Biomedical subjects

Yasuhiro Nishiyama

Publications and source records attributed to Yasuhiro Nishiyama.

31 records · Page 2Linked to original sources

PubMed2003

Autoantibodies to the epidermal growth factor receptor in systemic sclerosis, lupus, and autoimmune mice.

Autoantibodies to the recombinant extracellular domain of epidermal growth factor receptor (exEGFR) were detected by ELISA in the serum of Fas-defective old MRL/MpJ/lpr and C3H/HeJ/gld mice, but not young mice from these strains, or nonautoimmune young and old BALB/c, MRL/MpJ/++, and C3H/HeJ/MMTV mice. Compared with control human subjects without autoimmune disease, the frequency of exEGFR-binding autoantibodies was increased in scleroderma (systemic sclerosis) patients and to a lesser extent in lupus patients. Phage autoantibodies (Fv fragments) isolated from a lupus library by selection on a linear epitope of EGFR (residues 294-310) displayed the ability to bind exEGFR. Treatment of EGFR-expressing A431 cells with autoantibodies purified by affinity chromatography on immobilized exEGFR resulted in specific staining of the cells. Short-lived but strong inhibition of cellular DNA synthesis was observed in the presence of the autoantibodies. We concluded that autoantibody responses to EGFR hold the potential of fulfilling a pathogenic role in autoimmune disease.

Animals↗

PubMed2003

VIPase autoantibodies in Fas-defective mice and patients with autoimmune disease.

The immunoregulatory neuropeptide vasoactive intestinal peptide (VIP) was cleaved by purified IgG from Fas-defective C3H/gld mice, lupus patients, and autoimmune thyroiditis patients. No VIPase activity was detected in IgG from control mice and humans. Kinetic analyses of VIPase IgG preparations suggested low-affinity recognition of VIP. Yet the VIPase activity was VIP selective, judged by lack of correlation with other protease activities expressed by the IgG and by noninterference of unrelated peptides in the activity. Recombinant Fv constructs selected from a human lupus phage show library displayed VIPase activity, confirming that the active site is located in the V domains. Inhibition of the VIPase activity by di-isopropylfluorophosphate suggested a serine protease-like mechanism of catalysis. Irreversible binding of a biotinyated phosphonate diester by the IgG and Fv preparations was observed, consistent with the presence of activated nucleophiles similar to those in enzymes capable of covalent catalysis. These observations show that VIP is a target for specific catalytic autoantibodies in autoimmune disease.

Animals↗

PubMed2003

Different long-term course between chest pain and exercise-induced ST depression in syndrome X.

The aim of the present study was to assess the long-term clinical course of patients with syndrome X, focusing on different courses between exercise-induced ST depression and chest pain. Forty-three patients with syndrome X were followed up for 6.4 +/- 3.8 years. They were divided into the 3 groups according to chest pain: disappeared (n = 24), improved (n = 14), or unchanged (n = 5). No patients had cardiac events and all had a favorable long-term prognosis. In patients showing disappearance of chest pain, exercise-induced ST depression and rate-pressure product (RPP) at peak exercise did not change during follow-up. However, ST depression and RPP decreased significantly in those with improved chest pain. These observations suggest that abnormal pain perception plays an important role in the development of chest pain.

Adult↗

PubMed2003

[Post-stroke depression].

The frequency of post-stroke depression (PSD) was evaluated in an ischemic stroke cohort four weeks after onset, and the relationship between self-rating depression scale (SDS)/and infarct size, number and location of the ischemic brain lesions was also studied. The effects of a newly developed antidepressant SSRI (selective serotonin reuptake inhibitor), fluvoxamine maleate, on PSD and cerebral blood flow (CBF) was investigated in other ischemic stroke patients. The frequency of patients who had more than 40 on SDS score was 46% (18/39), and that of patients who had more than 50 was 13% (5/39). There were no differences in SDS score in infarct size, number and location of ischemic brain lesions, however there were significant differences in the lesion side. The score of the patients who had lesions in the left hemisphere was significantly higher than that of those who had them in the right. Administration of fluvoxamine maleate for four weeks improved the score on the Hamilton rating scale for depression (HAM-D) from 16.6 +/- 4.7 (n = 5) to 8.4 +/- 4.3 (n = 5), however it did not influence the mean cortical CBF. This study shows that the patients frequently had depression after ischemic stroke, and that left side lesion had a significant relationship with PSD. Therefore it is important that psychiatric examination of post-stroke patients is conducted. This study also shows that a newly developed antidepressant, fluvoxamine maleate, was effective for PSD.

Aged↗

PubMed2002

A mechanism-based probe for gp120-Hydrolyzing antibodies.

An antigenic peptide analogue consisting of HIV gp120 residues 421-431 (an antigen recognition site probe) with diphenyl amino(4-amidinophenyl)methanephosphonate located at the C-terminus (a catalytic site probe) was synthesized and its trypsin and antibody reactivity characteristics were studied. Antibodies to the peptide determinant recognized the peptidyl phosphonate probe. Trypsin was inhibited equipotently by the peptidyl phosphonate and its simple phosphonate counterpart devoid of the peptide determinant. The peptidyl phosphonate inhibited the gp120-hydrolyzing activity of a catalytic antibody light chain. It was bound covalently by the light chain and the binding was inhibited by the classical active-site directed inhibitor of serine proteinase, diisopropyl fluorophosphate. These results reveal that the peptidyl phosphonate ester can serve as a probe for the antigen recognition and catalytic subsites of proteolytic antibodies.

Animals↗

PubMed2002

Prospects for immunotherapeutic proteolytic antibodies.

Monoclonal antibodies are suitable for therapeutic applications by virtue of their excellent target binding characteristics (specificity, affinity) and long half-life in vivo. Catalytic antibodies (CAbs) potentially represent a new generation of therapeutics with enhanced antigen inactivation capability. Here, we describe prospects for development of therapeutic CAbs to the envelope protein gp120 of HIV. The strategy consists of exploiting the natural tendency of the immune system to synthesize germline-encoded, serine protease-like CAbs. Lupus patients were found to develop antibodies to a conserved component of the CD4 binding site of gp120, potentially offering a means to obtain human antibodies expressing broad reactivity with various HIV strains. Covalently reactive antigen analogs (CRAs) capable of selective recognition of nucleophilic Abs were synthesized and applied to isolate Fv and L chain catalysts from lupus phage repertoires. CRA binding by the recombinant Ab fragments was statistically correlated with catalytic cleavage of model peptide substrates. A peptidyl CRA composed of residues 421-431 with a phosphonate diester moiety at its C terminus was validated as a reagent that combines noncovalent and covalent binding interactions in recognition of a gp120ase L chain. A general challenge in the field is the apparent instability of the catalytic conformation of the Abs. In reference to therapy of HIV infection, assurance is required that the Abs recognize the native conformation of gp120 expressed as a trimer on the virus surface.

Antibodies, Catalytic↗

PubMed2002

Covalent reactivity of phosphonate monophenyl esters with serine proteinases: an overlooked feature of presumed transition state analogs.

Phosphonate monoesters have been assumed to serve as noncovalent transition state analogs for enzymes capable of catalyzing transacylation reactions. Here, we present evidence for the covalent reaction of certain serine proteinases and peptidase antibody fragments with monophenyl amino(4-amidinophenyl)methanephosphonate derivatives. Stable adducts of the N-biotinylated monophenyl ester with trypsin and antibody fragments were evident under conditions that disrupt noncovalent interactions. The reaction was inhibited by the active-site-directed reagent diisopropyl fluorophosphate. Mass spectrometry of the fragments from monoester-labeled trypsin indicated phosphonylation of the active site. Irreversible inhibition of trypsin- and thrombin-catalyzed hydrolysis of model substrates was observed. Kinetic analysis of inactivation of trypsin by the N-benzyloxycarbonylated monoester suggested that the first-order rate constant for formation of covalent monoester adducts is comparable to that of the diester adducts (0.47 vs 2.0 min(-1)). These observations suggest that the covalent reactivity of phosphonate monoesters contributes to their interactions with serine proteinases, including certain proteolytic antibodies.

Antibodies↗

PubMed2002

Homeostatic regulation of intestinal villous epithelia by B lymphocytes.

The epithelial cell of the small intestine is one of the most rapidly regenerating cells in the body. However, the cellular mechanism and biological significance underlying this rapid regeneration remain elusive. In this study we examined the intestinal epithelia of mutant mice that lack B and/or T cells and those of normal littermates. The absence of B cells in Ig mu-chain mutant mice or B and T cells in recombination-activating gene (RAG)-2(-/-) as well as SCID mutant mice was associated with a marked acceleration of epithelial cell turnover and an up-regulation of the expression of MHC class II molecules. No such effects were observed in T cell-deficient TCR-delta and -beta double-mutant mice. As far as the goblet cells of villous epithelium are concerned, absolute numbers of them remained the same among these mutant mice that have no B and/or T cells. Alymphoplasia (aly/aly) mutant mice that lacked Peyer's patches and Ig-producing cells in the lamina propria, but harbored a large number of intestinal mucosal T cells, also displayed a significant acceleration of epithelial cell turnover and, to some extent, up-regulated expression of MHC class II molecules. Notably, the accelerated epithelial cell turnover was not observed and returned to normalcy in the Ig mu-chain mutant mice that had been given antibiotic-containing water. These findings indicate that B cells down-regulate the generation and differentiation of intestinal epithelial cells in the normal wild-type condition and suggest that enteric microorganisms are implicated in the accelerated generation of epithelial cells in mice that have no B cells.

Ampicillin↗

PubMed2002

Identification of multiple isolated lymphoid follicles on the antimesenteric wall of the mouse small intestine.

We have revealed that 100-200 clusters, filled with closely packed lymphocytes, can be found throughout the length of the antimesenteric wall of the mouse small intestine. They are composed of a large B cell area, including a germinal center, and epithelia overlying the clusters contain M cells. A large fraction of B cells displays B220+ CD19+ CD23+ IgM(low)IgD(high)CD5(-)Mac-1(-) phenotype, and the composition of IgA+ B cells is smaller but substantial. To our knowledge, these clusters are the first identification of isolated lymphoid follicles (ILF) in mouse small intestine. ILF can be first detected at 7 (BALB/c mice) and 25 (C57BL/6 mice) days after birth, and lymphoid clusters equivalent in terms of cellular mass to ILF are present in germfree, athymic nude, RAG-2(-/-), TCR-beta(-/-), and Ig mu-chain mutant (mu(-/-)) mice, although c-kit+ cells outnumber B220+ cells in germfree and athymic nude mice, and most lymphoid residents are c-kit+ B220(-) in RAG-2(-/-), TCR-beta(-/-), and mu(-/-) mice. ILF develop normally in the progeny of transplacentally manipulated Peyer's patch (PP)-deficient mice, and decreased numbers of conspicuously atrophied ILF are present in IL-7Ralpha(-/-) PP(null) mice. Neither ILF nor PP are detectable in lymphotoxin alpha(-/-) and aly/aly mice that retain well-developed cryptopatches (CP) and thymus-independent subsets of intraepithelial T cells, whereas ILF, PP, CP, and thymus-independent subsets of intraepithelial T cells disappear from common cytokine receptor gamma-chain mutant mice. These findings indicate that ILF, PP, and CP constitute three distinct organized gut-associated lymphoid tissues that reside in the lamina propria of the mouse small intestine.

Animals↗

PubMed2002

Vasoactive intestinal peptide binding autoantibodies in autoimmune humans and mice.

Autoantibodies capable of binding the immunoregulatory neuropeptide vasoactive intestinal peptide (VIP) were detected in the sera of a mouse strain prone to autoimmune disease due to the lpr mutation (MRL/lpr). The autoantibodies were not present in control wildtype MRL/lpr mice, but they were readily detected in humans without autoimmune disease. The binding was due to low affinity VIP recognition. Increased VIP binding activity was evident in patients with systemic lupus erythematosus but not systemic sclerosis, Sjögren's syndrome (SS), rheumatoid arthritis or autoimmune thyroiditis. Recombinant VIP binding Fv clones (fragment variable; the variable domains of the light and heavy chains antibody subunits joined with a peptide linker) were isolated from a phage display library prepared from lupus patients. One Fv clone displaying VIP-selective binding and several clones displaying cross-reactivity with unrelated peptides were identified. Replacement mutations in the VIP-selective clone were preferentially localized in the regions known to make contacts with the antigen, i.e. the complementarity determining regions, suggesting that the selective binding activity is due to immunological maturation of the antibodies. Frequent occurrences of autoantibody responses to VIP indicate that immunological tolerance to this neuropeptide can be readily broken. The depletion of VIP by specific antibodies in autoimmune disease may interfere with VIP regulation of T cells and inflammatory cells and result in further amplification of autoreactive immunological responses.

Animals↗

PubMed2002

Anti-HIV-1 peptides derived from partial amino acid sequences of CC-chemokine RANTES. Regulated upon activation, normal T-cell expressed and secreted.

Fifteen acetyl-peptide-amides with partial amino acid sequences of RANTES (regulated upon activation, normal T-cell expressed and secreted), all Cys residues of which were substituted by Ala, were synthesized, and screened for anti-HIV-1 activity. Peptides corresponding to 1-10, 37-46, and 57-68 showed marked activity against CC-chemokine receptor 5-using HIV-1(JR-CSF) (% inhibition at 100 nM 69, 82, 76%, respectively). The results indicate that multiple regions, including the N-terminal part responsible for chemotactic activity, are involved in anti-HIV-1 activity of RANTES, yielding possible lead compounds for anti-HIV-1 agents.

Amino Acid Sequence↗

PubMedDate not supplied

Broadly distributed nucleophilic reactivity of proteins coordinated with specific ligand binding activity.

Covalent nucleophile-electrophile interactions have been established to be important for recognition of substrates by several enzymes. Here, we employed an electrophilic amidino phosphonate ester (EP1) to study the nucleophilic reactivity of the following proteins: albumin, soluble epidermal growth factor receptor (sEGFR), soluble CD4 (sCD4), calmodulin, casein, alpha-lactalbumin, ovalbumin, soybean trypsin inhibitor and HIV-1 gp120. Except for soybean trypsin inhibitor and alpha-lactalbumin, these proteins formed adducts with EP1 that were not dissociated by denaturing treatments. Despite their negligible proteolytic activity, gp120, sEGFR and albumin reacted irreversibly with EP1 at rates comparable to the serine protease trypsin. The neutral counterpart of EP1 reacted marginally with the proteins, indicating the requirement for a positive charge close to the electrophilic group. Prior heating resulted in altered rates of formation of the EP1-protein adducts accompanied by discrete changes in the fluorescence emission spectra of the proteins, suggesting that the three-dimensional protein structure governs the nucleophilic reactivity. sCD4 and vasoactive intestinal peptide (VIP) containing phosphonate groups (EP3 and EP4, respectively) reacted with their cognate high-affinity binding proteins gp120 and calmodulin, respectively, at rates exceeding the corresponding reactions with EP1. Reduced formation of EP3-gp120 adducts and EP4-calmodulin adducts in the presence of sCD4 and VIP devoid of the phosphonate groups was evident, suggesting that the nucleophilic reactivity is expressed in coordination with non-covalent recognition of peptide determinants. These observations suggest the potential of EPs for specific and covalent targeting of proteins, and raise the possibility of nucleophile-electrophile pairing as a novel mechanism stabilizing protein-protein complexes.

Animals↗

PubMedDate not supplied

Towards irreversible HIV inactivation: stable gp120 binding by nucleophilic antibodies.

Conventional antibodies react with antigens reversibly. We report the formation of unusually stable complexes of HIV gp120 and nucleophilic antibodies raised by immunization with an electrophilic HIV gp120 analog (E-gp120). The stability of the complexes was evident from their very slow dissociation in a nondenaturing solvent (approximate t(1/2) 18.5 days) and their resistance to dissociation by a denaturant commonly employed to disrupt noncovalent protein-protein binding (sodium dodecyl sulfate). Kinetic studies indicated time-dependent and virtually complete progression of the antibody-gp120 complexes from the initial noncovalent state to a poorly dissociable state. The antibodies to E-gp120 displayed improved covalent reactivity with an electrophilic phosphonate probe compared to control antibodies, suggesting their enhanced nucleophilicity. One of the stably binding antibodies neutralized the infectivity of CCR5-dependent primary HIV strains belonging to clades B and C. These findings suggest the feasibility of raising antibodies capable of long-lasting inactivation of antigens by electrophilic immunization.

Animals↗