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S Yoshikawa

Publications and source records attributed to S Yoshikawa.

At least 91 records · Page 5Linked to original sources
PubMed1997

Design and characterization of the anion-sensitive coiled-coil peptide.

As a model for analyzing the role of charge repulsion in proteins and its shielding by the solvent, we designed a peptide of 27 amino acid residues that formed a homodimeric coiled-coil. The interface between the coils consisted of hydrophobic Leu and Val residues, and 10 Lys residues per monomer were incorporated into the positions exposed to solvent. During the preparation of a disulfide-linked dimer in which the two peptides were linked in parallel by the two disulfide bonds located at the N and C terminals, a cyclic monomer with an intramolecular disulfide bond was also obtained. On the basis of CD and 1H-NMR, the conformational stabilities of these isomers and several reference peptides were examined. Whereas all these peptides were unfolded in the absence of salt at pH 4.7 and 20 degrees C, the addition of NaClO4 cooperatively stabilized the alpha-helical conformation. The crosslinking of the peptides by disulfide bonds significantly decreased the midpoint salt concentration of the transition. The 1H-NMR spectra in the presence of NaClO4 suggested that, whereas the disulfide-bonded dimer assumed a native-like conformation, the cyclic monomer assumed a molten globule-like conformation with disordered side chains. However, the cyclic monomer exhibited cooperative transitions against temperature and Gdn-HCl that were only slightly less cooperative than those of the disulfide-bonded parallel dimer. These results indicate that the charge repulsion critically destabilizes the native-like state as well as the molten globule-like state, and that the solvent-dependent charge repulsion may be useful for controlling the conformation of designed peptides.

Amino Acid Sequence↗
PubMed1997

Beef heart cytochrome c oxidase.

During the past two years, the crystal structures of beef heart cytochrome c oxidase with 13 subunits and the bacterial enzyme with four subunits have been reported at atomic resolution, ushering in a new era for cytochrome c oxidase research. Different proton pumping mechanisms have been proposed for the two organisms.

Animals↗
PubMed1997

Oral/aboral ectoderm differentiation of the sea urchin embryo depends on a planar or secretory signal from the vegetal hemisphere.

A monoclonal antibody that recognizes oral ectoderm and esophagus of sea urchin larvae was newly produced. Distribution of the antigen, named Hpoe, was examined by indirect immunofluorescence microscopy. Hpoe did not exist in eggs and appeared during the cleavage stage. In hatched blastulae, Hpoe was detected on the apical surface of all cells. As embryogenesis progressed, Hpoe disappeared from the primary mesenchyme, archenteron and aboral ectoderm. Hpoe reappeared in foregut at the prism stage and was restricted to the oral ectoderm and esophagus at the pluteus stage. Using this antigen as a molecular marker of oral/aboral ectoderm differentiation, the role of the vegetal hemisphere in ectoderm differentiation was examined. All animal hemispheres isolated from 16-cell stage embryos, mesenchyme blastulae, early gastrulae and mid gastrulae developed into epithelial balls and every cell expressed Hpoe. These epithelial balls failed in oral/aboral ectoderm differentiation. Twenty millimolar LiCl-treated whole embryos developed into exo-gastrulae but Hpoe restriction in ectoderm occurred in these exo-gastrulae. These results show that oral/aboral ectoderm differentiation requires an inductive interaction from the vegetal hemisphere and indicate that the inductive interaction depends on a planar or secretory signal, rather than the contact of the esophagus and ectoderm.

Animals↗
PubMed1997

Intrinsic and extrinsic determinants regulating cell fate decision in developing nervous system.

Cell fate in the nervous system is determined by environmental signals and/or heredity. They can be referred to as extrinsic and intrinsic determinants, respectively. The molecular and cellular mechanisms underlying these processes are well conserved from invertebrates to vertebrates. We have identified both types of determinants through the intensive screening of P-element-induced Drosophila mutants affected in neural development. As a member of the first category, argos gene product which regulates cell-cell interaction in the developing nervous system gives a good example. As a member of the second category, we refer to Drosophila musashi gene encoding a neural RNA-binding protein. The fractions of these gene products and application to the mammalian nervous system will be discussed.

Animals↗
PubMed1997

ATP and ADP bind to cytochrome c oxidase and regulate its activity.

By equilibrium dialysis of cytochrome c oxidase from bovine heart with [35S]ATPalphaS and [35S]ADPalphaS, seven binding sites for ATP and ten for ADP were determined per monomer of the isolated enzyme. The binding of ATP occurs in a time-dependent manner, as shown by a filtration method, which is apparently due to slow exchange of bound cholate. In the crystallized enzyme 10 mol of cholate were determined and partly identified in the high resolution crystal structure. Binding of ADP leads to conformational changes of the Tween 20-solubilized enzyme, as shown by a 12% decrease of the gamma-band. The conformational change is specific for ADP, since CDP, GDP and UDP showed no effects. The spectral changes are not obtained with the dodecylmaltoside solubilized enzyme. The polarographically measured activity of cytochrome c oxidase is lower after preincubation with high ATP/ADP-ratios than with low, in the presence of Tween 20. This effect of nucleotides is due to interaction with subunit IV, because preincubation of the enzyme with a monoclonal antibody to subunit IV released the inhibition by ATP. In the presence of dodecylmaltoside the enzyme had a 2 to 3-fold higher total activity, but this activity was not influenced by preincubation with ATP or ADP.

Adenosine Diphosphate↗
PubMed1996

Solid-phase synthesis of caged peptides using tyrosine modified with a photocleavable protecting group: application to the synthesis of caged neuropeptide Y.

A simple method for the synthesis of caged peptides using a novel derivative of tyrosine, N-Fmoc-O-(2-nitrobenzyl)-tyrosine, is described. The derivative of tyrosine can be incorporated at any position in an amino acid sequence by solid-phase peptide synthesis under the condition for Fmoc chemistry, and caged peptides that contain nitrobenzyl group on the side chain of tyrosine residue can be obtained. The nitrobenzyl group can be photocleaved by UV irradiation and the half life of the intermediate during photolysis is approximately 7 microseconds. The method was successfully applied to the synthesis of caged neuropeptide Y (NPY). The binding affinity of the caged NPY for the Y1 receptor was one or two orders of magnitude lower than that of intact NPY, but it increased to the value for intact NPY upon irradiation by UV light.

Binding, Competitive↗
PubMed1996

Resonance Raman/absorption characterization of the oxo intermediates of cytochrome c oxidase generated in its reaction with hydrogen peroxide: pH and H2O2 concentration dependence.

Effects of pH and H2O2 concentration on the reaction of cytochrome c oxidase (CcO) with H2O2 were studied with the high-performance Raman/absorption simultaneous determination technique reported previously (Proshlyakov et al., 1996). This reaction generates two intermediates called 607- and 580-nm forms, and we found that they show the same oxygen-isotope-sensitive RR bands as those of the intermediates in O2 reduction by CcO. In transient absorption spectra obtained under single turnover conditions, the 607-nm form appeared as the primary intermediate and subsequently the 580-nm and resting forms, suggesting that H2O2 serves as an oxidant for the resting enzyme but as a reductant for both the 607- and 580-nm forms in the peroxide cycle. The rise rate of absorption at 607 nm was insensitive to the H2O/D2O exchange, but the decay was significantly slower in D2O than in H2O. With the microcirculating system, each intermediate was maintained at a constant level under steady-state conditions by supplying H2O2 continuously. In the pH range between 7.4 and 10.0, the population of the 607-nm form decreased at higher pH and at higher concentrations of H2O2. The Fe=O stretching (VFe=O) frequencies of the oxo heme of the 607-nm form, observed at 804/769 cm-1 for their H2(16)O2/H2(18)O2 derivatives, were unaltered in this pH range and exhibited a D2O/H2O shift even at pH 10.0. This indicates that the iron-bound oxygen is hydrogen-bonded to a distal residue in this pH range. When the 580-nm form is dominant under the nonsaturating level of H2O2, two other oxygen-isotope-sensitive Raman bands have been observed at 785/750 cm-1 and 355/340 cm-1 at neutral pH, but the former disappeared above pH 8.5 and the latter above pH 9.0 without significant changes of absorption spectra, suggesting the presence of two separate species in the name of the 580-nm form. However, under the saturating concentration of H2O2, these Raman bands were unaltered between pH 7.4 and 10.0. In contrast, in the absence of excess peroxide, no oxygen-isotope-sensitive RR bands were observed despite dominance of the 580-nm form. The disappearance of these Raman bands demonstrates the occurrence of oxygen exchange between the oxo heme and bulk water, whose rate surpasses the formation rate of the 580-nm form at alkaline pH and/or at low H2O2 concentration. Such an oxygen exchange did not take place in the 607-nm form. Under the identical experimental conditions for generating a particular steady state, the exchange of H2O with D2O caused significant depopulation of the 580-nm form and concomitant increase of the 607-nm form. This was satisfactorily interpreted in terms of the difference in the decay rate of the 607-nm form between H2O and D2O. Thus, the reduction of the 607-nm form to the 580-nm form is likely to be a key step of the redox-linked proton pumping in the O2 reduction.

Animals↗
PubMed1996

The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 A.

The crystal structure of bovine heart cytochrome c oxidase at 2.8 A resolution with an R value of 19.9 percent reveals 13 subunits, each different from the other, five phosphatidyl ethanolamines, three phosphatidyl glycerols and two cholates, two hemes A, and three copper, one magnesium, and one zinc. Of 3606 amino acid residues in the dimer, 3560 have been converged to a reasonable structure by refinement. A hydrogen-bonded system, including a propionate of a heme A (heme a), part of peptide backbone, and an imidazole ligand of CuA, could provide an electron transfer pathway between CuA and heme a. Two possible proton pathways for pumping, each spanning from the matrix to the cytosolic surfaces, were identified, including hydrogen bonds, internal cavities likely to contain water molecules, and structures that could form hydrogen bonds with small possible conformational change of amino acid side chains. Possible channels for chemical protons to produce H2O, for removing the produced water, and for O2, respectively, were identified.

Amino Acid Sequence↗
PubMed1996

Factors that affect the stabilization of alpha-helices in short peptides by a capping box.

It was reported recently that the capping box sequences of four N-terminal residues are very important for the stabilization of alpha-helices in proteins and peptides. To elucidate factors that affect the stabilization of alpha-helices in short peptides by this motif, we analyzed conformational properties of side chains of five N-terminal residues in several analogs of neuropeptide Y (NPY). The analysis revealed three previously unreported factors that appear to be important for stabilization of an alpha-helix: (a) a second capping box hydrogen bond for the side chains of Ser, Thr, and Cys; (b) long-range electrostatic interactions between the first (N-cap) and fifth (N4) residues; and (c) capping interactions of alpha-amino groups with the N4 residue. These factors were incorporated into the parameter set of a recently published, statistical mechanics approach that showed excellent accuracy in the prediction of the helical propensities of short peptides in water [Muñoz, V., & Serrano, L. (1995) J. Mol. Biol. 245, 275-296, 297-308]. A significant improvement in the agreement between theoretical predictions and experimental data was achieved. The present results also clarify the nature of capping box stabilization of alpha-helices in peptides and proteins, indicating that the total influence of hydrogen bonding, local interactions between side chains, helix macrodipole--charge/dipole interactions, and solvation possibilities must all be taken into account. All these factors are associated with approximately the same energy, but with different residues at the N-cap position, they may have opposite effects on the helix stability of peptides. Thus, a delicate balance of interactions of different types controls the stabilization properties of N-cap residues in alpha-helices.

Amino Acid Sequence↗
PubMed1996

Microcirculating system for simultaneous determination of Raman and absorption spectra of enzymatic reaction intermediates and its application to the reaction of cytochrome c oxidase with hydrogen peroxide.

A new high-performance device for Raman/absorption simultaneous determination was developed. This was combined with a newly designed microcirculating system and was successfully applied to study intermediates in the reaction of bovine oxidized cytochrome c oxidase (CcO) with hydrogen peroxide under steady state conditions at ambient temperatures. Measurements with this device made it possible to correlate directly the species defined in terms of the visible absorption characteristics with specific Raman bands. The "607 nm" form of the enzyme obtained with H2(16)O2 gave an oxygen isotope sensitive band at 804 cm-1 (769 cm-1 with H2(18)O2) in the Soret excited resonance Raman (RR) spectrum. Its frequency and isotope frequency shifts are exactly the same as those observed previously with 607 nm excitation in nonsimultaneous measurements for the 607 nm form, for which the presence of an oxoiron heme was demonstrated. The so-called " 580 nm" form of the enzyme obtained with H2(16)O2 gave the main oxygen isotope sensitive band at 785 cm-1 (750 cm-1 with H2(18)O2) but appeared to consist of multiple species. This band was assigned to the FeIV = O stretching mode of ferryloxo heme on the basis of its isotopic frequency shift. Another oxygen isotope sensitive band was found at 355 cm-1 (340 cm-1 for H2(18)O2), similar to the case of dioxygen reaction. Temporal behavior of this band did not agree with either that of the 804 cm-1 band or that of the 785 cm-1 band but seemed to grow between the two species. The RR spectra in the higher frequency region of the 607 nm and 580 nm forms excited at 427 nm were quite alike and did not support the formation of a porphyrin pi-cation radical.

Animals↗
PubMed1996

Functional expression of the type 1 inositol 1,4,5-trisphosphate receptor promoter-lacZ fusion genes in transgenic mice.

Inositol 1,4,5-trisphosphate receptor (IP3R) is an inositol 1,4,5-trisphosphate (InsP3)-gated Ca2+ release channel. Type 1 IP3R (IP3R1) is the neuronal member of the IP3R family in the CNS and is predominantly expressed in cerebellar Purkinje cells. To elucidate the molecular mechanisms responsible for coupling gene expression to neuronal InsP3/Ca2+ signaling, we have studied the structure and function of the 5'-flanking region of the mouse IP3R1 gene. The cloned 5'-flanking region has several sequences sharing identity with motifs for known transcriptional regulation. We have fused 5'-flanking regions 1N from -528 to +169 and 4N from -4,187 to +169 to a beta-galactosidase gene (lacZ) as a reporter marker and have characterized their in vivo gene expression. Both 1N and 4N fusion genes functioned as a strong promoter in a neuroblastoma-glioma hybrid cell line NG108-15. Moreover, both 1N and 4N transgenic mouse lines carrying these 1N and 4N fusion genes showed characteristic patterns of beta-galactosidase activity in the CNS that are almost consistent with that of the endogenous IP3R1 protein, thereby suggesting that the 1N region from -528 to +169 contains sequence elements responsible for regulating gene expression in neurons and for specifying predominant expression in cerebellar Purkinje cells.

Animals↗
PubMed1996

Acylation of the alpha-amino group in neuropeptide Y(12-36) increases binding affinity for the Y2 receptor.

Competition assays using three series of analogs of neuropeptide Y (NPY) ([Xaa11]NPY(11-36), [Xaa12]NPY(12-36), and [Xaa13]NPY(13-36) revealed that the binding affinity for the Y2 receptor was considerably lowered by truncation of residue 11. Upon acetylation or succinylation of the alpha-amino group, the binding affinity of [Xaa12]NPY(12-36) recovered to a level similar to that of [Xaa11]NPY(11-36). No significant difference was observed between the increases caused by acetylation and those caused by succinylation, suggesting that the increase in binding affinity cannot be explained by the change in the net charge at the N-terminus as a consequence of the modification. The scattered data points on a plot of the alpha-helix content vs. IC50 of all these analogs revealed the absence of any apparent relationship, an indication that prior formation of the alpha-helix is not necessary for binding to the Y2 receptor. It has been widely accepted that fewer than 12 residues from the C-terminus are directly involved in binding of NPY to the Y2 receptor, while the remaining part of NPY only assists in the adoption of a favorable conformation by the C-terminal hexapeptide for recognition by the receptor. However, the present results suggest that the region around residue 12 does not project from the Y2 receptor.

Acylation↗
PubMed1996

Eosinophilia-induced vascular and airway remodeling and hyperresponsiveness in rat lungs.

We evaluated the effects of pulmonary infiltration of eosinophils without exogenous activators on airway and vascular hyperresponsiveness to muscarinic challenge in the lungs of rats infected with Toxocara [correction of Toxicara] canis, the canine round worm. Bronchoalveolar lavage of infected lungs produced 4.26 x 10(7) cells with 85% eosinophils, 15% mononuclear cells, and essentially no neutrophils. Eosinophils were present in the air spaces and interstitial spaces surrounding airways and vessels. The smooth muscle thickness increased about fourfold in large airways and vessels, and medium and small vessels were muscularized in infected lungs. In the T. canis-infected lungs, baseline airway resistance increased 288%, total vascular resistance (RT) increased 202%, and capillary filtration coefficient increased 208% compared with uninfected control lungs. Lung compliance was 56% of control. The concentration of acetylcholine that produced 50% of maximal response was 18.4 times greater for airway resistance and 18.7 times greater for RT in uninfected controls than in infected lungs. Isoproterenol (10(-4) M) decreased RT and peak airway pressure by 21% in infected lungs but had no significant effect on controls. We conclude that pulmonary interstitial infiltrates of eosinophils cause airway and vascular remodeling and increase baseline resistances and muscarinic reactivities of airways and vessels in rat lungs infected with T. canis.

Acetylcholine↗
PubMed1996

Acute hypoxic pulmonary vascular response does not accompany plasma endothelin-1 elevation in subjects susceptible to high altitude pulmonary edema.

We have previously shown that high altitude pulmonary edema-susceptible subjects (HAPE-S) have an accentuated pulmonary vascular response to hypoxia. In this study, we investigated the relationship between plasma endothelin-1 (ET-1) levels and the acute hypoxic pulmonary vascular response in HAPE-S and control subjects. In six HAPE-S and seven healthy subjects, we evaluated acceleration time/right ventricular ejection time (AcT/RVET) using Doppler echocardiography, and measured plasma ET-1 levels by radioimmunoassay (RIA) before and after 5 minutes of breathing 10% oxygen. The HAPE-S showed a significantly increased pulmonary vascular response to hypoxia compared with healthy subjects. However, no statistically significant changes of plasma ET-1 levels were observed before and after hypoxia in both groups. We conclude that the increased pulmonary vascular response to acute hypoxia in HAPE-S may not be related to ET-1 release.

Acute Disease↗
PubMed1996

[Distance walked in 10 minutes, pulmonary function, and pulmonary hemodynamics during exercise, in patients with pulmonary emphysema].

We examined relationships among the distance walked in 10 minutes (10 MD), pulmonary function, and pulmonary hemodynamics during exercise on a bicycle ergometer, in patients with chronic pulmonary emphysema who had dyspnea of grade III to IV on the Hugh-Jones scale. The 10 MD did not correlate significantly with desaturation during the 10-minute walk, but it did correlate significantly with the percent of predicted maximum voluntary ventilation, and it correlated negatively with airway resistance. These findings indicate that ventilatory impairment is an important factor limiting 10 MD in these patients. Also 10 MD correlated significantly and positively with %DLco; and it correlated negatively with the index of pulmonary vascular resistance and with the ratio of the change in pulmonary arterial pressure to the change in cardiac index during exercise. (delta Ppa/delta CI). These findings indicate that impairment of pulmonary circulation may also limit the 10 MD and exercise tolerance in patients with chronic pulmonary emphysema.

Aged↗
PubMed1996

[Pulmonary malignant fibrous histiocytoma presenting as hemothorax].

A 39-year-old woman, who suffered from a sudden episode of severe left-sided back pain, was admitted to our hospital because of hemothorax. Chest CT scans revealed a mass extending from the left hilum to the posterior thoracic wall. The mass occluded the left pulmonary artery at the left main bronchus. Examination of a frozen section of a specimen obtained during a thoracotomy showed a sarcomatous tumor. A left pneumonectomy was then performed. Microscopic examination of a specimen revealed that the tumor was composed of spindle cells with cellular atypia and mitosis forming fascicles and storiform arrays. Malignant fibrous histiocytoma (storiform-pleomorphic pattern) was diagnosed. Chemotherapy was carried out six times during the year after surgery. No recurrence of the tumor has been seen during 20 months of follow-up.

Adult↗
PubMed1996

[Crystal structure of fully oxidized cytochrome c-oxidase from the bovine heart at 2.8 A resolution].

Structural and functional studies on cytochrome c oxidase before the crystal structures appeared, are summarized to show the importance of X-ray crystal structure at the atomic resolution for understanding mechanism of the enzyme reaction, O2 reduction coupled with proton pumping. Crystal structure of bovine heart enzyme at the fully oxidized state at 2.8 A resolution are reviewed to evaluate of the contribution for understanding the enzyme reaction mechanism. Effects of detergent structure on the crystallization conditions of the bovine heart cytochrome c oxidase, which was critical for obtaining the X-ray crystal structure, are presented to propose a possible mechanism of crystallization of multicomponent membrane proteins.

Animals↗