Search PubMed⌕ Search

Biomedical subjects

T Haga

Publications and source records attributed to T Haga.

At least 91 records · Page 5Linked to original sources
PubMed1994

Location of agonist-dependent-phosphorylation sites in the third intracellular loop of muscarinic acetylcholine receptors (m2 subtype).

Muscarinic acetylcholine receptors (mAChR, human m2 subtype) expressed in Sf9 (Spodoptera frugiperda) cells using the baculovirus system were purified and subjected to phosphorylation by a mAChR kinase, which was partially purified from porcine cerebrum. Two bands with apparent molecular masses of 59 kDa and 39 kDa as determined by SDS/PAGE were found to be phosphorylated in an agonist-dependent manner. Both bands were labeled by the irreversible muscarinic ligand [3H]propylbenzilylcholine mustard. Molecular masses of the [32P]phosphorylated or [3H]propylbenzilylcholine-mustard-labeled bands decreased following treatment with N-glycanase. The 59-kDa and 39-kDa bands were converted to 52-kDa and 32-kDa bands, respectively, indicating that both the 59-kDa and 39-kDa bands contain the amino-terminal region where glycosylation sites are present. The ratio of incorporated [32P]phosphate and bound [3H]propylbenzilylcholine mustard was essentially the same for the 59-kDa and 39-kDa bands, indicating that all the phosphorylation sites reside in the sequence of 39 kDa from the amino-terminal region. The amounts of incorporated [32P]phosphate were estimated to be 10-11/receptor, with 7-8 serine and 3-4 threonine, but no phosphorylated tyrosine residues. Further treatment of [32P]phosphorylated or [3H]propylbenzilylcholine-mustard-labeled receptors with V8 protease indicated that the phosphorylation sites were not present in 30-kDa amino-terminal segment. These results indicate that the phosphorylation sites are localized in the range 30-39 kDa from the amino terminus, which consists of primarily the central part of the third intracellular loop. Consistent with this conclusion, a fusion protein containing glutathione S-transferase linked to a peptide corresponding to residues 227-324 of the central part of the third intracellular loop was found to be phosphorylated by the mAChR kinase in a heparin-sensitive manner.

Amino Acid Sequence↗
PubMed1994

G protein-mediated inhibition of phosphoinositide metabolism evoked by metabotropic glutamate receptors in frog oocytes.

1. Metabotropic glutamate receptor subtype 1 (mGluR1), when expressed in Xenopus oocytes, activates phosphoinositide-specific phospholipase C (PLC) in a G protein-dependent manner. This reaction results in the activation of chloride channels in the oocytes, and can be monitored electrophysiologically. We expressed different G protein alpha-subunits together with mGluR1 in oocytes, and examined the effects of these G protein subunits on the PLC-mediated reaction. 2. The expression of the alpha-subunit of GL2, a bovine version of G11, which is a member of the Gq subgroup, potentiated the mGluR1-evoked reaction, whereas the alpha-subunit of GL1, a bovine G14, which is also a member of the Gq subgroup, strongly suppressed it. The expression of Gs alpha also suppressed this reaction. 3. We then expressed G beta 1 gamma 2-subunits in addition to the G alpha-subunits, and examined the mGluR1-evoked reactions. Both the potentiation and suppression by GL2 alpha and GL1 alpha, respectively, were more pronounced in the presence of the G beta 1 gamma 2-subunits. In contrast, the suppression by Gs alpha was completely reversed by G beta 1 gamma 2. 4. The direct activation of G proteins by the intracellular injection of either fluoride ions or guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) causes similar PLC-mediated reactions. The expression of GL2 alpha, GL1 alpha or Gs alpha caused potentiation, suppression and no change, respectively, on the fluoride- (or GTP gamma S-) evoked reactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Focal spasms in clusters, focal delayed myelination, and hypsarrhythmia: unusual variant of West syndrome.

We report a patient who began to have clusters of seizures characterized by brief elevation of the right arm at 6 months of age. An interictal electroencephalogram (EEG) at 7 months revealed hypsarrhythmia without definite asymmetry. Simultaneous EEG and video recording disclosed that these focal spasms were associated with fast wave bursts superimposed on slow waves most markedly in the left centro-midtemporal region. The patient became seizure-free after synthetic ACTH therapy. The patient is developmentally normal at 3 years 5 months, but magnetic resonance imaging studies revealed findings suggestive of delayed myelination in the left frontal region. This patient is considered to have had an unusual variant of West syndrome associated with focal delayed myelination.

Adolescent↗
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↗
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

Solubilization and characterization of atrial muscarinic acetylcholine receptors in sucrose monolaurate.

Muscarinic acetylcholine receptors (mAChRs) can be solubilized from porcine atrial membranes in sucrose monolaurate (SM-1200) with retention of up to 80% of N-[3H]methylscopolamine ([3H]NMS) binding activity. The mAChRs solubilized by SM-1200 were relatively stable at 4 degrees C with an estimated half-life (tau 1/2) of the ligand binding activity of 19 days. Inactivation of the ligand binding activity is dependent on the incubation temperature, and tau 1/2 was estimated to be 5.7 h at 20 degrees C, 28 min at 30 degrees C, and less than 1 min at 45 degrees C. The activation energy of the receptor inactivation was estimated to be 199 kJ/mol. Ligand binding characteristics of SM-1200-solubilized mAChRs were similar to those of digitonin/cholate-solubilized receptors. Sucrose density gradient centrifugation revealed a single peak with an apparent sedimentation coefficient of 5.7 S. The solubilized atrial mAChRs were purified approximately 1000-fold by using affinity chromatography with aminobenztropine as the ligand. The purified mAChRs were reconstituted with GTP binding regulatory proteins (G(o)) in lipid vesicles, and the reconstituted vesicles showed guanine nucleotide-sensitive, high-affinity agonist binding and agonist-stimulated GTP gamma S binding in the presence of GDP. Thus, sucrose monolaurate is a new detergent in which mAChRs can be solubilized in stable form with high yield and purified up to 1000 times with retention of the binding activity with muscarinic ligands and G-proteins.

Animals↗
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

The effect of acarbose on blood glucose profiles of type 2 diabetic patients receiving insulin therapy.

Nine patients with Type 2 diabetes receiving insulin therapy were treated with acarbose 100 mg thrice daily for 1 week to investigate the effect of acarbose on blood glucose control. Daily blood glucose profiles contained fewer excursions during acarbose administration and low levels were maintained. The M-value, an indicator of blood glucose fluctuation, decreased significantly from a run-in period value of 37.6 +/- 8.7 (SEM) to 16.7 +/- 4.0 during the acarbose period (p < 0.05) and rose again to 28.9 +/- 6.7 (p > or = 0.05) in the follow-up period. The 24-h urinary glucose excretion similarly decreased during acarbose administration. As expected, no decrease in HbA1C was observed due to the short treatment period. The 24-h urinary C-peptide excretions and serum lipids were not influenced by acarbose therapy. Frequent episodes of clinical hypoglycaemia were experienced while on acarbose therapy, indicating a decrease in insulin requirements. Adverse events such as flatulence and abdominal distention were observed in six out of nine cases. Symptoms were generally mild and well tolerated, only one patient dropped out because of diarrhoea and abdominal pain. We conclude that acarbose could usefully be administered to Type 2 diabetic patients treated with insulin to improve blood glucose control and reduce insulin requirement if the appropriate selection criteria were met.

Acarbose↗
PubMed1993

[A case of spontaneous bacterial peritonitis with ascites caused by hypoproteinemia after a massive bleeding from a gastric ulcer].

A case of spontaneous bacterial peritonitis (SBP) developed in an old man whose ascitic fluid was related neither to portal hypertension nor nephrotic syndrome, but with severe hypoalbuminemia emerged after a massive bleeding from a gastric ulcer in a malnutrition state. Ascitic fluid, increasing day by day, yielded Enterobacter cloacae and Bacteroides fragilis. Though autopsy was not carried out because of refusal of his family, neither liver necropsy, nor abdominal CT scan nor repeated abdominal ultrasonography showed findings suggesting existence of liver cirrhosis. In the presence of his ascites, the extent of a chemiluminescence (CL) response of polymorphonuclear cells from volunteers was significantly lower than that of his serum. This report shows that SBP can develop in a patient with ascites unrelated to portal hypertension when ascitic fluid induces little CL response.

Aged↗
PubMed1993

Improved oral absorption of a poorly water-soluble drug, HO-221, by wet-bead milling producing particles in submicron region.

N-[[[4-(5-Bromo-2-pyrimidinyloxy)-3-chlorophenyl]amino]carbonyl]-2 -nitrobenzamide (HO-221) is being developed as an anticancer agent with a novel mode of action. HO-221 shows poor oral absorption and is only slightly soluble in water (0.055 micrograms/ml at 37 degrees C). In this study, it was shown that the reduction in particle size of HO-221 to the submicron region (0.453 microm, mean by volume) could be achieved by a wet milling in a decaglycerin monolaurate aqueous solution with small glass beads. The wet milling suspension obtained showed improved dissolution rate and oral absorption in rats. A solid dosage form could also be made from that suspension with addition of sucrose palmitate which prevented aggregation caused by the hydrophobic interaction. The solid dosage form thus obtained showed twice as much oral absorption in dogs as the preparation made by dry milling.

Animals↗
PubMed1993

[A survey of the initial course of tuberculosis chemotherapy under compulsory hospitalization on the basis of the revised Tuberculosis Prevention Law--report of the 32nd C series of controlled trials of chemotherapy--Cooperative Study Unit of Chemotherapy of Tuberculosis of the National Sanatoria in Japan].

We studied 2247 patients with pulmonary tuberculosis who were hospitalized for the initial course of chemotherapy under the provision of the revised Tuberculosis Prevention Law issued by Japanese Ministry of Welfare. The results were as follows. 1) There were more males than females with a ratio of 2.6: 1. 2) There were two peaks in their ages, 20s and 60s. 3) The average admission periods were 5.2 +/- 2.7 months for men and 5.1 +/- 2.8 months for women. 4) The average compulsory admission periods were 6.8 +/- 2.5 months for men and 6.4 +/- 2.3 months for women, so that the actual admission periods were shorter in the majority of the patients. 5) The sputum cultures were converted negative in three months in about 80 percent of the patients who were smear and/or culture positive on admission. 6) The appearance rate of INH-resistant and RFP-resistant strains were 2% and 1.4% respectively. 7) Most of the death from tuberculosis was observed in the early stages of admission. 8) With regards to the cost, there seemed to be no difference whether treated under the new or the old provisions. 9) The newly approved extension of the admission periods for those who had complications such as diabetes mellitus or pneumoconiosis and those who had INH and/or RFP positive bacilli seemed to be appropriate.

Adult↗
PubMed1992

Effects of magnesium ion on the interaction of atrial muscarinic acetylcholine receptors and GTP-binding regulatory proteins.

Muscarinic acetylcholine receptors (mAChR) purified from porcine atrium were reconstituted into lipid vesicles with GTP-binding regulatory proteins (G proteins, Gi, Go, or Gn) purified from porcine cerebrum. Apparent affinities of the reconstituted mAChR and G proteins for carbachol and GDP, respectively, were estimated from the effects of these ligands on the binding of [3H]-L-quinuclidinyl benzilate ([3H]QNB) to mAChR and [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP gamma S) to G proteins in the presence of different concentrations of MgCl2. A total of 30-35% of reconstituted mAChRs exhibited low affinity for carbamylcholine, irrespective of the presence or absence of guanine nucleotides, and the remainder of the mAChRs showed high affinities for carbamylcholine in the absence of GTP or GDP and a low affinity in their presence. The affinity for carbamylcholine in the absence of guanine nucleotides, but not in their presence, increased with increases in MgCl2 concentration. Apparent Kd's for carbamylcholine were estimated to be approximately 100 microM in the presence of guanine nucleotides, 1.5 microM in the absence of guanine nucleotide and Mg2+ (< 0.1 microM), and 0.1 microM in the absence of guanine nucleotide and the presence of MgCl2 (10 mM). These results indicate that mAChRs may assume at least three different conformations that are characterized by different affinities for agonists. Furthermore, the data suggest that MgCl2 is not necessary for the formation of the mAChR-G protein complex, but can induce a conformational change in the complex. On the other hand, the presence of MgCl2 was necessary for carbamylcholine to influence the binding of guanine nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

[Silicone breast implants and collagen diseases].

Connective tissue disease has been reported to occur following implantation of silicone gel-filled prostheses to augment the breast. In this case report and review of the literature, two patients are described in whom connective tissue disease developed within one and three years respectively after cosmetic surgery. One developed dermatomyositis with lung fibrosis, and the other developed cutaneous lupus and a Sjögren-like syndrome. Both suffered serious complications due to vasculitis. Although evidence of a causal relationship between the implantation and the development of connective tissue disease is circumstantial, removal of the silicone prosthesis has been reported to result in subsequent remission.

Adult↗
PubMed1992

Complete nucleotide sequence of the phosphoprotein of the Yamagata-1 strain of a defective subacute sclerosing panencephalitis (SSPE) virus.

The complete nucleotide sequence of the phosphoprotein (P) gene of the Yamagata-1 strain of a defective subacute sclerosing panencephalitis (SSPE) virus was determined. Comparison with the P gene of the Edmonston strain of measles virus (MV) revealed 44 differences of which 23 nucleotides substitutions were identical with those revealed between other SSPE viruses and MV (Cattaneo et al. (1989) Virology 173, 415-425). The consensus sequence of the G insertion site was completely conserved, whereas mRNAs with one or three non-templated G residue insertions were found in addition to the mRNA of the exact genome copy. As a result of the frameshift downstream of the site of G insertion, the cysteine-rich V protein was predicted from the one G-inserted mRNA besides the P and C proteins predicted from the genome-copied mRNA.

Amino Acid Sequence↗
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↗