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S Kawabata

Publications and source records attributed to S Kawabata.

At least 109 records · Page 6Linked to original sources
PubMed1997

Oral immunization with simian immunodeficiency virus p55gag and cholera toxin elicits both mucosal IgA and systemic IgG immune responses in nonhuman primates.

Rhesus macaques were orally immunized with a mucosal vaccine consisting of two different concentrations (1 mg vs 250 microg) of recombinant SIV p55gag (p55) with or without cholera toxin (CT, 50 microg) as a mucosal adjuvant. The plasma from macaques receiving the higher dose of p55 (1 mg) and CT had higher p55-specific IgG and IgA Ab titers compared with macaques that received the lower dose of p55 (250 microg) and CT. Further, high levels of p55-specific IgG and IgA Abs were present in external secretions from both groups. The level of p55-induced T cell responses was elevated in PBMCs isolated from the high dose group compared with the low dose group. When culture supernatants from these p55-stimulated PBMCs were examined for Th1 (IFN-gamma) and Th2 (IL-4 and IL-10) cytokines, both IFN-gamma and IL-10 were present, but IL-4 was absent. CD4+ T cells isolated from these p55-stimulated PBMCs contained IFN-gamma spot-forming cells (SFCs) but not IL-4 SFCs. These results were further confirmed by cytokine-specific reverse transcriptase PCR analysis, where p55-specific CD4+ T cells expressed mRNA for IFN-gamma, IL-6, and IL-10, but not IL-4. These findings suggest that oral immunization of nonhuman primates induced both IFN-gamma-secreting Th1 and select Th2 cytokine (e.g., IL-6 and IL-10)-producing CD4+ Th cells, which accounted for the generation of p55-specific systemic and mucosal Ab responses.

Administration, Oral↗
PubMed1997

Purification and characterization of prophenoloxidase from the hemolymph of coleopteran insect, Holotrichia diomphalia larvae.

Prophenoloxidase (pro-PO), a precursor of phenol oxidase (PO), was purified from the hemolymph of coleopteran Holotrichia diomphalia larvae. The enzyme was purified to apparent homogeneity, in six steps of chromatography, using Sephadex G-100, CM-52, Dextran-sulfate Sepharose CL-6B, Phenyl Sepharose CL-4B, Sephacryl S-200, and a Mono-Q column. The preparation exhibited a single band on SDS-PAGE. The proenzyme had a molecular weight of 158 kDa, as estimated by gel filtration. On SDS-PAGE under reducing conditions, it gave a 79 kDa band, indicating that it forms a dimer of the 79 kDa protein. On the other hand, the purified pro-PO gave two well-separated peaks, named pro-PO-1 and pro-PO-2, on reverse-phase HPLC. Amino acid compositions of both proteins were indistinguishable, thereby suggesting the presence of an allelic variant or an isoprotein. On dextran-sulfate Sepharose CL-6B chromatography, a fraction containing prophenoloxidase activating enzyme(s) (PPAE fraction), free from pro-PO, was also separated. In reconstitution experiments, the activation of purified pro-PO by PPAE fraction, was observed in the presence of 5 mM Ca2+, with a specific limited proteolysis. The NH2-terminal sequence of generated PO was determined to be NH2-Phe-Gly-Glu-Asp-Asp-. The activated PO oxidized o-diphenols but did not oxidize mono-phenol and p-diphenol substrates. The purified pro-PO was not activated by trypsin, alpha-chymotrypsin, and SDS.

Amino Acid Sequence↗
PubMed1997

Analysis of glycosidic linkages in saccharide compounds by post-source decay fragment methods in matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy.

Maltotriosyl- and panosyl-alpha-cyclodextrins and the nonaose of pullulan were analyzed by post-source decay (PSD) fragment methods of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy. By the mass number analysis, it was found that all of the PSD fragment ions were produced by cleavages of glycosidic linkages. Comparison of the relative intensities of the ions in those compounds enabled us to distinguish two kinds of glycosidic linkages, alpha 1-4 and alpha 1-6, by MALDI-TOFMS with a new type of ion reflector: the curved field reflectron.

Carbohydrate Sequence↗
PubMed1997

Biochemical and physicochemical characterization of normal and variant forms of human MTH1 protein with antimutagenic activity.

8-Oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is produced during cellular metabolism, and its misincorporation into DNA causes mutation. Human cells possess an enzyme that hydrolyzes 8-oxo-dGTP to the corresponding nucleoside monophosphate, thereby preventing misincorporation of 8-oxo-7,8-dihydroguanine into DNA. Sequence analyses of the MTH1 gene, encoding the 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphatase (8-oxo-dGTPase) protein in human cell lines revealed that a G to A base substitution frequently occurs at codon 83, which causes a change of valine to methionine in the MTH1 protein [Wu, C. et al., Biochem. Biophys. Res. Commun. 214 (1995) 1239-1245]. Here we isolated cDNAs for the two types of MTH1 protein and expressed them in Escherichia coli mutT-. cells, devoid of their own 8-oxo-dGTPase activity. The two forms of proteins were purified to physical homogeneity, and amino acid analyses confirmed that the variant protein, Met83-MTH1, indeed carries the corresponding amino acid substitution. Met83-MTH1, but not normal type Val83-MTH1, was separated into two peaks in hydrophobic interacting chromatography. 8-Oxo-dGTPase activity of Met83-MTH1 is more thermolabile than that of Val83-MTH1. Circular dichroism (CD) and fluorescence spectroscopic analyses confirmed this conclusion. CD further indicated that Met83-MTH1 has a higher alpha-helix content.

Amino Acid Substitution↗
PubMed1997

Tissue distribution and subcellular localization of rabbit liver metalloendopeptidase.

We have previously isolated rabbit liver microsomal metalloendopeptidase (MEP) as a candidate for the processing enzyme of vitamin K-dependent plasma proteins. A cDNA coding for MEP has revealed that it is structurally related to metalloendopeptidase-24.15, which catalyzes the proteolytic processing of several bioactive peptides. In this study we examined the tissue distribution and subcellular localization of MEP by light and electron microscopic immunohistochemical methods, in addition to Northern blot analysis. Chicken polyclonal antibodies were raised by using synthetic peptides AG1 (Met31-Asn46) and AG3 (Asp537-Gly551) derived from the sequence of MEP. Both anti-AG1 and anti-AG3 antibodies reacted specifically with MEP, as judged by Western blotting and immunohistochemical methods. Both antibodies gave an identical staining distribution, which was localized on the luminal cell surfaces and in the cytoplasm of the following organs: liver, brain, lungs, kidneys, esophagus, stomach, duodenum, pancreas, placenta, epididymis, uterus, ovary, and oviduct. Northern blot analysis revealed that the expression of MEP mRNA is similar to its immunohistochemical distribution except in the heart. These results suggest that MEP may participate more closely in a degradation role in peptide metabolism in various tissues than in a processing role of the proprotein, like metalloendopeptidase-24.15.

Amino Acid Sequence↗
PubMed1997

Elevated serum lipoprotein (a) levels associated with ulcerative colitis in a young Japanese patient.

Thromboembolism has been shown to play a role in the pathogenesis of inflammatory bowel disease (IBD). A possibility exists that lipoprotein (a) [Lp(a)], a newly-discovered prothrombotic factor, also participates in the development of at least some cases of IBD. Marked elevation of serum Lp(a) levels was observed in a young patient with ulcerative colitis. A biopsy specimen of the rectal mucosa showed findings compatible with ulcerative colitis, as well as small vessel thrombus occurring within the muscularis mucosa in the rectum. Serum Lp(a) levels were markedly elevated on admission (71 mg/dl), with a gradual decrease to 46 mg/dl on discharge. Moreover, serum Lp(a) levels decreased in parallel with clinical improvement. In the quiescent clinical stage, no small vessel thrombus was observed in the mucosa on follow-up colonoscopy. The association between IBD and hyper-Lp(a)-emia would be presumable but it has been, to our knowledge, previously unreported. The case reported here would be the first young patient, suggesting the presence of hyper-Lp(a)-emia and small vessel thrombus formation occurring in association with the development of ulcerative colitis.

Adult↗
PubMed1997

[Acute eosinophilic pneumonia induced by cigarette smoke].

An 18-year-old man was admitted to our hospital because of a dry cough and high fever of acute onset. A chest radiograph revealed diffuse bilateral infiltrates, mainly in peripheral lung zones. Laboratory data showed hypoxemia and leukocytosis, and no eosinophilia. The diagnosis was acute eosinophilic pneumonia, and was based on examination of a transbronchial lung biopsy specimen and on the clinical course. The patient was treated with a corticosteroid and responded quickly. We suspected that the disease was related to smoking because the patient had started smoking two days before the onset of the symptoms. Therefore, a challenge test was done in which the patient smoked cigarettes. By 15 hours after the challenge, he had become severely hypoxemic and by 21 hours after the challenge his pulmonary function had decreased. In this patient, smoking cigarettes appeared to have induced acute eosinophilic pneumonia.

Acute Disease↗
PubMed1997

Functional gamma delta T-lymphocyte defect associated with human immunodeficiency virus infections.

BACKGROUND: Antiviral cellular immune responses may influence immunological homeostasis in HIV-infected persons. Recent data indicate that V gamma 9/V delta 2 T lymphocytes display potent cytotoxic activities against human cells infected with certain viruses including HIV. Understanding the role of gamma delta T cells in the course of HIV infection may be helpful for designing novel treatment strategies for HIV-associated disorders. MATERIALS AND METHODS: The constitutive recognition of Daudi cells and monoethyl pyrophosphate (Etpp) by peripheral blood V gamma 9/V delta 2 T cells was assessed using a proliferation assay. The cytotoxicity of Daudi-stimulated lymphocyte populations was measured by chromium release assays. The HIV infectivity for gamma delta T cell clones was determined by measuring the levels of HIV p24 in cell supernatants. The effect of in vitro HIV-infection on cytokine mRNA production by gamma delta T cell clones was assessed by PCR. RESULTS: The constitutive proliferative responses of peripheral blood V gamma 9/V delta 2 T cells and the lytic functions of Daudi-expanded lymphoid cells from HIV+ persons were substantially diminished in comparison with those of HIV-seronegative persons. These alterations were present in asymptomatic HIV+ persons prior to substantial alpha beta CD4+ T cell loss. Productive HIV infection of gamma delta T cells in vitro had no measurable effect either on their proliferative response to Daudi stimuli or on the expression of cytokine mRNAs for IFN-gamma, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-13. CONCLUSIONS: The constitutive responsiveness of V gamma 9/V delta 2 T lymphocytes to Daudi and Etpp is severely altered in HIV+ persons. HIV infection of gamma delta T cells in vitro does not substantially change their cytokine expression or antigenic response.

Cell Division↗
PubMed1996

Molecular cloning of Limulus alpha 2-macroglobulin.

The American horseshoe crab Limulus polyphemus contains alpha 2-macroglobulin (alpha 2M) in the hemolymph plasma and hemocytes. alpha 2M from Limulus shows many of the typical characteristics of mammalian alpha 2M, including the presence of an internal thiol-ester, reactivity with a diversity of endopeptidases, a unique proteinase-trapping mechanism, and reactivity with the mammalian alpha 2M receptor. Additionally, Limulus alpha 2M has the unique property that it regulates the limulin-based hemolytic system of the plasma. A cDNA encoding Limulus alpha 2M has been obtained from a hemocyte cDNA library. The open reading frame encodes an N-terminal signal sequence of 25 amino acid residues and a mature protein of 1482 residues. The entire amino acid sequence is similar to those of the mammalian alpha 2Ms (28-29% identity) and contains common features found in mammalian alpha 2Ms. a bait region, an internal thiol-ester site, and a receptor-binding domain. However, the N-terminal portion (positions 24-105) has no sequence similarity with those of mammalian alpha 2Ms, and it is structurally related to that of the human complement factor C8 chain, consistent with a role for Limulus alpha 2M in host defense. The component sugar analysis of Limulus alpha 2M showed the existence of a complex type of oligosaccharide chain similar to those of mammalian alpha 2M. However, unlike mammalian alpha 2M, no sialic acid was detected in Limulus alpha 2M and it contained approximately 3 mol/mol N-acetylgalactosamine, suggesting the presence of O-linked sugar chains, which have not been found in mammalian alpha 2M. Expression of alpha 2M was detected in hemocytes, but not in hepatopancreas, heart, stomach, intestine, coxal gland, brain and skeletal muscle. Furthermore, immunoblotting of large and small granules of the hemocytes with antiserum against alpha 2M indicated the presence of the alpha 2M in large granules. Trypsin-treated Limulus alpha 2M, but not the native alpha 2M, displaced methylamine-treated human 125I-alpha 2M from the human alpha 2M receptor with a Kd of 30 nM, suggesting conservation of the proteinase-clearance mechanisms between mammalian and arthropod evolutionary lineages.

Amino Acid Sequence↗
PubMed1996

Limulus factor D, a 43-kDa protein isolated from horseshoe crab hemocytes, is a serine protease homologue with antimicrobial activity.

A glycoprotein (Mr = 43,000) from horseshoe crab hemocytes with antimicrobial activity against Gram-negative bacteria was purified. The internal peptide sequences coincided exactly with the deduced amino acid sequence of a cDNA clone, designated limulus factor D, which was isolated by screening a hemocyte cDNA library with an anti-human plasminogen antibody. The open reading frame codes for a precursor of factor D of 394 amino acid residues, including an NH2-terminal signal sequence. The COOH-terminal domain of factor D has significant sequence homology with the catalytic domain of mammalian serine proteases, in particular with human tissue plasminogen activator (32% identity), except for the substitution of Ser of the active site triad to Gly. Factor D has a unique NH2-terminal domain with weak sequence homology with part of the mammalian interleukin-6 receptor alpha-chain. Factor D is likely to have an important role in host defense mechanisms.

Amino Acid Sequence↗
PubMed1996

Control of calcium oscillations by phosphorylation of metabotropic glutamate receptors.

Stimulation of two metabotropic glutamate-receptor subtypes, mGluR1 and mGluR5, triggers the release of Ca2+ from intracellular stores through the inositol-(1,4,5) trisphosphate (InsP3) pathway. Here we report that glutamate induces single-peaked intracellular Ca2+ mobilization in mGluR1alpha-transfected cells but elicits Ca2+ oscillations in mGluR5a-transfected cells. The response patterns of the intracellular Ca2+ increase depend upon the identity of a single amino acid, aspartate (at position 854) or threonine (at position 840), located within the G-protein-interacting domains of mGluR1alpha and mGluR5a, respectively. Pharmacological and peptide mapping analyses indicated that phosphorylation of the threonine residue at position 840 of mGluR5a by protein kinase C (PKC) is responsible for the generation of Ca2+ oscillations in mGluR5a-expressing cells. To our knowledge this is the first evidence that PKC phosphorylation of G-protein-coupled receptors is important in producing oscillations in intracellular Ca2+ signalling.

3T3 Cells↗
PubMed1996

Limulus kexin: a new type of Kex2-like endoprotease specifically expressed in hemocytes of the horseshoe crab.

A Kex2-like protease was identified in hemocytes of the horseshoe crab (Tachypleus tridentatus), named limulus kexin, and a full-length cDNA was obtained from a hemocyte cDNA library. The deduced amino acid sequence contains 752 residues, composed of five domains with a signal sequence, a propeptide, a catalytic domain, a Ser/Thr-rich domain, and a transmembrane domain. The domain organization is very similar to that of the yeast Kex2 except that limulus kexin does not have a cytoplasmic tail. The catalytic domain exhibits striking sequence identities with those of furins, especially Drosophila furin1 (79%). Northern blotting showed specific expression of limulus kexin in hemocytes, suggesting the involvement in proteolytic processing of the granule components of hemocytes.

Amino Acid Sequence↗
PubMed1996

Interleukin 2 (IL-2) and interleukin 7 (IL-7) reciprocally induce IL-7 and IL-2 receptors on gamma delta T-cell receptor-positive intraepithelial lymphocytes.

In this study, we describe the interaction between cytokine and cytokine receptor (R) for the activation and proliferation of gamma delta T-cell receptor-positive T cells (gamma delta T cells). gamma delta T cells isolated from murine intestinal intraepithelial lymphocytes (IELs) were separated into gamma delta (Dim) and gamma delta (Bright) fractions according to the intensity of gamma delta T-cell receptor expression. The gamma delta T cells express low levels of IL-2R and IL-7R as shown by flow cytometry and reverse transcriptase-PCR analysis, whereas gamma delta (Bright) T cells did not express either receptor. Our study also revealed that recombinant marine (rm)IL-2 and rmIL-7 reciprocally induced high expressions of IL-7R and IL-2R, respectively, on gamma delta (Dim) T cells but not on gamma delta (Bright) cells. Thus, treatment of gamma delta (Dim) T cells with rmIL-2 and rmIL-7 resulted in high proliferative responses, whereas gamma delta (Bright) T cells did not respond to these two cytokines. The sources of these two cytokines for gamma delta T cells were neighboring epithelial cells (IL-7) and alpha beta T cells (IL-2 and IL-7). Cytokine signaling by IL-2 and IL-7 from alpha beta T cells and epithelial cells was necessary for the expression of IL-7R and IL-2R, respectively, on a subset of gamma delta T cells (e.g., gamma delta (Dim) T cells) in mucosa-associated tissue for subsequent activation and cell division.

Animals↗
PubMed1996

Post-source decay fragment spectra of cyclomalto-octaose and branched cyclomalto-hexaose by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

gamma-Cyclodextrin, maltosyl-alpha-cyclodextrin and diglucosyl-alpha-cyclodextrin were analyzed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. All of these compounds have the same molecular weight (M.W. = 1297.15) and consist of only D-glucopyranose. From a comparison of the intensities in the post-source decay (PSD) fragment spectra of these cyclodextrin derivatives, correlation between the chemical structures and the relative intensities in the PSD fragment ions was found. The correlation is considered to be caused by the difference in the number of cleavage sites at the glycosyl binding. It was found that the intensity of the PSD ion resulting from one cleavage is higher than that resulting from two cleavages at a glycosyl bond. The results show that PSD fragment-ion spectrum method used in MALDI-TOF mass spectrometry is a very powerful technique for the structural analyses of the sugar-substituted cyclodextrins.

Carbohydrate Sequence↗
PubMed1996

Deletion and reintroduction of glucosyltransferase genes of Streptococcus mutans and role of their gene products in sucrose dependent cellular adherence.

Streptococcus mutans has three kinds of glucosyltransferases (GTases), i.e. GTase-I, GTase-SI and GTase-S. These enzymes co-operatively synthesize adhesive glucan from sucrose, which contributes to firm adherence of growing organisms to solid surfaces. In this study, the genes encoding GTase-I (gtfB) and GTase-SI (gtfC) of S. mutans MT8148 (serotype c) were inactivated by the insertion mutagenesis by allelic exchange. Three types of isogenic mutants lacking either GTase-I, GTase-SI or both, respectively were isolated. Sucrose dependent cellular adherence of these mutants were significantly lower than that of the parent. Adherence of GTase-SI deficient mutant was lower than that of GTase-I deficient mutant. We then generated Escherichia coli-Streptococcus shuttle vectors carrying the gtfB and gtfC gene. The shuttle vector containing the gtfB gene was transformed into the GTase-I deficient mutant. Western blot analysis of the transformant revealed that GTase-I protein was fully expressed. Sucrose dependent adherence of the transformant increased but did not reach that of the parent. Similarly, the shuttle vector containing the gtfC gene was transformed into the GTase-SI deficient mutant. The expression of GTase-SI and sucrose dependent adherence of the transformant was revealed to be at a level similar to those by the parent. These results indicated that GTase-SI does play an essential role in the production of adhesive glucan that can lead to firm cellular adherence to solid surfaces.

Bacterial Adhesion↗
PubMed1996

A cysteine protease inhibitor stored in the large granules of horseshoe crab hemocytes: purification, characterization, cDNA cloning and tissue localization.

A cysteine protease inhibitor with an apparent Mr = 12,600, designated limulus (L)-cystatin, was isolated from hemocyte lysates of the Japanese horseshoe crab (Tachypleus tridentatus), using two steps of chromatography, including dextran sulfate-agarose, and carboxymethylated papain-agarose. L-cystatin inhibits amidolytic activity of papain by forming a noncovalent 1:1 complex with an equilibrium constant (Ki) of 0.08 nM. It also inhibits cathepsin L (Ki = 0.17 nM) and ficin (Ki = 0.52 nM), but not argingipain (a bacterial cysteine protease) and calpains. A cDNA for L-cystatin was isolated and the open reading frame coded for a mature protein of 114 amino acids, of which 99 residues were confirmed by peptide sequencing. L-cystatin shows significant sequence identities to members of the family 2 cystatin, such as bovine colostrum cystatin (33%) and human cystatin S (31%). Northern blotting revealed expression of the mRNA in hemocytes and slightly in heart but expression was negligible in hepatopancreas, intestine, stomach, and muscle. Immunoblotting revealed the localization to be in the large granules of hemocytes. Furthermore, L-cystatin has an antimicrobial activity against Gram-negative bacteria, which is much stronger than that of chicken egg white cystatin. These data suggest that the large granule-derived L-cystatin serves synergistically to accomplish an effective defense against invading microbes, together with other defense molecules that are released in response to external stimuli.

Agglutinins↗