Biomedical subjects

Y Hagihara

Publications and source records attributed to Y Hagihara.

At least 19 recordsLinked to original sources

PubMed2004

[Change in the volume of extra cellular fluid in coronary artery bypass grafting (CABG); comparison between off-pump and on-pump CABG].

During the perioperative period for off-pump coronary artery bypass surgery (OPCAB) and on-pump coronary artery bypass surgery (on-pump CABG), the volume of extra cellular fluid (ECF) was measured. The subjects were elective adult coronary artery bypass surgery cases, consisting of 13 OPCAB cases and 7 on-pump CABG cases. The ECF volume was measured the day before surgery, immediately after surgery, and 2, 4, 6, 8, 12, 24 and 48 hours after surgery, with a bioimpedance analyzer (XITRON 4000 C). ECF volume variation was defined as the difference from the preoperative value divided by body weight, and was expressed in %BW. At the same time, respiratory-index and leukocyte count were measured. The maximum postoperative ECF volume was 3.13 +/- 2.6 %BW in the OPCAB group and 5.36 +/- 2.0 %BW in the on-pump CABG group, that is, significantly higher in the on-pump CABG group. The ECF volume started to increase in the on-pump CABG group immediately after surgery (4.38 +/- 1.8 %BW in the on-pump CABG group and 2.07 +/- 2.4 %BW in the OPCAB group), reaching its peak 6 hours after surgery in the on-pump CABG group and 4 hours after surgery in the OPCAB group. Thereafter, the volume gradually decreased, and 48 hours after surgery the volume decreased in the OPCAB group to 0.064 +/- 1.5 %BW, or to about the same value as the preoperative value, whereas in the on-pump CABG group the volume remained high: 1.9 +/- 2.9 % BW. There was no significant difference between the 2 groups in the change in respiratory-index. The leukocyte count remained significantly higher in the on-pump CABG group. The ECF volume was measured by the bioimpedance measuring method. This is a useful method of measuring the volume non-invasively and continuously. In the OPCAB group, the increase in postoperative ECF volume was less, and recovery to the preoperative level was faster than in the on-pump CABG group.

Aged↗

PubMed2001

Flexible loop of beta 2-glycoprotein I domain V specifically interacts with hydrophobic ligands.

Beta2-glycoprotein I (beta2-GPI), which consists of four complement control protein modules and a distinctly folded fifth C-terminal domain, is an essential cofactor for the binding to phospholipids of anti-cardiolipin antibodies, isolated from patients with anti-phospholipid antibody syndrome, and its fifth domain has attracted attention as a specific phospholipid-binding site. We focused on the fifth domain of beta2-GPI (Domain V) and examined the interaction of intact Domain V, Domains IV-V, and nicked Domain V with various hydrophobic ligands, as a model molecule of phospholipid. We found that electrostatic and hydrophobic interactions are important for Domain V binding to the ligand molecules. We also found that, while Domain IV has no significant effect on the interactions with ligands, the nicked Domain V with cleavage in the flexible loop decreases the affinity, indicating that the flexible loop region is the binding site of the hydrophobic ligands. The synthetic peptide corresponding to the loop region was disordered and interacted with bis-ANS, confirming the critical role of the loop region. To clarify the nature of the interaction between the loop region and hydrophobic compounds, we prepared the reduced and alkylated Domain V, which was denatured but was assumed to be a collapsed state. Alkylation by iodoacetic acid decreased the interaction of Domain V with bis-ANS, probably because the protein net charge was decreased by the six introduced carboxyl groups and consequently the electrostatic interactions were decreased. In contrast, Domain V alkylated by iodoacetamide, therefore retaining a high positive net charge, bound bis-ANS more strongly than the intact Domain V. These results suggested that the interaction of Domain V with hydrophobic compounds through the flexible loop is similar to the binding of hydrophobic compounds to the protein folding intermediate.

Alkylation↗

PubMed2000

Identification of the phospholipid-binding site of human beta(2)-glycoprotein I domain V by heteronuclear magnetic resonance.

To understand the mechanism of the interaction between human beta(2)-glycoprotein I (beta(2)-GPI) and negatively charged phospholipids, we determined the three-dimensional solution structure of the fifth domain of beta(2)-GPI by heteronuclear multidimensional NMR. The results showed that the molecule is composed of well-defined four anti-parallel beta-strands and two short alpha-helices, as well as a long highly flexible loop. Backbone dynamic analysis demonstrated significant mobility of the flexible loop on a subnanosecond time scale. Structural modeling of the nicked fifth domain, in which the Lys317-Thr318 peptide bond was specifically cleaved, revealed the importance of this long C-terminal loop for the interaction between beta(2)-GPI and negatively charged phospholipids. A titration experiment with the anionic surfactant SDS showed that this highly mobile loop, as well as the short beta-hairpin between betaC and betaD strands, which is rich in positively charged residues, specifically interact with the surfactant. The mobile loop, together with the surrounding positively charged residues, probably construct the binding site for negatively charged phospholipids such as cardiolipin.

Amino Acid Sequence↗

PubMed2000

Widespread gene transfection into the central nervous system of primates.

We attempted in vivo gene transfection into the central nervous system (CNS) of non-human primates using the hemagglutinating virus of Japan (HVJ)-AVE liposome, a newly constructed anionic type liposome with a lipid composition similar to that of HIV envelopes and coated by the fusogenic envelope proteins of inactivated HVJ. HVJ-AVE liposomes containing the lacZ gene were applied intrathecally through the cisterna magna of Japanese macaques. Widespread transgene expression was observed mainly in the neurons. The lacZ gene was highly expressed in the medial temporal lobe, brainstem, Purkinje cells of cerebellar vermis and upper cervical cord (29.0 to 59.4% of neurons). Intrastriatal injection of an HVJ-AVE liposome-lacZ complex made a focal transfection around the injection sites up to 15 mm. We conclude that the infusion of HVJ-AVE liposomes into the cerebrospinal fluid (CSF) space is applicable for widespread gene delivery into the CNS of large animals. Gene Therapy (2000) 7, 759-763.

Animals↗

PubMed2000

Inhibitors of protein phosphorylation including the retinoblastoma protein induce germination of Candida albicans.

It has been previously shown that the induction of germination in Candida albicans occurs following its cessation of growth as a yeast. Similarly, mammalian cells undergo a differentiation process that is preceded by a growth cessation associated with a hypophosphorylation of proteins of the retinoblastoma gene family. It is postulated that a similar type of mechanism may be operative in C. albicans and protein phosphorylation inhibitors: forskolin (stimulates cyclic adenosine monophosphate production), okadaic acid (phosphatase inhibitor) and D-erythro-sphingosine (retinoblastoma protein phosphorylation inhibitor) have been used to further strengthen this hypothesis. Okadaic acid (1-1000 nM) and D-erythro-sphingosine (100 microM) significantly inhibited the growth of yeast cells of C. albicans. D-Erythro-sphingosine at 1000 microM was candidicidal. Forskolin did not significantly affect growth. Exponentially grown C. albicans pretreated with forskolin (10 microM), okadaic acid (1000 nM) or D-erythro-sphingosine (100 microM) readily germinated. In comparison, when these inhibitors were incorporated in the same medium, germination of exponentially grown cells did not occur. These results suggest that protein dephosphorylation may be necessary at an early stage of the yeast-hyphae transition in C. albicans.

Candida albicans↗

PubMed1998

Plasmin can reduce the function of human beta2 glycoprotein I by cleaving domain V into a nicked form.

beta2-Glycoprotein I (beta2GPI) is a highly glycosylated plasma protein with the ability to bind negatively charged substances such as DNA, heparin, dextran sulfate, and negatively charged phospholipids. The most relevant physiological role of beta2GPI is supposed to be the regulation of the function of anionic phospholipids like cardiolipin (CL). beta2GPI consists of a single polypeptide chain (326 amino acid residues) with a molecular mass of about 50 kD and with five tandem repeated domains (I, II, III, IV, and V). In the previous study, we found that factor Xa can produce the nicked form by cleaving Lys 317-Thr 318, using recombinant human domain V (r-Domain V). However, the reaction was extremely slow. In the present paper, we found that plasmin can produce the nicked form of domain V, using recombinant domain V (r-Domain V) and beta2GPI from human plasma. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, r-Domain V was rapidly cleaved into a nicked form by plasmin, very slowly by factor Xa, but not by thrombin, tissue-type plasminogen activator, urokinase, and tissue factor/factor VIIa. The cleavage site of r-Domain V and beta2GPI by plasmin was proved to be Lys 317-Thr 318 by amino acid sequence analysis of the digest and of the C-terminal peptide isolated by high-performance liquid chromatography. The cleavage was completely inhibited by plasmin inhibitor (alpha2PI). The nicked form was demonstrated to show reduced affinity for CL with a dissociation constant of one order of magnitude larger than that of the intact beta2GPI. To determine whether the specific cleavage of beta2GPI by plasmin can occur also in plasma, human plasma was first acid-treated to inactivate alpha2PI and then incubated with urokinase. About 12% of beta2GPI in plasma was nicked when alpha2PI activity decreased to 80%. The nicked form was not generated in plasminogen-depleted plasma. These results suggest that plasmin can produce the nicked form of beta2GPI with the reduced ability to bind phospholipids in vivo.

Amino Acid Sequence↗

PubMed1998

Dose-dependent doxycycline-mediated adrenocorticotropic hormone secretion from encapsulated Tet-on proopiomelanocortin Neuro2A cells in the subarachnoid space.

We previously reported that polymer-encapsulated mouse neuroblastoma cells that are capable of secreting beta-endorphin may reduce pain sensitivity in rats after capsule implantation into the cerebrospinal fluid (CSF)-filled subarachnoid space of the spinal cord. The neuroblastoma cells carry the proopiomelanocortin (POMC) gene that encodes the precursor of adrenocorticotropic hormone (ACTH) and beta-endorphin. To control the expression of these hormones in the present study, a promoter that is inducible by administration of tetracycline derivatives such as doxycycline (Dox) was linked to the POMC gene. Encapsulated cells in the CSF space of rats stimulated by four intraperitoneal doses of Dox responded with ACTH expression as determined in a subsequence 36-hr in vitro incubation. The amount of ACTH released was dependent on the in vivo Dox dose. These findings indicate that gene expression in xenogeneic cells in the CSF space can be manipulated by injection of a relatively innocuous drug, and suggest that this system may be applicable to cell transplantation therapy in patients with central nervous system diseases that require temporary control of ligand delivery.

Adrenocorticotropic Hormone↗

PubMed1998

Chain-like conformation of heat-denatured ribonuclease A and cytochrome c as evidenced by solution X-ray scattering.

BACKGROUND: Although the characterization of heat-denatured proteins is essential for understanding the thermodynamic mechanism of protein folding, their structural features are still unclear and controversial. In order to address this problem, we studied the size and shape of the heat-denatured states of bovine ribonuclease A (RNase A) and horse ferricytochrome c (cytochrome c) by solution X-ray scattering. RESULTS: RNase A has four disulfide bonds, whereas cytochrome c, with a covalently bound heme group, has no disulfide bond. Guinier plots show that the heat-denatured RNase A is relatively compact, but the heat-denatured cytochrome c is expanded. On the other hand, the Kratky plots of the two proteins are similar, indicating that the heat-denatured proteins assume a chain-like disordered conformation. The X-ray scattering of RNase A and cytochrome c at various temperatures confirmed that their thermal transitions from a globular native state to a chain-like extended conformation can be approximated well by a two-state transition. CONCLUSIONS: These results indicate that the heat-denatured RNase A and cytochrome c are substantially unfolded according to the criteria of solution X-ray scattering, although the heat-denatured RNase A remains compact because of the presence of the disulfide bonds. The results also confirm that the thermal denaturation occurs cooperatively with the breakdown of secondary and tertiary structure.

Animals↗

PubMed1997

Trifluoroethanol-induced conformational transition of hen egg-white lysozyme studied by small-angle X-ray scattering.

The trifluoroethanol (TFE)-induced conformational transition of hen lysozyme was studied with the combined use of far-UV circular dichroism (CD) and small-angle X-ray scattering. At pH 2.0 and 20 degrees C, the addition of TFE to the native lysozyme induced a cooperative transition to an intermediate state with an increased helical content (TFE state). Small-angle X-ray scattering measurements indicated that the TFE state has a radius of gyration which is 20% larger than that of the native state and assumes a chain-like conformation with some remaining globularity. The TFE-induced transition curves obtained by CD and the small-angle X-ray scattering measurements agreed well, consistent with a two-state transition mechanism. A singular value decomposition analysis of Kratky plots of the small-angle X-ray scattering profiles indicated that two basic scattering functions reproduce the observed spectra, further confirming the validity of a two-state approximation.

Animals↗

PubMed1997

Structure and function of the recombinant fifth domain of human beta 2-glycoprotein I: effects of specific cleavage between Lys77 and Thr78.

In order to elucidate the mechanism of binding of beta 2-glycoprotein I (beta 2-GPI) to cardiolipin (CL), we constructed a high-level expression system for the C-terminal domain (Domain V) of beta 2-GPI using Pichia pastoris and studied its conformation and liposome-binding activity. Purified Domain V was found to have the native disulfide bonds. It had a compactly folded conformation, judging from the circular dichroism spectrum, and exhibited a cooperative unfolding transition induced by pH or urea. Also, it bound liposomes containing CL. Commercially available human beta 2-GPI is known to be selectively cleaved between Lys 317 and Thr 318. We found that bovine factor Xa weakly but specifically cleaves the corresponding site of recombinant Domain V, i.e., the peptide bond between Lys 77 and Thr 78. The conformation of the "nicked" Domain V, which was cleaved at this site, was examined by circular dichroism and fluorescence measurements, and concluded to be similar to that of the intact protein. The stability of the nicked Domain V to urea was slightly lower than that of the intact protein. Although both Domains V bound to liposomes containing CL, the affinity of the nicked Domain V was greatly reduced in comparison with the intact protein, indicating that the cleavage of the peptide bond between Lys 77 and Thr 78 controls the binding to CL. In addition, analysis of the fluorescence spectra in the presence and absence of CL liposomes indicated that Trp 76 is involved in the binding site. These results suggest that the region including Trp 76, Lys 77, and Thr 78 has a critical role in binding to CL.

Amino Acid Sequence↗

PubMed1995

Role of the N- and C-terminal domains of bovine beta 2-glycoprotein I in its interaction with cardiolipin.

beta 2-Glycoprotein I (beta 2-GPI) is a cofactor in the recognition of the phospholipid antigen cardiolipin by anti-cardiolipin antibodies in autoimmune diseases such as systemic lupus erythematosus. We examined the interactions of various forms of bovine beta 2-GPI, such as its intact form, desialylated form (Asialo-beta 2-GPI), N-terminal domain (Domain I), and modified forms of beta 2-GPI and Asialo-beta 2-GPI with nicks in their C-terminal domains, with phospholipid liposomes under different conditions of pH and ionic strength. We found that at neutral pH and low ionic strength, beta 2-GPI became bound to liposome membranes containing cardiolipin, phosphatidylglycerol, phosphatidylserine, phosphatidylserine, phosphatidic acid, or phosphatidylinositol, but not phosphatidylcholine alone. The number of phospholipids involved in the binding seemed to depend on the head group structure of the negatively charged phospholipids, but the dissociation constant did not, being about 10(-8) M, except that for the interaction with phosphatidylinositol, which was one order of magnitude lower. We also found that Domain I and Asialo-beta 2-GPI bound to liposome membranes containing negatively charged phospholipids, and that in the interaction with cardiolipin, their dissociation constants were about 10(-6) and 10(-8) M, respectively. At neutral pH and both low and high ionic strengths, the affinities of the nicked forms of beta 2-GPI and Asialo-beta 2-GPI for cardiolipin were both lower than those of their intact forms but similar to that of Domain I.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Degradation of humoral host defense by Candida albicans proteinase.

The effect of an extracellular proteinase from the pathogenic yeast Candida albicans on the bactericidal and opsonizing activities of human serum was studied. The ability of human polymorphonuclear leukocytes to kill Staphylococcus aureus was greatly reduced when the bacteria were opsonized with human serum treated with the proteinase. The reduction in the opsonizing activity of human serum was attributed to degradation of the Fc portion of immunoglobulin G by the action of C. albicans proteinase as determined by immunoprecipitation reaction. However, the Fab portion of immunoglobulin G was resistant to proteolysis by the proteinase. A clear reduction in the bactericidal activity of human serum against Escherichia coli was observed when the serum was treated with C. albicans proteinase. The reduction of serum bactericidal activity was attributed to the degradation of complement C3 by proteolysis by the proteinase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while C5 resisted the action of the proteinase. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteinase also degrades endogenous proteinase inhibitors, such as alpha 2 macroglobulin and alpha 1 proteinase inhibitor, which are involved in regulating inflammation. These results suggest that destruction of a host's defense-oriented or regulatory proteins facilitates debilitation of the infected host.

Aspartic Acid Endopeptidases↗

PubMed1995

Structure and function of beta 2-glycoprotein I: with special reference to the interaction with phospholipid.

beta 2-Glycoprotein I (beta 2-GPI) is a cofactor in the recognition of a phospholipid antigen, cardiolipin, by anticardiolipin antibodies in autoimmune diseases such as systemic lupus erythematosus. We examined the interaction of various forms of bovine beta 2-GPI, such as its intact form, desialylated form (Asialo beta 2-GPI), N-terminal domain (domain I) and the modified forms of beta 2-GPI and Asialo beta 2-GPI with nicks in their C-terminal domains (domain 5), with phospholipid liposomes under different conditions of pH and ionic strength. We found that at neutral pH and low ionic strength beta 2-GPI bound to liposome membranes containing cardiolipin with a dissociation constant (Kd) of 10(-8) M. Phosphatidylglycerol, phosphatidylserine, phosphatidic acid or phosphatidylinositol bound to beta 2-GPI, but phosphatidylcholine did not. We also found that domain I and Asialo beta 2-GPI bound to cardiolipin with Kd values of 10(-6) and 10(-8) M, respectively. At neutral pH and both low and high ionic strengths, the affinities of nicked forms of beta 2-GPI and Asialo beta 2-GPI for cardiolipin were lower than those of intact forms but similar to that of domain 1.

Amino Acids↗

PubMed1994

Activation of blood clotting factors by microbial proteinases.

There are very few reports on the involvement of bacterial proteinases on the blood clotting system using both human plasma and purified clotting factors. We studied whether microbial proteinases from the opportunistic pathogens Candida albicans, Pseudomonas aeruginosa and Serratia marcescens activate the blood clotting cascade by using normal human plasma, human plasmas deficient in clotting factor XII or X, and also by using purified clotting factors XII, X and prothrombin. All proteinases tested activated either clotting factor XII or prothrombin in vitro, thus resulting in generation of thrombin. Clotting factor X was converted to the active form (Xa) by both Candida and Pseudomonas proteinases, but not by Serratia proteinase. These results suggest that peripheral and systemic blood circulation may be impaired by activation of the blood clotting cascade by microbial infections, especially in septic patients, which would enhance disseminated intravascular coagulation and multi-organ failure.

Amino Acid Sequence↗

PubMed1994

Comparison of the conformational stability of the molten globule and native states of horse cytochrome c. Effects of acetylation, heat, urea and guanidine-hydrochloride.

The molten globule state has been assumed to be a major intermediate of protein folding. We compared the stability of the native and acidic molten globule states of horse ferricytochrome c against heat, urea and guanidine hydrochloride (Gdn-HCl) using the intact species and species modified by various degrees of acetylation of the lysyl epsilon-amino groups. After acetylation, the amino groups cannot protonate at acidic pH. Thermal and urea-induced unfolding transitions measured by far-UV circular dichroism and differential scanning calorimetry showed that, whereas acetylation stabilizes the molten globule state at pH 2, it destabilizes the native state at pH 7, suggesting a difference in their mechanisms of conformational stability. On the other hand, the effects of Gdn-Hcl were remarkable. Contrary to what was expected from the thermal and urea-induced unfolding transitions, the Gdn-HCl-induced unfolding transition of the native state at pH 7 was insensitive to the extent of acetylation. At pH 2, Gdn-HCl at low concentrations stabilized the molten globule state and, at high concentrations, destabilized it. Consideration of the difference in the effects of Gdn-HCl from those of urea or heat indicated that, whereas the net positive charge repulsion destabilizes the molten globule state at pH 2, the local negative charge repulsion produced by acetylation of amino groups, and not the net charge, critically destabilizes the native state at pH 7. These results predict that, because of its ionic nature, Gdn-HCl will produce substantially different effects on the conformational states of some proteins compared with those of urea.

Acetylation↗

PubMed1994

Thermal unfolding of tetrameric melittin: comparison with the molten globule state of cytochrome c.

Whereas melittin at micromolar concentrations is unfolded under conditions of low salt at neutral pH, it transforms to a tetrameric alpha-helical structure under several conditions, such as high peptide concentration, high anion concentration, or alkaline pH. The anion- and pH-dependent stabilization of the tetrameric structure is similar to that of the molten globule state of several acid-denatured proteins, suggesting that tetrameric melittin might be a state similar to the molten globule state. To test this possibility, we studied the thermal unfolding of tetrameric melittin using far-UV CD and differential scanning calorimetry. The latter technique revealed a broad but distinct heat absorption peak. The heat absorption curves were consistent with the unfolding transition observed by CD and were explainable by a 2-state transition mechanism between the tetrameric alpha-helical state and the monomeric unfolded state. From the peptide or salt-concentration dependence of unfolding, the heat capacity change upon unfolding was estimated to be 5 kJ (mol of tetramer)-1 K-1 at 30 degrees C and decreased with increasing temperature. The observed change in heat capacity was much smaller than that predicted from the crystallographic structure (9.2 kJ (mol of tetramer)-1 K-1), suggesting that the hydrophobic residues of tetrameric melittin in solution are exposed in comparison with the crystallographic structure. However, the results also indicate that the structure is more ordered than that of a typical molten globule state. We consider that the conformation is intermediate between the molten globule state and the native state of globular proteins.

Anilino Naphthalenesulfonates↗

PubMed1994

The relationship between the glucose uptake system and growth cessation in Candida albicans.

It is thought that dimorphic Candida albicans undergoes changes in its intracellular metabolic state prior to yeast-mycelial transformation. Cells grown in budding form to mid-exponential phase could not be induced to form germ tubes when grown in glucose medium. However, cells in which growth was initially inhibited by either starvation or inhibitors (0.1% hydroxyurea, 4% sodium malonate or 4% 2-deoxy-D-glucose) could be induced to form germ tubes in the same medium. The effects of these initial treatments on the intracellular state in mid-exponential phase cells were analysed by measuring the kinetics of D-glucose uptake. D-glucose uptake in mid-exponential phase and stationary phase cells was measured. The untreated mid-exponential phase cells exhibited only a high Km (6.9 mM). However, mid-exponential phase cells, in which growth was initially inhibited, exhibited both a high Km (3.2-6.2 mM) and a low Km (0.40-0.78 mM) simultaneously. In addition, the stationary phase cells exhibited both a high Km (5.6 mM) and a low Km (0.56 mM). These results suggest that there are two kinetically distinct systems of glucose transport in C. albicans and that changes in the glucose uptake system in C. albicans may be related to intracellular changes prior to transition from the budding to the mycelial form.

Candida albicans↗

PubMed1993

Vascular permeability enhancing activity of Porphyromonas gingivalis protease in guinea pigs.

Porphyromonas gingivalis protease, which had been isolated from a culture supernatant, caused vascular permeability enhancement in a dose-dependent manner when injected into guinea pig skin. The permeability-enhancing reaction caused by the protease was not affected by treatment with antihistamine, but was greatly augmented by simultaneous injection of a kinin potentiator, carboxypeptidase N inhibitor. However, the reaction was inhibited by soybean trypsin inhibitor or alpha 2-antiplasmin, although both of these inhibitors could not inhibit P. gingivalis protease at all by themselves. A bradykinin-degrading enzyme, carboxypeptidase B, weakened this vascular reaction. Results described indicate that the permeability-enhancing reaction induced by the protease is caused by activation, of the kallikrein-kinin cascade in the tissue.

Animals↗