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Biomedical subjects

M Kikuchi

Publications and source records attributed to M Kikuchi.

At least 469 records · Page 26Linked to original sources
PubMed1996

[Successful treatment with combination of all-trans retinoic acid (ATRA) and rhG-CSF a relapsed acute promyelocytic leukemia with umbilical tumor].

A 48-year-old female was admitted to our hospital because of pancytopenia and pneumonia in February, 1993. The increase of abnormal promyelocytes with t (15; 17) and PML-RAR mRNA was detected in bone marrow aspirate and a diagnosis of acute promyelocytic leukemia was made. She obtained complete remission after the administration of all-trans retinoic acid (ATRA) and following chemotherapy. Then she received peripheral blood stem cell transplantation in September, 1993. However she noticed a umbilical tumor in June, 1995. Abnormal promyelocytes were demonstrated not only in bone marrow aspirate but also in the umbilical tumor. Because of the poor response to ATRA and development of fever, a side effect of ATRA, G-CSF and prednisolone were administrated together with ATRA. After the combined therapy, umbilical tumor disappeared and she obtained complete remission again. These findings suggest that combined therapy of ATRA and G-CSF is effective to the low responder to ATRA and that combined use of ATRA and prednisolone reduces the side effect of ATRA without diminishing the favorable effect on differentiation.

Drug Therapy, Combination↗
PubMed1996

Cytotoxicity of synthetic barium hydroxyapatite.

Barium hydroxyapatite (Ba10(PO4)6(OH)2, Ba-HAp) was synthesized by a wet method using Ba(OH)2.8H2O and (NH4)2HPO4 as starting materials. The Ba-HAp obtained had a Ba/P atomic ratio of 1.76 and contained CO3 groups. The Ba-HAp was sintered at 1073 K for 12 hours. The sintered Ba-HAp had a three point bending strength of 29 MPa and Young's modulus of 27 GPa. Cytotoxicity of the sintered bodies and particles was tested using L-cells. The sintered Ba-HAp showed no cytotoxicity, and the cells were closely in contact with the surfaces of sintered Ba-HAp. Morphological observation of the cell around the Ba-HAp particles also showed no cytotoxicity. However, cell growth was inhibited by Ca adsorption on the Ba-HAp particles. These results suggested that the Ba-HAp had no cytotoxicity and can be applied as a bioactive X-ray opaque material.

Adsorption↗
PubMed1995

Protein disulfide isomerase mutant lacking its isomerase activity accelerates protein folding in the cell.

We investigated the effect of protein disulfide isomerase (PDI) on in vivo protein folding of human lysozyme (h-LZM) in a specially constructed yeast coexpression system. Coexpression with PDI increased the amounts of intracellular h-LZM with the native conformation, leading to an increase in h-LZM secretion. The results indicated that PDI is a real catalyst of protein folding in the cell. The secretion of h-LZM increased even when both active sites of PDI were disrupted, suggesting that the effect of PDI resulted from a function other than the formation of disulfide bonds. This is the first finding that PDI without isomerase activity accelerates protein folding in vivo.

Base Sequence↗
PubMed1995

Contribution of hydrophobic residues to the stability of human lysozyme: calorimetric studies and X-ray structural analysis of the five isoleucine to valine mutants.

In order to understand the contribution of hydrophobic residues to the conformational stability of human lysozyme, five Ile mutants (Ile --> Val) in the interior of the protein were constructed. The thermodynamic parameters characterizing the denaturation of these mutant proteins were determined by scanning calorimetry, and the three-dimensional structure of each mutant protein was solved at high resolution by X-ray crystallography. The thermodynamic analyses at 64.9 degrees C and at pH 2.7 revealed the following. (1) The stabilities of all the mutant proteins were decreased as compared with that of the wild-type protein. (2) The changes in the calorimetric enthalpies were larger than those in the Gibbs energies, and were compensated by entropy changes. (3) The destabilization mechanism of the mutant proteins differs, depending on the location of the mutation sites. X-ray analyses showed that the overall structures of all the mutant human lysozymes examined were identical to that of the wild-type protein, and only small structural rearrangements were observed locally around some of the mutation sites. The most striking change among the mutant proteins was found in the mutant protein, 159V, which contains a new water molecule in the cavity created by the mutation. The thermodynamic stabilities of the mutant proteins are discussed in light of the high-resolution X-ray structures of the wild-type and five mutant human lysozymes examined.

Calorimetry, Differential Scanning↗
PubMed1995

Construction of a divalent cell adhesive lysozyme by introducing the Arg-Gly-Asp sequence at two sites.

To increase the cell adhesion activity of 74RGD4, an RGDS-inserted mutant between Val74 and Asn75 of human lysozyme, one more site for the RGD introduction was investigated in the lysozyme molecule. We found that 47RGD4 with RGDS in place of AGDR (residues 47 to 50) in a beta-turn region possesses the same level of adhesion activity as that of 74RGD4. The acceptance of the RGD introduction in the beta-turn region of human lysozyme is in good agreement with recent studies on the functional conformation of RGD. We constructed (47,74)RGD4, a mutant containing RGD at two sites, by combining the N-terminal domain of 47RGD4 and the C-terminal domain of 74RGD4. The (47,74)RGD4 lysozyme, with two functional RGD sequences, exhibits even higher cell adhesion activity than that of 74RGD4 or 47RGD4.

Animals↗
PubMed1995

Cloning and sequencing of the cDNA encoding human P5.

The cDNA encoding human P5 was cloned and sequenced. The predicted 440-amino-acid (aa) sequence of human P5 contains two thioredoxin-like domains, which are also found in members of the protein disulfide isomerase superfamily. The human and hamster P5 genes reveal 87 and 93% similarity in their nucleotide and deduced aa sequences, respectively.

Amino Acid Sequence↗
PubMed1995

Molecular cloning of the cDNA encoding a novel protein disulfide isomerase-related protein (PDIR).

We isolated the cDNA of a novel protein disulfide isomerase (PDI)-related protein, designated PDIR, from a human placental cDNA library. Deduced from its nucleotide sequence, PDIR has the three CXXC-like motifs (Cys-Ser-Met-Cys, Cys-Gly-His-Cys and Cys-Pro-His-Cys), which are found in proteins within the PDI superfamily and are responsible for oxidoreductase activity. PDIR has a hydrophobic stretch at its amino terminus, which may serve as a signal sequence, and the putative endoplasmic reticulum (ER) retention signal 'Lys-Glu-Glu-Leu' at its carboxy terminus, indicating that PDIR is an ER resident protein. Northern blots showed that PDIR is preferentially expressed in cells actively secreting proteins and that the expression of PDIR is stress-inducible. These results suggested that PDIR has oxidoreductase activity of disulfide bonds against polypeptides and that it acts as a catalyst of protein folding in the lumen of the ER.

Amino Acid Sequence↗
PubMed1995

Inhibition of pancreatic beta-cell glucokinase by antisense RNA expression in transgenic mice: mouse strain-dependent alteration of glucose tolerance.

We have generated transgenic mice, in either C57BL/6 or C3H background, expressing antisense glucokinase mRNA in beta-cells. The glucose phosphorylating activity at 60 mM glucose in transgenic islets was significantly lower than that in controls, and the insulin secretory response to glucose was lower in transgenic islets than in those of controls in both strains. Following i.p. glucose challenge, higher blood glucose levels were observed in transgenic mice than in controls in the C57BL/6 but not the C3H background. These data suggest that a beta-cell secretory defect, in combination with other undefined genetic factors, causes impaired glucose homeostasis in mice.

Animals↗
PubMed1995

Structure of a glutathionylated human lysozyme: a folding intermediate mimic in the formation of a disulfide bond.

The three-dimensional structure of a mutant human lysozyme, C77A-a, in which the residue Cys77 is replaced by alanine, has been refined to an R value of 0.125 using 8230 reflections in the resolution range 10.0-1.8 A. It has been shown that C77A-a, in which the counterpart of Cys77 (Cys95) is modified with glutathione, has been shown to mimic an intermediate in the formation of the disulfide bond Cys77-Cys95 during the folding of human lysozyme [Hayano, Inaka, Otsu, Taniyama, Miki, Matsushima & Kikuchi (1993). FEBS Lett. 328, 203-208]. An earlier structure demonstrates that its overall structure is essentially identical to that of the wild-type protein and served as the starting model. The refined model includes atoms for all protein residues (1-130), 20 glutathione atoms and 113 water atoms. Further refinement shows more clearly the details of the protein, the bound glutathione molecule and solvent structure. However, the main-chain folding and the atomic thermal factors of the loop region from Thr70 to Leu79 were highly affected by the binding of the glutathione molecule, as compared with those of the wild-type protein. The bound glutathione shifted the main-chain atoms from Va174 to Ala77 by more than 6.0 A, and the temperature factors of the atoms in the loop region were quite high (more than 40 A(2)), indicating that the backbone conformation of this region is highly flexible and that the loop region is not folded in the specific conformation observed in the wild-type protein. These results strongly suggest that the loop structure in human lysozyme is folded later than the other regions of the protein in vivo, as observed in in vitro folding. Since the bound glutathione is efficiently and irreversibly dissociated by protein disulfide isomerase, the glutathione molecule may act as a protecting group to prevent the formation of an incorrect disulfide bond in the protein folding process in vivo.

Journal Article↗
PubMed1995

DsbA-DsbB interaction through their active site cysteines. Evidence from an odd cysteine mutant of DsbA.

Formation of disulfide bonds in Escherichia coli envelope proteins is facilitated by the Dsb system, which is thought to consist of at least two components, a periplasmic soluble enzyme (DsbA) and a membrane-bound factor (DsbB). Although it is believed that DsbA directly oxidizes substrate cysteines and DsbB reoxidizes DsbA to allow multiple rounds of reactions, direct evidence for the DsbA-DsbB interaction has been lacking. We examined intracellular activities of mutant forms of DsbA, DsbA30S and DsbA33S, in which one of its active site cysteines (Cys30 or Cys33, respectively) has been replaced by serine. The DsbA33S protein was found to dominantly interfere with the disulfide bonds formation and to form intermolecular disulfide bonds with numerous other proteins when cells were grown in media containing low molecular weight disulfides such as GSSG. In the absence of added GSSG, DsbA33S protein remained specifically disulfide-bonded with DsbB. These in vivo results not only confirm the previous findings that Cys30 of DsbA is hyper-reactive in vitro but provide evidence that DsbA indeed interacts selectively with DsbB. We propose that the Cys30-mediated DsbA-DsbB complex represents an intermediate state of DsbA-DsbB recycling reaction that has been fixed because of the absence of Cys33 on DsbA.

Amino Acid Sequence↗
PubMed1995

Human and viral interleukin-10 in Hodgkin's disease, and its influence on CD4+ and CD8+ T lymphocytes.

The Epstein-Barr virus (EBV) is closely related to Hodgkin's disease (HD), while the BCRF-I (viral [v] IL-10) gene of the EBV is highly homologous to the human interleukin-10 (h IL-10) gene. To investigate the relationship of IL-10 and HD, we performed both immunostaining and in situ hybridization (ISH) in 30 cases of HD. The presence of EBV in Hodgkin (H) and Reed-Sternberg (RS) cells was seen in 16 of the 30 cases, by ISH of the EBV EBER-I region and/or immunostaining of latent membrane protein (LMP-I). Of the 16 EBV-positive cases, 12 also showed IL-10 antigen (Ag) in H and RS cells by immunostaining, 5 of the 16 demonstrated hIL-10 RNA by ISH and 14 of the 16 showed vIL-10 RNA. But only 2 of the 14 EBV-negative cases showed IL-10 Ag, and one of them showed hIL-10 RNA, while none demonstrated vIL-10 RNA. The T cells in the HD-involved tissues were found to be mainly CD4-positive T cells, and had no association with EBV infection. However, the lymphocytes surrounding H and RS cells were more frequently CD4 cells and rarely CD8 cells in the EBV-positive cases, in contrast with the EBV-negative cases. The above results indicate that an EBV infection influenced both cytokine synthesis and the response of T cells in HD.

CD4-Positive T-Lymphocytes↗
PubMed1995

A possible role of ER-60 protease in the degradation of misfolded proteins in the endoplasmic reticulum.

Wild-type human lysozyme (hLZM) is secreted when expressed in mouse L cells, whereas misfolded mutant hLZMs are retained and eventually degraded in a pre-Golgi compartment (Omura, F., Otsu, M., Yoshimori, T., Tashiro, Y., and Kikuchi, M. (1992) Eur. J. Biochem. 210, 591-599). These misfolded mutant hLZMs are associated with protein disulfide isomerase (Otsu, M., Omura, F., Yoshimori, T., and Kikuchi, M. (1994) J. Biol. Chem. 269, 6874-6877). From the observation that this degradation is sensitive to cysteine protease inhibitors, such as N-acetyl-leucyl-leucyl-norleucinal and N-acetyl-leucyl-leucyl-methioninal, but not to the serine protease inhibitors, 1-chloro-3-tosylamido-7-amino-2-heptanone and (p-amidinophenyl)methanesulfonyl fluoride, it was suggested that some cysteine proteases are likely responsible for the degradation of abnormal proteins in the endoplasmic reticulum (ER). ER-60 protease (ER-60), an ER resident protein with cysteine protease activity (Urade, R., Nasu, M., Moriyama, T., Wada, K., and Kito, M. (1992) J. Biol. Chem. 267, 15152-15159), was found to associate with misfolded hLZMs, but not with the wild-type protein, in mouse L cells. Furthermore, denatured hLZM is degraded by ER-60 in vitro, whereas native hLZM is not. These results suggest that ER-60 could be a component of the proteolytic machinery for the degradation of misfolded mutant hLZMs in the ER.

Animals↗
PubMed1995

O-glycosylation of the Thr70 residue of cell-adhesive lysozyme in yeast.

The cell-adhesive protein Cys-RGD4 has been constructed using a yeast expression system by inserting the sequence Cys-Arg-Gly-Asp-Ser-Cys (CRGDSC) between Val74 and Asn75 of human lysozyme [Yamada, T., Uyeda, A., Kidera, A. & Kikuchi, M. (1994b) Biochemistry 33, 11678-11683]. The Cys74a, Arg74b, Gly74c, Asp74d, Ser74e, Cys74f-lysozyme mutant, purified from the yeast culture supernatant contained glycosylated variants, in addition to the unglycosylated form. Peptide mapping analyses suggested that the glycosylation occurred at the Thr70 residue in the Cys-RGD4 molecule. Electrospray ionization mass spectrometric analysis demonstrated the presence of two hexose residues in the major variant, and one, three, four, or five hexose residues in the minor variants. All of these hexose residues were identified as mannose by analysis of the oligosaccharide mixture obtained by mild alkaline treatment of the variants. No other glycosylation was observed, although the Cys-RGD4 molecule possesses a total of 12 threonine and serine residues. In addition, the Thr70 residue is not glycosylated in either native lysozyme or the Arg-Gly-Asp-Ser (RGDS)-inserted mutant, RGD4 [Yamada, T., Matsushima, M., Inaka, K., Ohkubo, T., Uyeda, A., Maeda, T., Titani, K., Sekiguchi, K. & Kikuchi, M. (1993) J. Biol. Chem. 268, 10588-10592]. Thus, this O-glycosylation seems to be specific for both the mutant lysozyme molecule and the site of the threonine residue. Structural analyses of these lysozymes by X-ray crystallography suggest that the conformation of the serine-containing or threonine-containing region can affect the specificity of yeast O-glycosylation.

Amino Acid Sequence↗
PubMed1995

Peptide ligands for integrin alpha v beta 3 selected from random phage display libraries.

The integrin alpha v beta 3 binds promiscuously to cell-adhesive proteins: vitronectin, fibronectin, and several others containing the RGD motif. We have explored molecular recognition by alpha v beta 3 through selection of ligands from large random libraries of peptides displayed on phage. Ligands bound by alpha beta 3 consisted primarily of RGD peptides; however, these peptides showed considerable heterogeneity with respect to the identities of amino acids flanking RGD. The tolerance of alpha v beta 3 for RGD peptides of diverse composition is consistent with its role in vivo as a versatile receptor for RGD-containing extracellular matrix proteins. Peptide ligands for alpha v beta 3 also included a novel binding sequence, identical to a tetrapeptide found in vitronectin, which is a candidate for a synergistic site in this adhesive protein that may act in concert with RGD to promote molecular recognition.

Amino Acid Sequence↗
PubMed1995

Structure of a conformationally constrained Arg-Gly-Asp sequence inserted into human lysozyme.

To examine the effect of a conformational constraint introduced into the Arg-Gly-Asp (RGD) sequence on cell adhesion activity, we constructed a mutant protein by inserting an RGD-containing sequence flanked by two Cys residues between Val74 and Asn75 of human lysozyme. The CRGDSC-inserted lysozyme was expressed in yeast, purified, and designated as Cys-RGD4. Using baby hamster kidney cells, Cys-RGD4 was shown to possess even higher cell adhesion activity than that of the RGDS-inserted lysozyme, RGD4. The Cys-RGD4 protein was co-crystallized with a lysozyme inhibitor, tri-N-acetylchitotriose, and the three-dimensional structure was determined at 1.6-A resolution by x-ray crystallography. In contrast to RGD4, the inserted RGD-containing region of Cys-RGD4 was well defined. The structural analysis revealed that the two inserted Cys residues form a new disulfide bond in Cys-RGD4, as expected, and that the RGD region assumes a type II' beta-turn conformation of Gly-Asp with a hydrogen bond between the C = O of Arg and the H-N of Ser. In addition, it was confirmed that two more hydrogen bonds are present in the RGD region of the Cys-RGD4 lysozyme. These results suggest that the conformation of the RGD-containing region is rigid and stable in the Cys-RGD4 molecule and that the type II' beta-turn structure of RGD is essential for binding to integrins with high affinity.

Asparagine↗
PubMed1995

A novel isoform of syntaxin-binding protein homologous to yeast Sec1 expressed ubiquitously in mammalian cells.

munc-18/n-Sec1/rbSec1, a brain homologue of the yeast Sec1p protein, is thought to participate in regulating the docking and fusion of synaptic vesicles. We have screened the mouse cDNA library of an MIN6 cell line, derived from pancreatic beta cells, for its novel isoform and have identified a cDNA encoding a 593-amino acid protein having 63, 53, and 30% identity with munc-18/n-Sec1/rbSec1, Caenorhabditis elegans unc18, and Saccharomyces cerevisiae Sec1p, respectively. While munc-18/n-Sec1/rbSec1 expression has been reported to be neural-specific, RNA blot analysis has revealed that the novel isoform, which we refer to as muSec1 (mammalian ubiquitous Sec1), is expressed ubiquitously. We have also identified mouse munc-18/n-Sec1/rbSec1 from the MIN6 cDNA library, indicating that different isoforms of a protein participating in vesicular transport exist in a single cell. muSec1 bound to glutathione S-transferase-syntaxin 1A and, although with lower affinity, to glutathione S-transferase-syntaxin 4 fusion protein. These findings suggest that muSec1 is, via its binding to the syntaxin family, involved in the protein trafficking from the Golgi apparatus to the plasma membrane and that the fundamental mechanisms of protein trafficking have been conserved from yeast through virtually all mammalian cells.

Amino Acid Sequence↗
PubMed1995

Structural and functional significance of cysteine residues of glutathione-independent prostaglandin D synthase. Identification of Cys65 as an essential thiol.

Glutathione-independent prostaglandin D synthase in rat brain is composed of 189 amino acid residues and catalyzes the isomerization of prostaglandin H2 to prostaglandin D2, an endogenous sleep-promoting substance. This enzyme is the only enzyme among members of the lipocalin superfamily composed of various secretory lipophilic ligand-carrier proteins and is recently identified to be a beta-trace protein, a major constituent of human cerebrospinal fluid. We expressed the active enzyme in Escherichia coli and then systematically substituted all cysteine residues of the delta 1-29 enzyme at positions of 65, 89, and 186 with alanine or serine. The parent and mutant enzymes were purified to apparent homogeneity with a recovery of approximately 30% by chromatography with Sephadex G-50 and S-Sepharose, by which all the enzymes showed identical elution profiles. The purified enzymes, irrespective of the mutation, showed almost the same circular dichroism spectral characteristics as displayed by a highly ordered beta-structure. The recombinant enzymes containing Cys65 showed the activity comparable with that of the enzyme purified from rat brain (approximately 3 mumol/min/mg of protein) in the presence, but not in the absence, of sulfhydryl compounds. However, all of the single, double, and triple mutants without Cys65 lost the enzyme activity. The purified delta 1-29 Ala89,186 enzyme was inactivated reversibly by conjugation with glutathione at Cys65 and irreversibly by the stoichiometric chemical modification with N-ethylmaleimide. These results indicate that Cys65 is an essential thiol of the enzyme and that both the intrinsic and extrinsic sulfhydryl groups are necessary for nonoxidative rearrangement of 9,11-endoperoxide of prostaglandin H2 to produce prostaglandin D2 catalyzed by the enzyme.

Amino Acid Sequence↗