Biomedical subjects

H Kihara

Publications and source records attributed to H Kihara.

At least 37 records · Page 2Linked to original sources

PubMed1998

Kinetic refolding of beta-lactoglobulin. Studies by synchrotron X-ray scattering, and circular dichroism, absorption and fluorescence spectroscopy.

beta-Lactoglobulin (beta LG) is a predominantly beta-sheet protein with a markedly high helical propensity and forms non-native alpha-helical intermediate in the refolding process. We measured the refolding reaction of beta LG with various techniques and characterized the folding kinetics and the structure of the intermediate formed within the burst phase of measurements, i.e. the burst-phase intermediate. Time-resolved stopped-flow X-ray scattering measurements using the integral intensity of scattering show that beta LG forms a compact, globular structure within 30 ms of refolding. The averaged radius of gyration within 100 ms is only 1.1 times larger than that in the native state, ensuring that the burst-phase intermediate is compact. The presence of a maximum peak in a Kratky plot shows a globular shape attained within 100 ms of refolding. Stopped-flow circular dichroism, tryptophan absorption and fluorescence spectroscopy show that pronounced secondary structure regains rapidly in the burst phase with concurrent non-native alpha-helix formation, and that the subsequent compaction process is accompanied by annealing of non-native secondary structure and slow acquisition of tertiary structure. These findings strongly suggest that both compaction and secondary structure formation in protein folding are quite rapid processes, taking place within a millisecond time-scale. The structure of the burst-phase intermediate in beta LG refolding was characterized as having a compact size, a globular shape, a hydrophobic core, substantial beta-sheets and remarkable non-native alpha-helical structure, but little tertiary structure. These results suggest that both local interactions and non-local hydrophobic interactions are dominant forces early in protein folding. The interplay of local and non-local interactions throughout folding processes is important in understanding the mechanisms of protein folding.

Animals↗

PubMed1998

Unfolding and refolding of dimeric creatine kinase equilibrium and kinetic studies.

Equilibrium and kinetic studies of the guanidine hydrochloride induced unfolding-refolding of dimeric cytoplasmic creatine kinase have been monitored by intrinsic fluorescence, far ultraviolet circular dichroism, and 1-anilinonaphthalene-8-sulfonate binding. The GuHCl induced equilibrium-unfolding curve shows two transitions, indicating the presence of at least one stable equilibrium intermediate in GuHCl solutions of moderate concentrations. This intermediate is an inactive monomer with all of the thiol groups exposed. The thermodynamic parameters obtained by analysis using a three-state model indicate that this intermediate is similar in energy to the fully unfolded state. There is a burst phase in the refolding kinetics due to formation of an intermediate within the dead time of mixing (15 ms) in the stopped-flow apparatus. Further refolding to the native state after the burst phase follows biphasic kinetics. The properties of the burst phase and equilibrium intermediates were studied and compared. The results indicate that these intermediates are similar in some respects, but different in others. Both are characterized by pronounced secondary structure, compact globularity, exposed hydrophobic surface area, and the absence of rigid side-chain packing, resembling the "molten globule" state. However, the burst phase intermediate shows more secondary structure, more exposed hydrophobic surface area, and more flexible side-chain packing than the equilibrium intermediate. Following the burst phase, there is a fast phase corresponding to folding of the monomer to a compact conformation. This is followed by rapid assembly to form the dimer. Neither of the equilibrium unfolding transitions are protein concentration dependent. The refolding kinetics are also not concentration dependent. This suggests that association of the subunits is not rate limiting for refolding, and that under equilibrium conditions, dissociation occurs in the region between the two unfolding transitions. Based upon the above results, schemes of unfolding and refolding of creatine kinase are proposed.

Anilino Naphthalenesulfonates↗

PubMed1997

Characterization and evolution of a gene encoding a Trimeresurus flavoviridis serum protein that inhibits basic phospholipase A2 isozymes in the snake's venom.

The proteins that bind phospholipase A2 (PLA2) isozymes of Trimeresurus flavoviridis (habu snake, crotalinae) venom were fractionated from sera on four columns, each conjugated with one of four PLA2 isozymes. Five proteins, termed PLA2 inhibitors (PLI) I-V, were obtained as the binding components. The combinations of the binding components differed depending on the PLA2 isozymes. PLI-IV and PLI-V correspond to PLI-A and PLI-B, respectively, which were known to bind to a major [Asp49]PLA2, PLA2, and contained a segment similar to the carbohydrate-recognition domain of C-type lectins. PLI-I, which is a major component of inhibitory proteins against three basic PLA2 isozymes, PLA-B (a basic [Asp49]PLA2) and basic proteins I and II (both [Lys49]PLA2s), has been isolated, and its partial amino acid sequence has been determined. A cDNA encoding PLI-I was isolated from a T. flavoviridis liver cDNA library and sequenced. PLI-I cDNA encoded 200 amino acid residues, including a signal peptide of 19 amino acid residues. One sugar chain was predicted to occur at position 157. A gene coding for PLI-I was isolated. It is 9.6-kb long and consists of five exons and four introns. Comparison of the exon-intron structure of the PLI-I gene with those of genes encoding urokinase-type-plasminogen-activator receptor (uPAR), Ly-6, CD59 and neurotoxins showed that they have characteristic unit encoding approximately 90 amino acid residues, which is divided over two exons. This strongly suggests that the PLI-I gene belongs to the uPAR, Ly-6, CD59 and neurotoxin gene family. There are two types of structurally different inhibitors against PLA2 isozymes in T. flavoviridis serum with different evolutionary origins.

Amino Acid Sequence↗

PubMed1997

Unfolding of dimeric creatine kinase in urea and guanidine hydrochloride as measured using small angle X-ray scattering with synchrotron radiation.

The denaturation of dimeric creatine kinase (CK) induced by urea and guanidine hydrochloride (GuHCl) has been studied by small angle X-ray scattering (SAXS), which is a direct way to measure the changes in the overall dimensions of a protein molecule. The radii of gyration (Rg) of CK are 29+/-0.4 angstroms in the native state and 46+/-1.5 angstroms in the unfolded state in either 8 M urea or 3 M GuHCl. The transition curves of urea denaturation derived from the Rg values and the zero angle intensity (I(0)) are similar to that from intrinsic fluorescence, indicating that the changes in the molecular shape, the tertiary structure and the dissociation of the subunits proceed simultaneously. In the case of GuHCl-induced denaturation, the dramatic increases both in Rg and in I(0) in 0.3-0.5 M GuHCl suggest clearly that soluble aggregates form at low GuHCl concentrations. The aggregates dissociate and the molecule unfolds at higher GuHCl concentrations. The results suggest that the mechanisms of CK denaturation in urea and in GuHCl are somewhat different and the intermediate in GuHCl denaturation can much more easily form soluble aggregates.

Animals↗

PubMed1997

Structures of genes encoding phospholipase A2 inhibitors from the serum of Trimeresurus flavoviridis snake.

Inhibitors (PLIs) against snake venom gland phospholipases A2 (PLA2s) have been found in their sera. A cDNA encoding a PLI from Trimeresurus flavoviridis (Tf, habu snake, Crotalinae) serum, cPLI-A, was isolated from the Tf liver cDNA library and sequenced. Northern blot analysis with cPLI-A showed that PLIs are expressed only in liver. Genes for PLIs, gPLI-A and gPLI-B, were isolated from the Tf genomic DNA library and their nucleotide (nt) sequences were determined. The genes consisted of four exons and three introns, and exon 4 encoded the carbohydrate recognition domain (CRD)-like motif. Comparison of the nt sequences between gPLI-A and gPLI-B showed that these genes are highly homologous, including introns, except that exon 3 is rich in nonsynonymous nt substitutions which are almost four times as frequent as synonymous nt substitutions. This evolutionary feature of PLI genes is different from that of venom gland PLA2 isozyme genes in which nonsynonymous nt substitutions are spread over the entire mature protein-coding region.

Amino Acid Sequence↗

PubMed1997

[Pheochromocytoma of the left retroperitoneal paraganglion associated with torsade de pointes: a case report].

A 54-year-old woman developed torsade de pointes with secondary QT prolongation due to hypokalemia and hypomagnesemia. Her serum K and Mg levels were 2.5 mEq/l and 1.5 mg/dl, respectively. This electrolyte imbalance was due to intentional overdosing of metolazone. Attacks of torsade de pointes occurred three times in the intensive care unit and were corrected by intravenous lidocaine administration. Her serum K level was corrected using KCl infusion, restoring the normal QT interval. Routine computed tomography found a left retroperitoneal paraganglioma. Urinary and serum catecholamine examination revealed extremely high values of epinephrine and norepinephrine. The diagnosis was pheochromocytoma in the left retroperitoneal paraganglion. The tumor which was removed measured 70 x 65 x 60 mm in size. Microscopic examination revealed the characteristic patterns of pheochromocytoma.

Diuretics↗

PubMed1996

Downstream signaling molecules bind to different phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) peptides of the high affinity IgE receptor.

The cytoplasmic tails of both the beta and gamma subunits of the high affinity IgE receptor (FcepsilonRI) contain a consensus sequence termed the immunoreceptor tyrosine-based activation motif (ITAM). This motif plays a critical role in receptor-mediated signal transduction. Synthetic peptides based on the ITAM sequences of the beta and gamma subunits of FcepsilonRI were used to investigate which proteins associate with these motifs. Tyrosine-phosphorylated beta and gamma ITAM peptides immobilized on beads precipitated Syk, Lyn, Shc, Grb2, and phospholipase C-gamma1 from lysates of rat basophilic leukemia RBL-2H3 cells. Syk was precipitated predominantly by the tyrosine-diphosphorylated gamma ITAM peptide, but much less by the diphosphorylated beta ITAM peptide or by the monophosphorylated peptides. Phospholipase C-gamma1, Shc, and Grb2 were precipitated only by the diphosphorylated beta ITAM peptide. Non-phosphorylated ITAM peptides did not precipitate these proteins. In membrane binding assays, fusion proteins containing the Src homology 2 domains of phospholipase C-gamma1, Shc, Syk, and Lyn directly bound the tyrosine-phosphorylated ITAM peptides. Although the ITAM sequences of the beta and gamma subunits of FcepsilonRI are similar, once they are tyrosine-phosphorylated they preferentially bind different downstream signaling molecules. Tyrosine phosphorylation of the ITAM of the gamma subunit recruits and activates Syk, whereas the beta subunit may be important for the Ras signaling pathway.

Amino Acid Sequence↗

PubMed1996

Protein globularization during folding. A study by synchrotron small-angle X-ray scattering.

Various conformational states of polypeptide chains were investigated by synchrotron small-angle X-ray scattering (SAXS). SAXS patterns of proteins and model polypeptides in globular states (native and "molten globule") and in non-globular states (unfolded protein as well as randomly coiled, partially alpha-helical and partially beta-structural synthetic polypeptides) were analyzed in terms of Guinier and Kratky plots. Large differences in the SAXS pattern have been found between globular and non-globular conformations of the polypeptide chains, and they have been interpreted in terms of differences in the shape and size of the globular and non-globular scatterers with the same molecular mass. The equilibrium and time-resolved unfolding curves of bovine carbonic anhydrase and yeast phosphoglycerate kinase were monitored by integrated SAXS intensity, and were found to be coincident with the curves measured by other physicochemical techniques, such as tryptophan fluorescence and peptide circular dichroism spectra. The intermolecular association of the protein "molten globule"-like intermediates accumulated during the guanidine hydrochloride-induced unfolding of bovine carbonic anhydrase has been investigated by various SAXS parameters. It has been shown that the integrated SAXS intensity is much less sensitive to the protein intermolecular association than the zero angle intensity and the radius of gyration. We propose the integrated SAXS intensity as a global parameter which is particularly appropriate for fast kinetic studies of protein coil to globule transitions. Time-resolved refolding curves of the above proteins were monitored by the integrated SAXS intensity to investigate the globularization process in protein folding. Two fast kinetic processes for bovine carbonic anhydrase and two fast (each within two seconds) as well as two slow (within 500 seconds) kinetic processes for yeast phosphoglycerate kinase have been recorded. The kinetic processes reflect both protein intramolecular globularization and its intermolecular association.

Animals↗

PubMed1996

Accelerated evolution of Trimeresurus okinavensis venom gland phospholipase A2 isozyme-encoding genes.

Three Trimeresurus okinavensis (To; himehabu snake, Crotalinae) venom gland phospholipase A2 (PLA2) isozymeencoding genes, gPLA2-o1, gPLA2-o2 and gPLA2-o3, were isolated from its genomic DNA library. The nucleotide (nt) sequence analysis revealed that two of the three genes (gPLA2-o2 and gPLA2-o3) occasionally have been converted to inactivated genes by introduction of one base insertion or substitution. It was confirmed from Southern blot analysis that the To haploid genome contains only three venom gland PLA2 isozyme genes herein isolated. Comparison of these genes showed that nonsynonymous nt substitutions have occurred more frequently than synonymous nt substitutions in the protein-coding regions, except for the signal-peptide coding domain, implying that To venom gland PLA2 isozyme genes have evolved via accelerated evolution. Such an evolutionary feature of To venom gland PLA2 isozyme genes proves the general universality of accelerated evolution previously drawn for venom gland PLA2 isozyme genes of other crotalinae snakes. The variability in the mature protein-coding regions of three To venom gland PLA2 isozyme genes appears to have been brought about by natural selection for point mutations.

Amino Acid Sequence↗

PubMed1996

The effect of dorsally angulated distal radius fractures on distal radioulnar joint congruency and forearm rotation.

A biomechanical cadaver study was performed to evaluate the effect of dorsally angulated distal radius fractures on the distal radioulnar joint. Frykman I distal radius fractures were simulated, and laxity measurements were taken with and without sectioning the triangular fibrocartilage complex and the interosseous membrane. The findings of this study were threefold. First, measured in terms of radial diastasis, incongruency of the distal radioulnar joint occurred with increasing dorsal tilt of the distal radius. It became most dramatic with a change of more than 20 degrees of dorsal angulation of the distal radius. This corresponds to approximately 10 degrees of dorsal tilt of the articular surface of the distal radius, as measured on an x-ray film. Second, increased dorsal angulation caused interosseous membrane tightness and limited maximum pronation and maximum supination. Third, distal radioulnar joint dislocation did not occur until both the triangular fibrocartilage complex and interosseous membrane were sectioned. These results reveal the importance of anatomic reduction of the distal radius fracture and evaluation of damaged soft tissue structures.

Biomechanical Phenomena↗

PubMed1996

Dose-response curve for anaesthetics based on the Monod-Wyman-Changeux model.

We have analysed the dose-response curve for halothane and isoflurane according to the Monod-Wyman-Changeux (MWC) model. This model describes the nature of the physiological data reported by Wakamori, Ikemoto and Akaike for inhibitory Cl currents induced by GABA or glycine in dissociated rat brain neurones and by Herrington and colleagues for Ca2+ currents in clonal pituitary cells. With some assumptions on the difference in response to anaesthetics between patients, the model is applicable in vivo, and it also describes well the human dose-response curve for isoflurane reported by Mather, Raftery and Prys-Roberts. However, the steeply sigmoidal dose-response curve in humans for halothane presented by deJong and Eger is difficult to understand with the same model, because it gives rise to unrealistic MWC variables.

Anesthetics, Inhalation↗

PubMed1996

Pharmacologic study of basophil histamine release induced by monocyte chemotactic protein-1 with kinase inhibitors.

Monocyte chemotactic protein-1 (MCP-1)/monocyte chemotactic activating factor has a potent histamine-releasing activity for basophils and is a major component of IgE-independent histamine-releasing factors (HRF). In this study, we examined the effect of a panel of kinase inhibitors on MCP-1-induced histamine release from human basophils to characterize the signaling pathway used by this chemokine. Genistein (3 micrograms/ml), an inhibitor of tyrosine kinase, inhibited MCP-1-induced histamine release by 44%. Wortmannin is a specific inhibitor of phosphatidylinositol 3 kinase (PI-3 kinase). It blocked MCP-1-induced histamine release with an IC50 of 3.3 x 10(-8) M indicating a role of PI-3 kinase in this reaction. KT5926, an inhibitor of myosin light chain kinase, also inhibited histamine release in response to MCP-1 with an IC50 of 10(-6) M. Staurosporine, a potent inhibitor of protein kinase C, although being not specific, augmented MCP-1-induced histamine release by 31.9% at 10(-6) M. These results indicate the possible involvement of a series of kinases, including PI-3 kinase, in the signal transduction pathway used by MCP-1.

Alkaloids↗

PubMed1996

Purification and characterization of a cellulase from the giant snail Achatina fulica.

A cellulase with activity as a cellobiohydrolase [EC3.2.1.91] was purified from the intestinal juice of the giant snail Achatina fulica. The enzyme (M(r) = about 23,000, and pI = about 5.3) also had a weak beta-glucosidase activity. The amino acid sequence of the N-terminal 20 amino acids was analyzed, and no similarity was noted for sequences of other known cellobiohydrolases.

Amino Acid Sequence↗

PubMed1996

[Primary right atrial hemangiosarcoma manifesting as cardiac tamponade: a case report with transesophageal echocardiography].

A 39-year-old woman presented with a right atrial hemangiosarcoma manifesting as cardiac tamponade with complaints of chest discomfort and dyspnea. Transthoracic echocardiography revealed remarkable pericardial effusion and a right atrial mass. Transesophageal echocardiography disclosed the tumor extending into the right atrial cavity. Surgery found the tumor was poorly demarcated, immobile and adhered to the adjacent right atrial wall and septum. The echocardiographic findings correlated well with the surgical and autopsy findings.

Adult↗

PubMed1995

Pharmacological study of stem-cell-factor-induced mast cell histamine release with kinase inhibitors.

Stem cell factor (SCF) is a ligand for c-kit receptor and has a critical role in the development of mast cells. In this study, we investigated the effect of a panel of kinase inhibitors on SCF-induced histamine release from rat peritoneal mast cells. Genistein, an inhibitor of tyrosine kinases, inhibited SCF-induced histamine release with IC50 of 1.6 x 10(-5) M. Wortmannin, an inhibitor of phosphatidylinositol 3'-kinase (PI3 kinase), inhibited histamine release stimulated with SCF dose-dependently with IC50 of 4 x 10(-9) M. KT5926, an inhibitor of myosin light chain (MLC) kinase, reduced histamine release with IC50 of 1.8 x 10(-7) M. Staurosporine, an inhibitor of protein kinases, also inhibited SCF-induced histamine release with IC50 of 6.5 x 10(-8) M. These results show the early involvement of tyrosine kinase and PI3 kinase and the possible role of MLC kinase in the late secretory phase in the signaling pathway used by SCF.

Alkaloids↗

PubMed1995

A serine protease zymogen in insect plasma. Purification and activation by microbial cell wall components.

A protease zymogen present in the plasma fraction of the hemolymph of silkworm, Bombyx mori, was purified to homogeneity as judged by SDS/PAGE and IEF/PAGE. An activating system for the zymogen was also isolated from the plasma fraction and was shown to be triggered by zymosan (yeast cell wall polysaccharide containing beta-1,3-glucan) or peptidoglycan. Using this system, the purified zymogen was activated and the active enzyme was purified to homogeneity. The physiological function of the zymogen or its active form is not yet known, but the active form was shown to have narrower substrate specificity than trypsin. Among 33 peptide derivatives examined, Boc-Gln-Arg-Arg-NH-Mec and Boc-Val-Pro-Arg-NH-Mec (Boc = tert-butoxycarbonyl, NH-Mec = 4-methylcoumaryl-7-amide) were the best and the second best substrates, respectively. The purified zymogen was determined to be a 39-kDa protein consisting of a single polypeptide. The active form of the zymogen was labeled with [3H]diisopropylfluorophosphate and was completely inactivated by (p-amidinophenyl)methanesulfonyl fluoride. The molecular mass of the [3H]-labeled enzyme was determined to be 38 kDa in SDS/PAGE under reducing conditions. These results indicate that the 39-kDa protein purified in the present study is a zymogen of a serine-type protease and that the activation of the zymogen occurs by limited proteolysis.

Amino Acid Sequence↗

PubMed1995

Localization and expression of phospholipases A2 in Trimeresurus flavoviridis (habu snake) venom gland.

The localization and expression profiles of phospholipases A2 (PLA2s) in Trimeresurus flavoviridis venom gland were studied by means of in situ hybridization and immunohistochemical techniques. Venom gland cells are tightly arrayed in a single layer along the inlet-like lumens in which venom proteins are stored. mRNAs for PLA2s were detected at the high level in cytoplasm. Using the immunohistochemical technique with polyclonal anti-Asp-49-PLA2 antibody, Asp-49-PLA2 and, possibly, its isozymes were detected in intracellular granules and in venom lumens. The intracellular granules containing PLA2 proteins appear to be transferred from the nucleus towards the outer membrane facing the lumen, and then to be secreted.

Amino Acid Sequence↗

PubMed1995

Solution X-ray scattering study on the chaperonin GroEL from Escherichia coli.

The molecular architecture of native GroEL has been studied by solution X-ray scattering. The radius of gyration for the native molecule was estimated to be 66.0 A in 50 mM Tris-HCl, 100 mM KCl at pH 7.5 and 25 degrees C. The maximum dimension was estimated to be 170 A, based on the pair distance distribution function. A cylindrical structure or two heptameric rings was found to be the best for native GroEL among structures examined by using a multi-sphere model analysis in which the radius of constituent sphere was 6 A. The results of the model analysis show that the radius of GroEL is 68.0 A and the height is 150.7 A. Unexpectedly, the central penetrating hole through GroEL was not confirmed in the best-fit structure.

Chaperonin 60↗