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Y Hagihara

Publications and source records attributed to Y Hagihara.

At least 37 records · Page 2Linked to original sources
PubMed1993

Acid-induced unfolding and refolding transitions of cytochrome c: a three-state mechanism in H2O and D2O.

Whereas the salt-dependent conformational transition of acid-denatured horse ferricytochrome c at pH 2 is approximated by a two-state mechanism from the acid-unfolded state to the molten globule state [Kataoka, M., Hagihara, Y., Mihara, K., & Goto, Y. (1993) J. Mol. Biol. 229, 591-596], the corresponding transition in D2O has been proposed to involve a noncompact, alpha-helical intermediate state (the pre-molten globule state) [Jeng, M.-F., & Englander, S. W. (1991) J. Mol. Biol. 221, 1045-1061]. To examine the proposed difference in the conformational transitions, we carried out the HCl and DCl titrations of cytochrome c in H2O and D2O, respectively, measured by far-UV circular dichroism, tryptophan fluorescence, and Soret absorption. In both D2O and H2O, unfolding from the native state to the acid-unfolded state and subsequent refolding to the molten globule state were observed. In either solvent, the conformational transitions were well approximated by a minimal three-state mechanism consisting of the native, molten globule, and acid-unfolded states. Thus, our results did not substantiate the presence of a pre-molten globule state in D2O. Acetylation of amino groups of cytochrome c is known to stabilize the molten globule state at pH 2. On the basis of the three-state mechanism, we constructed a conformational phase diagram for the effect of pH and the degree of acetylation. This phase diagram was similar to that of the pH- and salt-dependent conformational transition of cytochrome c, suggesting that the effects of acetylation on the conformational states are similar to those of salt.

Animals↗
PubMed1993

Guanidine hydrochloride-induced folding of proteins.

Guanidine hydrochloride (Gdn-HCl) is the most commonly used denaturant for proteins. Contrary to expectation, we found that Gdn-HCl at low concentrations refolds acid-unfolded apomyoglobin and cytochrome c, stabilizing the molten globule state, i.e. a compact denatured state with a significant amount of secondary structure, but substantially disordered tertiary structure. A further increase in Gdn-HCl concentration, above 1 M, caused co-operative unfolding of the molten globule state. Similar sequential folding and unfolding transitions were also observed at neutral pH with a synthetic amphiphilic peptide consisting of Lys and Leu residues, indicating the generality of the phenomenon. Although the Gdn-HCl-induced refolding and unfolding transitions were puzzling at first glance, we show that they are readily interpreted in terms of the differential action of Gdn-HCl. We also show that the comparison of the unfolding curves for the molten globule and native states provides a measure of the buried surface area upon formation of the molten globule state.

Amino Acid Sequence↗
PubMed1993

Molten globule of cytochrome c studied by small angle X-ray scattering.

Recent advances in the small angle X-ray scattering technique have made it possible to obtain information on the spatial distribution of protein structure in solution, such as the radius of gyration and shape of the molecule, in a relatively short time. We have studied the various conformational states of horse ferricytochrome c by small angle X-ray scattering. Whereas Guinier plots showed that the radius of gyration of the molten globule state is slightly larger than that of the native state, Kratky plots indicated that their shape is globular. To clarify the mechanism of the formation of the molten globule state, we measured, by the small angle X-ray scattering, the equilibrium transition at pH 2 from the acid-unfolded state to the molten globule state, which was induced either by the addition of NaCl or the acetylation of lysyl amino groups. The Kratky plots at various stages of folding showed an isoscattering point, strongly suggesting a two-state mechanism. For both the NaCl and acetylation-induced transitions, the transition curve determined from Kratky plots agreed well with the transition curves from far-ultraviolet circular dichroism, tryptophan fluorescence, and Soret absorption, further supporting the two-state mechanism.

Acetylation↗
PubMed1992

Charge repulsion in the conformational stability of melittin.

Electrostatic repulsion between positively charged groups has been suggested to be critical in determining the conformation of melittin. To clarify the role of repulsive forces, we prepared a series of succinylated melittins, an acetylated melittin, and a synthetic melittin mutant, with various degrees of charge repulsion. The conformation of the melittin derivatives was examined by far-UV circular dichroism under various conditions of pH and salt at 20 degrees C. The stability of the tetrameric helical state was found to be dependent on the net charge of the peptides. The charge repulsive forces destabilized the helical state of intact melittin by 600 cal/(charge.mol of tetramer). This value was close to the corresponding one (450 cal/(charge.mol)) obtained for the acidic molten globule of horse cytochrome c [Goto, Y., & Nishikiori, S. (1991) J. Mol. Biol. 222, 679-686], which has a molecular weight and a net charge comparable to those of the tetrameric melittin. Small-angle X-ray scattering of the tetrameric melittin and the molten globule of cytochrome c showed that the two states are also comparable to each other in the radius of gyration. These results suggest that the contribution of electrostatic repulsion to the conformational stability of melittin is similar to that of the molten globule.

Amino Acid Sequence↗
PubMed1992

Mechanism of the conformational transition of melittin.

It is known that, while melittin at micromolar concentrations is unfolded under conditions of low ionic strength at neutral pH, it adopts a tetrameric alpha-helical structure under conditions of high ionic strength, at alkaline pH, or at high peptide concentrations. To understand the mechanism of the conformational transition of melittin, we examined in detail the conformation of melittin under various conditions by far-UV circular dichroism at 20 degrees C. We found that the helical conformation is also stabilized by strong acids such as perchloric acid. The effects of various acids varied largely and were similar to those of the corresponding salts, indicating that the anions are responsible for the salt- or acid-induced transitions. The order of effectiveness of various monovalent anions was consistent with the electroselectivity series of anions toward anion-exchange resins, indicating that the anion binding is responsible for the salt- or acid-induced transitions. From the NaCl-, HCl-, and alkaline pH-induced conformational transitions, we constructed a phase diagram of the anion- and pH-dependent conformational transition. The phase diagram was similar in shape to that of acid-denatured apomyoglobin [Goto, Y., & Fink, A.L. (1990) J. Mol. Biol. 214, 803-805] or that of the amphiphilic Lys, Leu model polypeptide [Goto, Y., & Aimoto, S. (1991) J. Mol. Biol. 218, 387-396], suggesting a common mechanism of the conformational transition. The anion-, pH-, and peptide concentration-dependent conformational transition of melittin was explained on the basis of an equation in which the conformational transition is linked to proton and anion binding to the titratable groups.

Anions↗
PubMed1992

The relationship between cyclic adenosine 3',5'-monophosphate and morphology in exponential phase Candida albicans.

The relationship between changes in cyclic AMP content and germ tube formation in exponential phase Candida albicans was investigated using two simple media containing glucose plus ammonium chloride, or N-acetyl-D-glucosamine (GlcNAc). The glucose medium did not promote germ tube formation unless the cells were starved before inoculation, whereas the GlcNAc medium promoted germ tube formation in both non-starved and starved cells. The cyclic AMP content of exponential phase cells, non-starved cells and starved cells was 0.21, 0.34 and 0.64 pmol mg-1 dry wt, respectively. In glucose medium, cyclic AMP content in both non-starved cells and starved cells increased for a period of 60 min after inoculation, but then decreased for a further 120 min. The cyclic AMP content of non-starved cells and starved cells was 0.16 and 0.29 pmol mg-1 dry wt, respectively, after 180 min. The maximum percentage of non-starved cells with germ tubes was around 20%. Starved cells with germ tubes were observed after 40 min and reached a maximum (around 90%) after 140 min. The number of germ tubes remained constant for the next 40 min. In GlcNAc medium, the cyclic AMP content of both non-starved cells and starved cells showed a tendency to increase for 180 min. The content of non-starved cells and starved cells was 2.02 and 1.75 pmol mg-1 dry wt, respectively, after 180 min. Germ tube formation in non-starved cells started after 70 min, reached around 80% after 150 min, and remained stable for the next 30 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylglucosamine↗
PubMed1992

[Late results of coronary artery bypass grafting with the internal thoracic artery].

Between November 1981 and December 1991, 755 patients underwent isolated coronary artery bypass grafting. We compared postoperative events, graft patency and survival rates of the patients who received at least one internal thoracic artery graft (n = 517) with those of the patients who had only saphenous vein bypass grafts (n = 238). No significant difference was found between the 2 groups in patient's age at the operation, female ratio and the incidence of emergency operation. The operative mortality was not significantly different in either group. Graft patency was better for internal thoracic artery grafts than for SVG (ITA grafts 97.1%, SVG 85.6% p less than 0.0001). Graft-LAD patency rate was also better for ITA grafts than for SVG (ITA grafts 97.7%, SVG 86.8%, p less than 0.0001). The incidence of late cardiac events (late cardiac death, reoperation, myocardial infarction) is lower in patients with ITA. Furthermore, patients who received ITA grafts had a better survival rate at 8 years (91.1% versus 85.3%, p = 0.048) than those who had vein bypass grafts alone. We conclude that the use of ITA grafts enhances the long-term benefits including long-term survival rate. This is the first observation in Japanese population.

Adult↗
PubMed1992

Synthesis and antitumor activity of poly(ethylene glycol)s linked to 5-fluorouracil via a urethane or urea bond.

In order to provide a macromolecular prodrug of 5-fluorouracil (5FU) with reduced side-effects and exhibiting strong antitumor activity, 5FU was covalently linked to poly(ethylene glycol) (PEG) via a urethane or urea bond. For the purpose of evaluating the release behavior of 5FU, the hydrolysis of the urethane or urea bond in the obtained conjugate of PEG-end capped with 5FU was investigated in vitro at 37 degrees C in aqueous solution media. The survival effect for the conjugate was assessed in vivo against p388 lymphocytic leukemia in female CDF1 mice by intraperitoneal (i.p.) transplantation/i.p. injection. The effects of a hydrophobic hexamethylene spacer group, the end group and the number n of ethylene oxide (EO) units in PEG on the release behavior of 5FU and the survival effect were investigated. The release rate of 5FU from the 5FU-terminated PEG conjugates via urethane or urea bond was very fast. However, it became slow with increasing n of EO units in PEG and was depressed by the introduction of hydrophobic spacer group. The 5FU-terminated PEG conjugates obtained exhibited significant survival effects against p388 leukemia mice i.p./i.p. Especially, the methoxy PEG (n = 113)/urethane/hexamethylene/urea/5FU conjugate showed the strongest survival effect among the synthesized 5FU-capped PEG conjugates via urethane or urea bond compared to free 5FU against p388 leukemia mice. These conjugates obtained did not display an acute toxicity even in high dose ranges.

Animals↗
PubMed1990

Activation of the plasma kallikrein-kinin system by Candida albicans proteinase.

An extracellular carboxyl proteinase produced by the yeast Candida albicans enhanced vascular permeability when injected into the dorsal skin of guinea pigs. The character and mechanism of the permeability-enhancing reaction were studied in vivo and in vitro. Permeability was not enhanced when the C. albicans proteinase was heat treated (100 degrees C, 5 min) or when it was treated with pepstatin, a specific carboxyl proteinase inhibitor. The permeability reaction induced by the C. albicans proteinase was not affected by pretreatment with antihistamine but was greatly augmented by simultaneous injection of a kinin potentiator, carboxypeptidase N inhibitor. However, the simultaneous injection of a kinin-degrading enzyme, carboxypeptidase B, interfered with the reaction. Furthermore, in vitro conversion of plasma prekallikrein to kallikrein by the C. albicans proteinase was observed, and the reaction was inhibited by corn trypsin inhibitor, an inhibitor of activated Hageman factor, and soybean trypsin inhibitor, a well-known inhibitor of plasma kallikrein. These results indicate that C. albicans proteinase enhances vascular permeability through activation of the plasma kallikrein-kinin system, which generates bradykinin.

Animals↗
PubMed1989

Local cerebral glucose utilization in the postictal phase of amygdaloid kindled rats.

Local cerebral glucose utilization was measured by means of the quantitative autoradiographic 2-[14C]deoxyglucose method during the postictal phase of various seizure stages of amygdaloid kindling in conscious rats. The partially kindled animals exhibited a partial seizure such as chewing and/or head nodding, and the fully kindled animals, a generalized tonic-clonic convulsion. The control animals were implanted with an electrode, but not electrically stimulated. Cerebral glucose utilization of the fully kindled animals was deeply depressed in the postictal phase as compared to the control, and that of the partially kindled animals was moderately decreased. The side-to-side differences of cerebral glucose utilization were observed only in the partially kindled group in which glucose utilization was more depressed on the side of stimulation. Among the structures with depressed glucose utilization, only one structure, the interpeduncular nucleus, showed a relative increase in glucose utilization during the postictal phase of the kindled groups. As the postictal phase has been considered as a period of inhibition, these results may indicate that the neural networks linking the interpeduncular nucleus play an active role in the mechanisms of termination of a seizure and postictal refractoriness.

Amygdala↗
PubMed1989

Geographutoxin-sensitive and insensitive sodium currents in mouse skeletal muscle developing in situ.

1. The whole-cell voltage-clamp technique was used to examine developmental changes of Na+ current properties in single fibres of mouse flexor digitorum brevis muscles developing in situ from birth to 20 days post-natal. 2. Geographutoxin II (GTX II), a novel polypeptide toxin from the marine snail Conus geographus, distinguished two different types of voltage-sensitive Na+ currents: GTX II-sensitive and GTX II-insensitive currents, which corresponded respectively to currents with high or low TTX sensitivity. 3. Voltage-dependent activation and inactivation of the GTX II-insensitive currents occurred at membrane potentials 10-20 mV more negative than those for the GTX II-sensitive currents. 4. The GTX II-insensitive current in fibres from mice older than 8 days inactivated more slowly than the GTX II-sensitive current. However, in fibres from younger mice, the two currents decayed with similar speed. 5. The mean specific Na+ conductance (gNa) for the total (GTX II-sensitive plus GTX II-insensitive) Na+ channels was 0.22 mS/muF at a Na+ concentration of 5 mM at birth. The total gNa increased 6-fold to 1.32 mS/muF during the first 20 days after birth. 6. The mean specific gNa for the GTX II-insensitive channels was 0.15 mS/muF at birth, remained at approximately the same level for the first 8 days, and then decreased progressively to become undetectable by day 16. 7. In muscle fibres denervated 12 days after birth, the GTX II-insensitive gNa increased over the next 8 days, whereas the total gNa increased less than normal. 8. By contrast, in fibres denervated on day 4, the total gNa increased more than normal in the following 8 days, and the GTX II-insensitive specific gNa increased above the level seen at birth. 9. Half-maximal activation and inactivation potentials of the total and the GTX II-insensitive currents shifted in the negative direction by 9-17 mV in the first 8 days after birth. 10. We conclude that the regulatory effects of innervation on the total gNa are either suppressive or enhancing depending on the stage of development. On the other hand, denervation elicits an increase in GTX II-insensitive Na+ currents at all ages studied.

Aging↗
PubMed1989

Starvation and germ tube formation in the exponential phase Candida albicans.

Two chemically defined media were developed for the induction of germ tubes in exponential phase cells of Candida albicans. One medium was N-acetyl-D-glucosamine medium which is composed of L-thiazolidine-4-carboxylic acid, L-proline, NaHCO3, sodium acetate, NaH2PO4 and N-acetyl-D-glucosamine. The other one was glucose medium in which N-acetyl-D-glucosamine is exchanged for glucose plus NH4Cl in N-acetyl-D-glucosamine medium. In these media, a high percentage of germ tube forming cells was obtained without a temperature shift. However, starvation of the cells in water at 37 degrees C was a necessary pretreatment to consistently obtain a high percentage of germ tube forming cells. The effect of starvation was remarkable in glucose medium, the percentages of germ tube forming cells among the normal cells and starved cells were 20 and 80, respectively. As for intracellular changes during starvation, a decrease in adenosine triphosphate concentration and an increase in adenosine 3',5'-cyclic monophosphate concentration were observed.

Acetylglucosamine↗
PubMed1988

Degradation of human dentine collagen by an enzyme produced by the yeast Candida albicans.

An extracellular collagenolytic enzyme separated from a culture medium of this pathogenic yeast was found to attack undenatured predentine collagen as seen in scanning electron micrographs. After treatment with the enzyme at pH 4.0, but not by that acidity alone, dentine tubules were less easily distinguished and the collagen fibres were less well-organized.

Candida albicans↗
PubMed1988

Degradation of bovine achilles tendon collagen by Candida albicans proteinase.

Candida albicans, when cultivated in a medium containing insoluble bovine achilles tendon as a nitrogen source, was able to produce a collagen degrading proteinase. The degradation of achilles tendon collagen by the proteinase was verified by morphological change and the release of hydroxyproline. The proteinase activity was inhibited by pepstatin.

Achilles Tendon↗