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Biomedical subjects

H Kihara

Publications and source records attributed to H Kihara.

At least 55 records · Page 3Linked to original sources
PubMed1995

The stabilizing mechanism of the distal radioulnar joint during pronation and supination.

A biomechanical cadaver study was performed to determine the roles of the stabilizing structures of the distal radioulnar joint during pronation and supination. Subluxation and dislocation of the radius with respect to the ulna were evaluated in seven cadaver forearms placed in supination, pronation, and neutral forearm rotation. The amount of subluxation was measured with all structures intact, and after sectioning in various sequences the dorsal and palmar radioulnar ligaments, the distal portion of the interosseous membrane including the pronator quadratus, and the entire interosseous membrane. After sectioning two of any four structures, the distal radioulnar joint remained stable. When the interosseous membrane was disrupted first, the dorsal radioulnar ligament was found to be more important than the palmar radioulnar ligament in stabilizing the distal radioulnar joint in pronation, and conversely the palmar radioulnar ligament was more important than the dorsal radioulnar ligament in supination. Dislocation, and frequently diastasis, occurred only with sectioning of all four structures. This suggests that all four structures contribute to stability of the distal radioulnar joint.

Biomechanical Phenomena↗
PubMed1995

ATP-hydrolyzing excitation state of the reconstituted alpha 3 beta 3 complex of ATP synthase from the thermophilic bacterium PS3: structural characteristics shown by time-resolved small-angle X-ray scattering with synchrotron radiation.

The ATP-hydrolyzing excitation state of the alpha 3 beta 3 complex of the ATP synthase from the thermophilic bacterium PS3 was investigated using time-resolved small-angle X-ray scattering with synchrotron radiation. The results showed the presence of the alpha 3 beta 3 complex at a steady state during ATP hydrolysis when the alpha 3 beta 3 hexamer reacted with Mg-ATP. The radius of gyration of the complex in the steady state was significantly larger than that of the Mg-AMP-PNP-hexamer complex, indicating a conformational change to an expanded structure during catalysis. This alpha 3 beta 3 complex dissociated into alpha 1 beta 1 heterodimers with apparent first-order reaction kinetics after all the ATPs were converted to ADPs. In contrast, when the alpha 3 beta 3 complex reacted with Mg-ADP, the complex dissociated into dimers with apparent first-order reaction kinetics without showing the steady state of the complex. The dimers, however, re-associated into the hexamer when Mg-ATP was added. The results were well-explained by a computer simulation based on non-linear chemical dynamics, in which a reaction mechanism that incorporates the dynamic structure of the hexamer in the steady state was considered.

Adenosine Triphosphate↗
PubMed1995

Crystal structure analysis of phospholipase A2 from trimeresurus flavoviridis (Habu snake) venom at 1.5 A resolution.

The crystal structure of dimeric phospholipase A2 (PLA2) from the venom of Habu snake, Trimeresurus flavoviridis, has been determined by the molecular replacement method, and has been refined at 1.5 A resolution to an R-factor of 0.175. In the crystal, T. flavoviridis PLA2 forms a dimer using two 14 kDa subunits related by a pseudo 2-fold axis. Along the axis, the dimer has a narrow channel passing through it. Although no calcium ion is present in the calcium binding site, the peptide-chain folding of the subunits, the conformation of the catalytic residues, and the hydrogen-bonding network around the active sites are almost identical to those of the group I/II monomeric or dimeric PLA2s. The catalytic residues in both subunits are buried in the interior of the dimer and are inaccessible to substrate from the bulk solvent. In addition, the subunits of the dimer interact with each other at the hydrophobic region of the molecular surface where the entrance to the active site opens and where PLA2 is presumed to interact with the phospholipid of the substrate. Therefore, it is inferred that dimerization of T. flavoviridis PLA2 is the result of free-energy minimization by excluding the hydrophobic molecular surface from the aqueous solvent, rather than being required for the enzymatic function.

Amino Acid Sequence↗
PubMed1995

[A case of aortic bicuspid valvular endocarditis with congenital left ventricular diverticulum].

A 29-year-old man had a prolonged fever and painful Osler's nodes on his right foot. The aortic valve was bicuspid with vegetation mass and the left ventricular diverticulum was additionally present connecting with the left ventricular outflow tract. An operation was performed after intravenous administration of antibiotics for 3 weeks. The aortic bicuspid valve and the vegetation were removed and replaced by an artificial valve (SJM-HP 19 mm). The left ventricular diverticulum was resected. The echocardiographic findings correlated well with the intraoperative observation.

Adult↗
PubMed1994

Stopped-Flow Apparatus for X-ray Scattering and XAFS.

A stopped-flow apparatus is described for X-ray scattering and X-ray absorption studies. It can be successfully operated over a wide temperature range down to 253 K with highly viscous solutions (20 cP). The characteristics of the stopped-flow apparatus and its application to studies of biological reactions are reviewed.

Journal Article↗
PubMed1994

Src homology 2 domains of Syk and Lyn bind to tyrosine-phosphorylated subunits of the high affinity IgE receptor.

Aggregation of the high affinity IgE receptor (Fc epsilon RI) on rat basophilic leukemia RBL-2H3 cells results in activation of the protein tyrosine kinases Syk and Lyn. Here, we report that fusion proteins containing the Src homology 2 (SH2) domains of Syk and Lyn precipitated tyrosine-phosphorylated proteins from RBL-2H3 cell lysates. There was no detectable precipitation with the N-terminal Syk SH2 domain, minimal with the C-terminal Syk SH2, and strong when the two SH2 domains were expressed in tandem. The Syk SH2 domains showed pronounced selectivity in the binding of tyrosine-phosphorylated proteins. Thus, Syk SH2 precipitated only three phosphoproteins, two of which were the beta and gamma subunits of Fc epsilon RI. In contrast, the fusion protein with Lyn SH2, in addition to precipitating the same Fc epsilon RI components, bound to many other phosphoproteins. Tyrosine phosphorylation of the beta and gamma subunits of Fc epsilon RI was essential for their precipitation with the SH2 fusion proteins. By direct binding studies, there was more binding of Syk SH2 to Fc epsilon RI gamma than to Fc epsilon RI beta, whereas Lyn SH2 bound only to Fc epsilon RI beta. The Syk SH2 fusion protein competitively inhibited the association of gamma and beta with Syk in lysates of activated cells. The SH2-mediated association of these two protein tyrosine kinases with Fc epsilon RI could play an important role in receptor signaling.

Amino Acid Sequence↗
PubMed1994

Kinetics of the quaternary structure change of aspartate transcarbamylase triggered by succinate, a competitive inhibitor.

The quaternary structural change of Escherichia coli aspartate transcarbamylase (ATCase) was studied by time-resolved X-ray solution scattering following the binding of carbamoyl phosphate and of succinate, a competitive inhibitor of the natural substrate L-aspartate. Stopped-flow experiments at sub-zero temperatures in the presence of 30% ethylene glycol allowed us to monitor the evolution of the scattering pattern, including the characteristic scattering peak in an s (=2 sin theta/lambda) range of 0.01-0.06 A-1. The inhibitor binding promotes a quaternary structure change from the T state toward the R state, and as expected for a simple ligand binding process, ATCase remains in the R state, unlike the physiological enzyme reaction [Tsuruta, H., et al. (1990) FEBS Lett. 263, 66-68]. After equilibrium had been established, the final scattering pattern was recorded. When the succinate concentration was sufficiently high, this pattern was the same as that given by ATCase saturated with the bisubstrate analogue N-phosphonoacetyl-L-aspartate (PALA). This implies that, under cryogenic conditions, succinate and carbamoyl phosphate promote the same quaternary structure change as PALA, which is in good agreement with the crystallographic studies of Gouaux and Lipscomb [Gouaux, J.E., & Lipscomb, W.N. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 4205-4208]. Scattering patterns recorded during the course of the structural transition were satisfactorily reproduced by a linear combination of the initial and final patterns, suggesting that there is no significant concentration of quaternary structure intermediates between the T and R states. This is consistent with a concerted structural transition of ATCase.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspartate Carbamoyltransferase↗
PubMed1994

The anti-ganglioside monoclonal antibody AA4 induces protein tyrosine phosphorylations, but not degranulation, in rat basophilic leukemia cells.

The monoclonal antibody AA4 (mAb AA4) recognizes novel alpha-galactosyl derivatives of GD1b on rat basophilic leukemia (RBL-2H3) cells. The binding of mAb AA4 induced protein tyrosine phosphorylations without histamine release. Several of the same proteins including Fc epsilon RI beta, Fc epsilon RI gamma, p72syk, and phospholipase C-gamma 1 were tyrosine-phosphorylated by mAb AA4 binding and by the activation of the high affinity IgE receptor, Fc epsilon RI. There was also activation of the p53/56lyn and p72syk protein-tyrosine kinases, but compared to direct Fc epsilon RI activation, mAb AA4 did not result in increased tyrosine phosphorylation of pp105-115 or pp125FAK, and the receptor subunits (Fc epsilon RI beta and Fc epsilon RI gamma) were more heavily phosphorylated. Furthermore, the time course of the phosphorylations with mAb AA4 was slower than that induced by Fc epsilon RI aggregation. By immunofluorescence, the tyrosine-phosphorylated proteins after mAb AA4 stimulation were localized in patches at the cell membrane and in areas of cell-cell contact, whereas after Fc epsilon RI activation, there was a reticular cytoplasmic pattern. There were no protein tyrosine phosphorylations either when Fc epsilon RI was saturated with IgE or when F(ab')2 fragments of mAb AA4 were used, although the F(ab')2 fragments still induced morphological changes. There was also coprecipitation of the beta and gamma subunits of Fc epsilon RI with the anti-ganglioside antibody. These data strongly suggest the involvement of Fc epsilon RI in the antiganglioside-induced protein tyrosine phosphorylations. Moreover, phosphorylations of these proteins including the beta and gamma chains of Fc epsilon RI and activation of p53/56lyn and p72syk did not result in histamine release.

Animals↗
PubMed1994

The kinetics of conformational changes of alpha 2-macroglobulin determined by time resolved X-ray solution scattering.

The rate of gross conformational change of alpha 2-macroglobulin (alpha 2M) during its proteinase trapping was directly determined for the first time using time-resolved X-ray solution scattering. Decrease of radius of gyration was observed under pseudo-first-order conditions with excess proteinases, which exhibited a monophasic time-course. The rate constants were 0.5 +/- 0.1 s-1 and 0.8 +/- 0.2 s-1 for the reaction with chymotrypsin and trypsin, respectively. There was no concentration dependence of the observed rate constants. Therefore, the rate-limiting step of the gross conformational change was not the bimolecular encounter reaction between alpha 2M and proteinases, which requires a new proposal of pre-trapping of proteinases before the gross conformational change.

Chymotrypsin↗
PubMed1994

Kinetic study on myoglobin refolding monitored by five optical probe stopped-flow methods.

The refolding kinetics of horse cyanometmyoglobin induced by concentration jump of urea was investigated by five optical probe stopped-flow methods: absorption at 422 nm, tryptophyl fluorescence at around 340 nm, circular dichroism (CD) at 222 nm, CD at 260 nm, and CD at 422 nm. In the refolding process, we detected three phases with rate constants of > 1 x 10(2) s-1, (4.5-9.3) s-1, and (2-5) x 10(-3) s-1. In the fastest phase, a substantial amount of secondary structure (approximately 40%) is formed within the dead time of the CD stopped-flow apparatus (10.7 ms). The kinetic intermediate populated in the fastest phase is shown to capture a hemindicyanide, suggesting that a "heme pocket precursor" recognized by hemindicyanide must be constructed within the dead time. In the middle phase, most of secondary and tertiary structures, especially around the captured hemindicyanide, have been constructed. In the slowest phase, we detected a minor structural rearrangement accompanying the ligand-exchange reaction in the fifth coordination of ferric iron. We present a possible model for the refolding process of myoglobin in the presence of the heme group.

Animals↗
PubMed1994

Purification and characterization of a coagulant enzyme, okinaxobin II, from Trimeresurus okinavensis (himehabu snake) venom which release fibrinopeptides A and B.

A coagulant enzyme, okinaxobin I, which was purified from Trimeresurus okinavenis (himehabu snake) venom, released specifically fibrinopeptide B from fibrinogen to form fibrin clots. In the present study, its isozyme denoted as okinaxobin II has been purified to homogeneity from the same venom by chromatographies on Sephadex G-100, CM-Toyopearl 650M, and FPLC Mono-Q columns. Differently from okinaxobin I, okinaxobin II specifically cleaved fibrinopeptides A and B from fibrinogen similarly as found for alpha-thrombin. The enzyme acted on fibrinogen with specific activity of 42 NIH units/mg at optimum pH of 8.0. Okinaxobin II was a monomeric glycoprotein with a mol. wt of 37,500 on SDS-PAGE, which was reduced to 29,500 after treatment with N-glycanase. Okinaxobin II was much more basic (pI = 8.1) than okinaxobin I (pI = 5.4). The N-terminal sequence was highly similar to those of okinaxobin I and some other snake venom coagulant enzymes such as flavoxobin (Trimeresurus flavoviridis), batroxobin (Bothrops atrox and Bothrops moojeni), and catroxobin (Crotalus atrox). Okinaxobin II hydrolyzed tosyl-L-arginine methyl ester and benzoyl-L-arginine p-nitroanilide. The esterase activity was strongly inhibited by diisopropylfluorophosphate and to a lesser extent by tosyl-L-lysine chloromethyl ketone, indicating that the enzyme is a serine protease like alpha-thrombin. In terms of amino acid composition, okinaxobin II was similar to okinaxobin I and dissimilar to alpha-thrombin.

Amino Acid Sequence↗
PubMed1994

Src family tyrosine kinase Lyn binds several proteins including paxillin in rat basophilic leukemia cells.

Aggregation of the high affinity IgE receptors on rat basophilic leukemia (RBL-2H3) cells results in protein tyrosine phosphorylation although the receptor has no intrinsic enzymatic activity. The Src related protein tyrosine kinase p53/56lyn present in RBL-2H3 cells could play a role in this reaction. Here we have isolated the cDNA for rat Lyn and found it to be very homologous at the amino acid level to both the human and mouse proteins. A bacterially expressed maltose binding protein-Lyn (MBP-Lyn) fusion protein was already tyrosine phosphorylated and had tyrosine kinase activity. In a filter-binding assay, MBP-Lyn fusion protein (at 0.1 microM) specifically bound to several proteins of RBL-2H3 cells. In lysates of IgE receptor-activated cells, there was increased binding of MBP-Lyn to 65, 72, 78 and 110 kDa tyrosine phosphorylated proteins. The 72, 78 and 110 kDa tyrosine phosphorylated proteins were precipitated by a fusion protein containing the Lyn Src Homology 2 (SH2) domain. The 72 kDa Lyn binding protein was different from p72syk. Furthermore, paxillin, a cytoskeletal protein, was identified as one of the Lyn binding proteins. Thus Fc epsilon RI mediated signal transduction in RBL-2H3 cells may result from the interaction of p53/56lyn with paxillin, pp72, pp110 and other proteins.

Amino Acid Sequence↗
PubMed1994

Polymorphisms of Trimeresurus flavoviridis venom gland phospholipase A2 isozyme genes.

Trimeresurus flavoviridis venom gland phospholipase A2 (PLA2) genes pgPLA 1a and pgPLA 2a encode Asp-49-PLA2 and genes pgPLA 1b and pgPLA 2b encode an isozyme of Asp-49-PLA2. Polymorphisms were found in pairs of pgPLA 1a and pgPLA 2a and of pgPLA 1b and pgPLA 2b for individuals of T. flavoviridis. The occurrence of both homozygotes and heterozygotes was demonstrated.

Animals↗
PubMed1993

Protein-tyrosine kinase p72syk in high affinity IgE receptor signaling. Identification as a component of pp72 and association with the receptor gamma chain after receptor aggregation.

Protein-tyrosine phosphorylation plays a critical role in the high-affinity IgE receptor (Fc epsilon RI) signaling. Here we investigated the involvement of the tyrosine kinase p72syk in Fc epsilon RI signaling in the rat mast cell line RBL-2H3. Specific antibodies were raised against peptides synthesized on the basis of the deduced peptide sequence of an essentially full-length rat syk cDNA. The expression of p72syk in RBL-2H3 cells was demonstrated with these antibodies. The aggregation of Fc epsilon RI led to the tyrosine phosphorylation of p72syk that was detected after 15 s of stimulation, reached a plateau by 5 min, and was not induced by calcium influx or protein kinase C activation. Association of p72syk with the tyrosine phosphorylated Fc epsilon RI gamma chain was detected only after receptor aggregation. We previously demonstrated that aggregation of the Fc epsilon RI on mast cells results in the tyrosine phosphorylation of a 72-kDa protein (pp72) involved in IgE signaling. The depletion of p72syk from RBL-2H3 cell lysates resulted in only a slight decrease in the amount of pp72. These results demonstrate that pp72 is composed of several phosphoproteins and identify p72syk as one component of pp72. These data, together with recent observations in T cells, indicate that the interaction between p72syk-related tyrosine kinases and zeta-related proteins could play an important role in signal transduction.

Amino Acid Sequence↗
PubMed1993

Accelerated evolution of Trimeresurus flavoviridis venom gland phospholipase A2 isozymes.

Six Trimeresurus flavoviridis (Habu snake) venom gland phospholipase A2 (PLA2) isozyme genes were found to consist of four exons and three introns and to encode proteins of 138 amino acid residues, including the signal sequence of 16 amino acid residues. Comparison of the nucleotide sequences showed that the introns are much more homologous than the protein-coding regions of exons except for the signal peptide-coding region of the first exon. The numbers of nucleotide substitutions per site (KN) for introns are approximately one-fourth of the numbers of nucleotide substitutions per synonymous site (KS) for the protein-coding regions, indicating that the introns are unusually conserved. The absence of an apparent functional role for the introns suggests that the protein-coding regions, except for the signal peptide-coding domains, have evolved at greater substitution rates than introns. The fact that the numbers of nucleotide substitutions per nonsynonymous site (KA) are close to or larger than KS values for relevant pairs of genes revealed that Darwinian-type accelerated substitutions have occurred in the protein-coding regions or exons. This is compatible with the presence of PLA2 species with diverse physiological activities in the venom.

Amino Acid Sequence↗
PubMed1993

Evidence of an associative intermediate on the myoglobin refolding pathway.

Time-resolved small-angle x-ray scattering using the stopped-flow method has been applied successfully to investigate the refolding of myoglobin. This is the only method to date that yields direct information on protein physical dimensions during the folding process. It has the potential to detect and probe important processes, such as protein compaction and association, on a millisecond time scale. Initial experiments were performed with horse myoglobin denatured in high concentrations of urea. The denatured protein was diluted rapidly into a buffer containing no urea or low concentrations of urea. The time-course of the forward-scattered intensity shows a decrease in amplitude which is clearly not engendered by the compaction of the protein, but does correspond well to a dimer dissociation process. Initial and final radii of gyration correspond well to a dimer and a monomer, respectively. Kratky plots of the initial and final states also support the transient dimerization model. The apparent dissociation rate constant was obtainable directly from the data. An association rate constant and an equilibrium constant could be estimated. The dimerizing intermediate is speculated to be a globular non-native state with an exposed hydrophobic surface.

Animals↗