine nucleotide free G proteins. The stimulation by G protein beta gamma subunits was also observed for the light- or agonist-dependent phosphorylation of rhodopsin and beta AR by the mAChR kinase but not for the light-dependent phosphorylation of rhodopsin by rhodopsin kinase. The phosphorylation by beta ARK 1 was also found to be stimulated by G protein beta gamma subunits. The beta gamma subunit is considered to interact with the extra 130 amino acid residue carboxyterminal tail of beta ARK, which does not exist in rhodopsin kinase, and the interaction results in the activation of the kinase. We may assume that the G protein coupled receptor kinase is an effector of G protein beta gamma subunits and that one of the functions of beta gamma subunits is to stimulate the phosphorylation of G protein coupled receptors thereby facilitating their desensitization.

Acetylcholine↗

PubMed1993

[Effect of Y-25130 on gastric motility in anesthetized rats].

We developed an automatic analyzing system using a personal computer to evaluate gastric motility objectively. Gastric motility was divided into three elements: motility index, amplitude and frequency. These elements analyzed with both computer and manual methods closely correlated with each other (R2 = 0.943, 0.985 and 0.986, respectively). The effect of Y-25130 on gastric motility was investigated in anesthetized rats with the automatic analyzing system. Intravenous administration of Y-25130 increased both spontaneous and cisplatin-reduced gastric motility. The effect of Y-25130 on spontaneous motility was superior to that of ondansetron and metoclopramide, and the effect on cisplatin-reduced motility was almost the same as that of ondansetron and superior to that of metoclopramide. In vagotomized rats, Y-25130 did not increase the gastric motility not only in spontaneous conditions but also in efferent vagus stimulated conditions. Atropine completely blocked the gastric motility induced by Y-25130. In capsaicin pre-treated rats, Y-25130 had no effect. On the other hand, Y-25130 increased the gastric motility induced by afferent vagus stimulation. These results suggest that Y-25130 increases gastric motility through the vagus nerve and acts on afferent neurons.

Animals↗

PubMed1993

[Effect of Y-25130, a selective 5-HT3 receptor antagonist, on the intestinal fluid secretion in rats].

We studied the effect of Y-25130 on the intestinal fluid secretion induced by 5-HT and cholera toxin in rats. 1) Net fluid secretion was increased dose-dependently by intramesenteric artery infusion of 5-HT (1-10 micrograms/min). 2) Y-25130 (0.01-1 mg/kg, i.v.) inhibited the net fluid secretion induced by 5-HT (3 micrograms/min). Granisetron and ondansetron also inhibited the net fluid secretion induced by 5-HT. 3) Methysergide did not inhibit the fluid secretion, and neither atropine nor tetrodotoxin inhibited it. 4) Cholera toxin (1-10 micrograms/2 ml into jejunal loops) caused profuse net fluid secretion. 5) Y-25130 (1 mg/kg, i.v.) inhibited the net fluid secretion induced by cholera toxin (3 micrograms/2 ml). Granisetron was inhibitory and ondansetron tended to inhibit the cholera toxin-induced secretion. These results suggest that 5-HT may increase the net fluid secretion through 5-HT3 receptors, and cholera toxin-induced fluid secretion may be at least partially mediated by 5-HT3 receptors. It is expected that Y-25130 may be useful for treating secretory diarrhea in humans.

Animals↗

PubMed1993

The effects of orally administered Y-25130, a selective serotonin3-receptor antagonist, on chemotherapeutic agent-induced emesis.

The antiemetic effects of orally administered Y-25130, a potent and selective 5-HT3-receptor antagonist, were compared with those of ondansetron, granisetron, metoclopramide and domperidone. Y-25130 (0.1-1.0 mg/kg) dose-dependently prolonged the latency to the first vomiting and decreased the number of vomitings induced by cisplatin in dogs. The antiemetic effect of Y-25130 against cisplatin-induced vomiting was more potent than that of metoclopramide and ondansetron, but it showed little difference from that of granisetron. The emesis induced by the combined treatment of doxorubicin and cyclophosphamide was also inhibited by Y-25130 (0.1-1 mg/kg) in ferrets. The antiemetic effect of Y-25130 was more potent than that of metoclopramide, almost the same as that of granisetron and less potent than that of ondansetron. Because of a notable difference of potency ranking between Y-25130 and ondansetron in these two tests, a third test was performed to evaluate the inhibitory effect of Y-25130 in ferrets on cisplatin-induced emesis in comparison with that of ondansetron. The antiemetic effect of Y-25130 on cisplatin-induced emesis in ferrets was very similar to that of ondansetron. Domperidone did not inhibit these cytotoxic agents-induced emeses. These results suggest that Y-25130 is an orally active antiemetic compound against cisplatin and doxorubicin/cyclophosphamide-induced emeses; and its the antiemetic potency is similar to those of granisetron and ondansetron, but superior to those of metoclopramide and domperidone.

Administration, Oral↗

PubMed1992

Activation by G protein beta gamma subunits of agonist- or light-dependent phosphorylation of muscarinic acetylcholine receptors and rhodopsin.

We have partially purified a protein kinase that phosphorylates muscarinic receptors (mAChR) in the presence of agonists and have shown that the phosphorylation is stimulated by the beta gamma subunits of the GTP binding protein Go (Haga, K., and Haga, T. (1990) FEBS Lett. 268, 43-47). We report here that rhodopsin is also phosphorylated in a light-dependent manner by the same kinase preparation and that beta gamma subunits derived from Gs, Gi, and Go stimulate the phosphorylation of both rhodopsin and mAChRs. The rhodopsin- and mAChR-phosphorylating activities were eluted in the same fractions using a purification procedure that is essentially the same as that used for the purification of beta-adrenergic receptor kinase (Benovic, J.L., Strasser, R.H., Caron, M.G., and Lefkowitz, R.J. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2797-2801) and were inhibited by low concentrations of heparin, an inhibitor of beta-adrenergic receptor kinase, (IC50 = 15 nM), suggesting that both mAChR and rhodopsin are phosphorylated by the same or very similar kinase(s) belonging to the beta-adrenergic receptor kinase family. G protein beta gamma subunits increased the Vmax of the phosphorylation of rhodopsin 12-fold. Kinetic data were consistent with the assumptions that the protein kinase (mAChR kinase) binds rhodopsin and beta gamma subunits in a random order and that the reaction rate is proportional to concentration of the ternary complex. By contrast, the light-dependent phosphorylation of rhodopsin by the rhodopsin kinase was not stimulated by the beta gamma subunits. These results indicate that beta gamma subunits may interact with and activate the mAChR kinase but not rhodopsin kinase and suggest that the beta gamma subunit of G proteins may take part in the desensitization of G protein-linked receptors.

Animals↗

PubMed1992

Functional relationships between cyclodextrin glucanotransferase from an alkalophilic Bacillus and alpha-amylases. Site-directed mutagenesis of the conserved two Asp and one Glu residues.

Comparison of the amino acid sequences of cyclodextrin glucanotransferases (CGTases) with those of alpha-amylases revealed that two Asp and one Glu residues, which are considered to be the catalytic residues in alpha-amylases, were also conserved in CGTases. To analyze the function of the three conserved amino acid residues in CGTases, site-directed mutagenesis was carried out. The three mutant CGTases, in which Asp229, Glu257 and Asp328 were individually replaced by Asn or Gln, completely lost both their starch-degrading and beta-cyclodextrin-forming activities, whereas another mutant CGTase, in which Glu264 was replaced by Gln, retained these activities. The three inactive enzymes retained the ability to be bound to starch. These results suggest that Asp229, Glu257 and Asp328 play an important role in the enzymatic reaction catalyzed by CGTase and that a similar catalytic mechanism is present in both CGTases and alpha-amylases.

Aspartic Acid↗

PubMed1992

Effect of glutathione on lambda deoxyribonucleic acid strand breaks in the reaction system of glutathione-alloxan in the presence of Fe(3+)-ethylenediaminetetraacetic acid.

Alkaline sucrose density gradient and agarose gel electrophoresis methods were used to observe lambda deoxyribonucleic acid (DNA) strand breaks by the reaction system of reduced glutathione (GSH) with alloxan in the presence of Fe(3+)-ethylenediaminetetraacetic acid (EDTA). When DNA was incubated in the reaction system for 10 min, DNA strand breaks were easily induced. The increasing concentrations of GSH up to 1.0 mM in the reaction system in the presence of 1.0 mM alloxan caused DNA strand breaks in a concentration-dependent fashion and GSH beyond 2.0 mM caused in the strand breaks of DNA by which the fragments with multiple ranges of molecular weight were produced. The strand breaks of DNA in the reaction system containing low concentrations of GSH were protected by catalase and hydroxyl radical (HO.) scavengers but superoxide dismutase (SOD) did not, indicating that such breaks were induced by HO.generated from the Fenton reaction. On the other hand, the strand breaks of DNA at high concentrations of GSH were protected by ethanol and desferrioxamine, but not effectively by SOD and HO.scavengers, suggesting the possible participation of some oxidizing species of iron rather than HO.. These results indicate that HO.or oxidizing species of iron generated in the GSH-alloxan system depending on the concentration of GSH attacks DNA to produce strand breaks.

Alloxan↗

PubMed1992

Enzymatic generation of alloxan radicals in rat liver microsomes: possible participation of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P-450 reductase.

Electron spin resonance studies showed that addition of rat liver microsomes to the reaction system of alloxan with reduced nicotinamide adenine dinucleotide phosphate (NADPH) resulted in a marked increase in the generation of alloxan radicals (AH.), whereas heat-denatured microsomes were without such effect. Oxidation of NADPH by alloxan was also stimulated by microsomes. The microsomes from rats treated with phenobarbital, an inducer of cytochrome P-450 reductase, greatly stimulated both the AH.generation and the NADPH oxidation. However, the microsomes from rats treated with 3-methylcholanthrene, an inducer of DT-diaphorase, did not have stimulative effect greater than the control microsomes. These results suggest the possibility that NADPH-linked AH.generations in microsomal membranes is catalyzed by NADPH-cytochrome P-450 reductase.

Alloxan↗

PubMed1992

Antagonistic activity of Y-25130 on 5-HT3 receptors.

This paper describes the 5-hydroxytryptamine3 (5-HT3) receptor antagonism of Y-25130 ((+-)-N-(1-azabicyclo[2.2.2]oct-3-yl)-6-chloro-4-methyl-3-oxo-3,4-dih yd ro- 2H-1,4-benzoxazine-8-carboxamide monohydrochloride) in the rat cerebral cortex, isolated rabbit heart and isolated guinea pig ileum. In an in vitro binding assay, Y-25130 inhibited the specific binding of [3H]quipazine to 5-HT3 receptors at the synaptic membranes of the rat cerebral cortex with a Ki value of 2.9 nM, the same as that of ondansetron. Metoclopramide, 5-HT and 2-methyl-5-HT also showed an inhibitory effect, but their affinities for 5-HT3 receptors were lower than that of Y-25130. Y-25130 showed low affinity for histamine H1 receptors (IC50 = 4.4 microM) but it could not reveal any affinities for the other receptors (5-HT1A, 5-HT2, dopamine D1, dopamine D2, alpha 1-adrenoceptor, alpha 2-adrenoceptor, muscarine and benzodiazepine) even at a 10 microM concentration. In the isolated rabbit heart, Y-25130 antagonized the indirect sympathomimetic responses to 5-HT (pA2 value = 10.06) and this effect was more potent than that of metoclopramide. In the isolated longitudinal smooth muscle of the guinea pig ileum, concentration-contraction effect curves for 5-HT were biphasic in the presence of ketanserin. Y-25130 shifted to the right only in the second phase of concentration-effect curves for 5-HT (pA2 value = 7.04) and its activity was more potent than that of metoclopramide. These results indicate that Y-25130 is a potent and selective 5-HT3 receptor antagonist.

Acetylcholine↗

PubMed1991

Purification and properties of a xylanase from Cellvibrio gilvus that hydrolyzes p-nitrophenyl cellooligosaccharides.

An enzyme component that hydrolyzes pNP-G2 but not CMC has been isolated from a culture broth of Cellvibrio gilvus by a multi-step procedure involving Butyl-Toyopearl, DEAE-Toyopearl, and CM-Toyopearl chromatographies. The purified enzyme gave a single protein band on native, SDS-, and IEF-PAGE. The enzyme had a molecular weight of 40,000, an isoelectric point of 5.0, an optimum pH of 6.5, and an optimum temperature of 55 degrees C. It was stable from pH 4.0 to 9.0 at 37 degrees C for 1 hr and below 50 degrees C for 30 min. It hydrolyzed agluconic bonds not only of pNP-G2 but also of pNP-G3, pNP-G4, and pNP-G5. Cellooligosaccharides with D.P. of 3 to 5 were not hydrolyzed at all. Instead, the enzyme hydrolyzed xylan 4 times as fast as pNP-G2. Both HgCl2 and p-chloromercuribenzoic acid inhibited the two activities completely. Xylan inhibited the hydrolysis of pNP-G2 competitively. From these results, the purified enzyme was considered to be a unique xylanase that hydrolyzed the agluconic bonds of pNP-Gn.

Amino Acid Sequence↗

PubMed1990

Dual regulation by G proteins of agonist-dependent phosphorylation of muscarinic acetylcholine receptors.

Muscarinic acetylcholine receptors purified from porcine atrium were phosphorylated, depending on the presence of agonists, by a protein kinase partially purified from porcine brain, which had similar properties to the beta-adrenergic receptor kinase. GTP-binding regulatory proteins (Go) had dual effects on the phosphorylation of muscarinic receptors, i.e. stimulation at lower concentrations and inhibition at higher concentrations. The stimulatory effect was reproduced with the beta gamma subunit of Go and the inhibitory effect with the combination of the alpha and beta gamma subunits.

Adenosine Triphosphate↗

PubMed1990

Interaction of atrial muscarinic receptors with three kinds of GTP-binding proteins.

Purified porcine atrial muscarinic acetylcholine receptors were reconstituted into lipid vesicles with three different G proteins (Gi, Go and Gn)1 purified from porcine cerebrum. All the G proteins interacted with the receptor as evidenced by GTP-sensitive high affinity binding with acetylcholine, and stimulation by acetylcholine of GTP gamma S binding and GTPase activities. The curves of displacement by acetylcholine of [3H]QNB binding were explained by assuming two sites with the same affinity for [3H]QNB but different affinities for acetylcholine. The proportion of the high affinity site increased from 3 to 7% up to 82 to 83% of total binding sites with increasing G protein concentration, and essentially the same results were obtained with the three G proteins. The GTPase activities of Gi, Go and Gn in the reconstituted vesicles were 2.7-, 1.7- and 1.6-times higher, respectively, in the presence of 1 mM acetylcholine than those in the presence of 10 microM atropine. An obvious enhancement by acetylcholine of the GTP gamma S binding was observed in the presence of 10 to 100 microM GDP, while the enhancement was minimal, if at all, in the absence of GDP. When the molar ratios of reconstituted Gi, Go and Gn to muscarinic receptors were 54, 84 and 107, respectively, the acetylcholine-induced increase in the [35S]GTP gamma S binding was as much as 12, 35 and 27 mol with Gi, Go and Gn, respectively, per mole of the receptor molecule, indicating that the muscarinic receptors interact with G proteins catalytically.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗

PubMed1990

Phosphorylation by protein kinase C of the muscarinic acetylcholine receptor.

Muscarinic acetylcholine receptors purified from porcine cerebrum were phosphorylated by protein kinase C purified from the same tissue. More than 1 mol of phosphate was incorporated per mole of receptor, with both serine and threonine residues being phosphorylated. Neither the degree nor the rate of the phosphorylation was affected by the presence or absence of acetylcholine. GTP-sensitive high-affinity binding with acetylcholine was observed for muscarinic receptors reconstituted with GTP-binding proteins (Gi or Go), irrespective of whether muscarinic receptors or the GTP-binding proteins had been phosphorylated by protein kinase C or not. This indicates that the interaction between purified muscarinic receptors and purified GTP-binding proteins in vitro is not affected by their phosphorylation.

Animals↗