PubMed Health⌕ Search

Biomedical subjects

T Nukada

Publications and source records attributed to T Nukada.

At least 37 records · Page 2Linked to original sources
PubMed1995

Characterization of Gq family G proteins GL1 alpha (G14 alpha), GL2 alpha (G11 alpha), and Gq alpha expressed in the baculovirus-insect cell system.

The alpha subunits of Gq family G proteins, GL1 alpha (G14 alpha), GL2 alpha(G11 alpha), and Gq alpha were expressed with G protein beta 1 and gamma 2 subunits in insect cells using a baculovirus system. The trimeric forms of G proteins, GL1 (GL1 alpha beta gamma), GL2 (GL2 alpha beta gamma), and Gq (Gq alpha beta gamma), were solubilized by 1% sodium cholate and purified by sequential chromatography on three kinds of columns. GL1, GL2, and Gq activated phospholipase C-beta purified from bovine brain in the presence of aluminum fluoride to the same extent. Muscarinic acetylcholine receptor m1 subtype stimulated the guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding to GL1, GL2, and Gq in the presence of similar concentrations of carbamylcholine. When m1 receptor, G protein, and phospholipase C-beta were reconstituted in lipid vesicles, each subtype of Gq family G proteins mediated the activation of phospholipase C-beta by carbamylcholine in the presence of either 1 microM GTP gamma S or 1 mM GTP. Phospholipase C-beta stimulated the GTPase activity of GL1, GL2, and Gq in the presence of m1 receptor and carbamylcholine but did not stimulate the GTPase activity of GO. Protein kinase C phosphorylated m1 receptor and phospholipase C-beta, but the phosphorylation did not significantly affect the ability of the m1 receptor to stimulate phospholipase C-beta in the reconstitution system of purified proteins.

Animals↗
PubMed1995

Synthesis of a glycopeptide carrying a N-linked core pentasaccharide.

A glycopeptide carrying a pentasaccharide core structure of asparagine-linked glycoproteins was synthesized. The synthesis of the carbohydrate part was performed starting from monosaccharide components in an unambiguous manner. The resultant pentaglycosyl azide was reduced into corresponding glycosyl amine and coupled with an aspartic acid derivative to furnish an Asn-linked oligosaccharide in a protected form. Subsequent coupling with a dipeptide, followed by deprotection gave the target compound.

Amino Acid Sequence↗
PubMed1994

Tyrosine kinases Lyn and Syk regulate B cell receptor-coupled Ca2+ mobilization through distinct pathways.

Stimulation of B lymphocytes through their antigen receptor (BCR) results in rapid increases in tyrosine phosphorylation on a number of proteins and induces both an increase of phosphatidylinositol and mobilization of cytoplasmic free calcium. The BCR associates with two classes of tyrosine kinase: Src-family kinase (Lyn, Fyn, Blk or Lck) and Syk kinase. To dissect the functional roles of these two types of kinase in BCR signaling, lyn-negative and syk-negative B cell lines were established. Syk-deficient B cells abolished the tyrosine phosphorylation of phospholipase C-gamma 2, resulting in the loss of both inositol 1,4,5-trisphosphate (IP3) generation and calcium mobilization upon receptor stimulation. Crosslinking of BCR on Lyn-deficient cells evoked a delayed and slow Ca2+ mobilization, despite the normal kinetics of IP3 turnover. These results demonstrate that Syk mediates IP3 generation, whereas Lyn regulates Ca2+ mobilization through a process independent of IP3 generation.

Animals↗
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

A region of the muscarinic-gated atrial K+ channel critical for activation by G protein beta gamma subunits.

Complementary DNAs encoding two types of inwardly rectifying K+ channels, GIRK1 and IRK1, have been cloned from rat atrium and mouse macrophage, respectively. GIRK1 expressed in Xenopus oocytes was activated by acetylcholine when m2 muscarinic acetylcholine receptor was coexpressed. The acetylcholine-induced activation of GIRK1 was enhanced by coexpression with the G protein beta 1 gamma 2 subunit but not the beta 1 gamma 1 or alpha subunits. Deletion of the C-terminus of GIRK1 impaired the channel activation associated with the beta 1 gamma 2 subunit. Moreover, replacement of the C-terminus of IRK1 with that of GIRK1 produced a chimera channel that was activated by the beta 1 gamma 2 subunit, whereas intact IRK1 was not activated by the beta 1 gamma 2 subunit. These findings define the C-terminus of GIRK1 as a regulatory region for the G protein beta gamma subunit.

Animals↗
PubMed1992

Synthesis of an octasaccharide fragment of high-mannose-type glycans of glycoproteins.

O-(alpha-D-Mannopyranosyl)-(1----2)-O-(alpha-D-mannopyranosyl)-(1----3)- O- [(alpha-D-mannopyranosyl)-(1----2)-O-(alpha-D-mannopyranosyl)-(1----6)]- O- (alpha-D-mannopyranosyl)-(1----6)-O-(beta-D-mannopyranosyl)-(1----4)-O-( 2- acetamido-2-deoxy-beta-D-glucopyranosyl)-(1----4)-2-acetamido-2-deoxy- glucopyranose, an octasaccharide fragment of high-mannose type glycan of glycoproteins, was synthesized. Crucial glycosylation of trisaccharide intermediate, benzyl O-(2,4-di-O-benzyl-beta-D-mannopyranosyl)-(1----4)-O-(2-acetamido-3,6-di -O- benzyl-2-deoxy-beta-D-glucopyranosyl)-(1----4)-2-acetamido-3,6-di-O-benz yl-2- deoxy-beta-D-glucopyranoside, was successful only with a di-O-acetyltetradeca-O-benzyl-D-mannopentaosyl chloride. The use of the corresponding hexadeca-O-acetyl-D-mannopentaosyl bromide did not give the desired product.

Acetylglucosamine↗
PubMed1992

[Assessment of the rate of successful transcranial Doppler recording through the temporal windows in Japanese with special reference to aging and sex].

Transcranial Doppler method (TCD), newly developed in 1982, is ultrasound technology that measures physiologic parameters of blood flow in the major intracranial arteries. A major limitation of TCD is that, in some individuals, satisfactory recording is not possible, especially through the temporal windows. The purpose of the present study is to assess the rate of successful recording in TCD through the temporal ultrasonic windows in Japanese with special reference to aging and sex. In 423 Japanese (244 males and 179 females) aged from 25 to 93 years, measurement of blood flow velocity in basal cerebral arteries was performed by transcranial Doppler velocimeter with a 2 MHz pulsed wave Doppler probe. The signal was successfully recorded in 299 of 423 Japanese (71%). In Japanese males, successful measurement was more frequent (206/244; 84%) than in Japanese females (93/179; 52%). The rate of successful recording was evaluated in every decade from the twenties to the eighties, and it was found to decrease significantly according to age (100%, 94%, 91%, 74%, 66%, 67%, and 46%, respectively). In females, it significantly decreased in those older than 50 years old (100%, 100%, 93%, 61%, 36% 43%, and 27%, respectively), but not in males (100%, 88%, 89%, 81%, 88%, 83%, and 69%, respectively). These findings suggest that this noninvasive method has clinical usefulness for evaluating intracranial circulation but has limitation for assessing the intracranial lesions of elderly Japanese women.

Adult↗
PubMed1992

[Platelet accumulation in abdominal aortic aneurysms and the effect of antiplatelet drugs: assessment by indium platelet scintigraphy].

A dual tracer technique using 111In labeled platelets and 99mTc labeled human serum albumin was applied to evaluate the in vivo thrombogenicity in 12 cases with abdominal aortic aneurysm (AAA) and the effect of antiplatelet drug on the thrombogenicity. The magnitude of platelet accumulation at AAA was expressed as the ratio of radioactivity of 111In platelets on the vascular wall to those in the blood pool (PAI; platelet accumulation index). Of the 12 patients with AAA, 11 had positive studies on baseline imaging and 1 had equivocally positive image. The PAI value (Mean +/- SD) over the AAA was 53.8 +/- 34.1% as compared to -8.6 +/- 4.4% in the control group (p less than 0.01). Seven patients with an AAA and positive baseline images were restudied during platelet active drug with 325 mg of aspirin. During treatment with aspirin, of 7 patients, 5 had positive images, of which 3 were decreased and others unchanged compared to baseline studies, 1 equivocally positive one and one negative one. The PAI value during treatment (21.9 +/- 18.6%) was significantly decreased compared to those in baseline study (52.1 +/- 23.9%). Our results suggest that the method used for platelet imaging in the present study may be useful for studying the in vivo thrombogenicity and the effect of platelet active drugs in AAA.

Aged↗
PubMed1991

Identification of two novel GTP-binding protein alpha-subunits that lack apparent ADP-ribosylation sites for pertussis toxin.

Molecular cloning of cDNAs encoding alpha-subunits of guanine nucleotide-binding regulatory proteins (G-proteins) has revealed the existence of nine species of alpha-subunits. We have identified two additional G-protein alpha-subunits, which we refer to as GL1 alpha and GL2 alpha, by isolating bovine liver cDNA clones that cross-hybridized at reduced stringency with bovine Gi1 alpha-subunit cDNA. The deduced amino acid sequences of GL1 alpha and GL2 alpha share 83% identity with each other and show 45-55% identity with those of other known G-protein alpha-subunits. Both GL1 alpha and GL2 alpha lack a consensus site for ADP-ribosylation by pertussis toxin. Messenger RNA corresponding to GL2 alpha was detected in all tissues examined, but GL1 alpha mRNA was detected only in liver, lung, and kidney. Antiserum prepared against a synthetic pentadecapeptide corresponding to the deduced carboxyl terminus of GL2 alpha specifically reacted with a 40-kDa protein in mouse liver, brain, lung, heart, kidney, and spleen. The amount of the 40-kDa protein was highest in brain and lung. We suggest that GL1 alpha and GL2 alpha are new members of a subfamily of pertussis toxin-insensitive G-proteins.

Adenosine Diphosphate Ribose↗
PubMed1991

Conformation of the glucotriose unit in the lipid-linked oligosaccharide precursor for protein glycosylation.

The conformation of the glucotriose unit of the protein glycosylation precursor Glc3Man9GlcNAc2 was assessed by deuterium exchange studies on the model tetrasaccharide alpha Glc----2 alpha Glc----3 alpha Glc----3 alpha Man----OCH2CH2CH3 dissolved in deuterated dimethyl sulfoxide. The hydroxyl proton on C-2 of the nonreducing end glucose and on C-4 of the glucose attached to mannose both show dramatic isotope shifts indicative of a strong hydrogen bond between these two hydroxyl groups. Such a hydrogen bond requires a fixed conformation of the glucotriose unit that brings these hydroxyl groups within 3 A of each other, a conformation that is supported by molecular modeling based on hard-sphere exo-anomeric (HSEA) calculations. The temperature dependence of the hydroxyl proton chemical shifts supports the postulated hydrogen bond, and the torsional angles between the three glucose units derived from the HSEA calculations are consistent with results from related studies on other saccharides. The results support a model for biochemical function in which the glucotriose unit could modulate the activity of the oligosaccharyltransferase by binding in a fixed conformation to a specific effector site in the enzyme.

Binding Sites↗
PubMed1990

Synthesis of an appropriately protected core glycotetraoside, a key intermediate for the synthesis of "bisected" complex-type glycans of a glycoprotein.

A stereocontrolled synthetic route to a glycotetraoside, allyl O-(3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1--- -4)-O- (3,6-di-O-allyl-2-O-benzyl-beta-D-mannopyranosyl)-(1----4)-O-3, 6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1----4)-3-O- benzyl- 2-deoxy-6-O-p-methoxy-phenyl-2-phthalimido-beta-D-glucopyranoside, an important intermediate for the synthesis of "bisected" complex type glycans of glycoproteins has been established by employing two glycosyl donors, 3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl trichloroacetimidate and 4-O-acetyl-3,6-di-O-allyl-2-O-benzyl-alpha-D-mannopyranosyl bromide, and a glycosyl acceptor, allyl O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1----4) -3-O- benzyl-2-deoxy-6-O-p-methoxyphenyl-2-phthalimido-beta-D-glucopyranoside.

Carbohydrate Conformation↗
PubMed1990

Synthesis of alpha-D-Manp-(1----3)-[beta-D-GlcpNAc-(1----4)]-[alpha-D-Manp+ ++-(1----6)] - beta-D-Manp-(1----4)-beta-D-GlcpNAc-(1----4)-[alpha-L-Fucp- (1----6)]-D- GlcpNAc, a core glycoheptaose of a "bisected" complex-type glycan of glycoproteins.

A synthesis of alpha-D-Manp-(1----3)-[beta-D-GlcpNAc-(1----4)]-[alpha-D-Manp++ +-(1----6)]- beta-D-Manp-(1----4)-beta-D-GlcpNAc-(1----4)-[alpha-L-Fucp-( 1----6)]-D- GlcpNAc was achieved by employing benzyl O-(3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1--- -4)-O- (2-O-benzyl-beta-D-mannopyranosyl)-(1----4)-O-(3,6-di-O-benzyl-2-deoxy-2 - phthalimido-beta-D-glucopyranosyl)-(1----4)-3-O-benzyl-2-deoxy-6-O-p- methoxyphenyl-2-phthalimido-beta-D-glucopyranoside as a key glycosyl acceptor. Highly stereoselective mannosylation was performed by taking advantage of the 2-O-acetyl group in the mannosyl donors. The alpha-L-fucopyranosyl residue was also stereoselectively introduced by copper(II)-mediated activation of methyl 2,3,4-tri-O-benzyl-1-thio-beta-L-fucopyranoside.

Carbohydrate Conformation↗