Biomedical subjects

A Miyama

Publications and source records attributed to A Miyama.

At least 19 recordsLinked to original sources

PubMed1997

Histidine-44 of the A subunit of Escherichia coli enterotoxin is involved in its enzymatic and biological activities.

We examined the role in toxicity of histidine-44 of the A subunit of Escherichia coli enterotoxin, which is located in the active site cavity close to glutamic acid-112. Although amino acid substitution of histidine-44 usually renders a mutant toxin unstable to trypsin, one mutant, alanine-44 (His44Ala) was found to be stable. His44Ala did not show any agmatine:ADP-ribosyltransferase activity in the presence or absence of recombinant ADP-ribosylation factor. It showed no diarrheal or rabbit skin permeability activity and was a competitor in enterotoxin-ADP-ribosyltransferase assays containing recombinant ADP-ribosylation factor. These results suggest that like glutamic acid-112, histidine-44 plays an essential role in toxicity. A tentative model, which explains NAD+ catalysis and the transfer of the ADP-ribosyl moiety to a target amino acid, is proposed for histidine-44 and glutamic acid-112.

ADP-Ribosylation Factors↗

PubMed1997

A unique DNA sequence of human enterotoxigenic Escherichia coli enterotoxin encoded by chromosomal DNA.

We detected Ent plasmids in 300 strains of human enterotoxigenic Escherichia coli, but one strain, E. coli 240-3, had neither a small nor a large plasmid and encoded the heat-labile enterotoxin (LTh(240-3)) gene on its chromosome. DNA sequences showed that LTh(240-3) differed by 12 and 14 base pairs from LT (LTh) and LT (LTp) from human H10407 and porcine EWD299 strains, respectively. In deduced precursor toxins, LTh(240-3), LTh and LTp differed from LTh, LTp and LTh(240-3) at nine, eight and eleven positions, respectively. These data suggest that although LTh(240-3) encoded in the chromosome is antigenically similar to LTh, it cannot be grouped with LTh due to differences in its DNA and amino acids sequences.

Amino Acid Sequence↗

PubMed1997

Flow cytometry analysis of the Fas ligand expression of activated lymph node T-cells.

There is increasing evidence for the role of the Fas/Fas ligand interaction in the immunoregulation of T-cells. We studied the expression of the Fas ligand (FasL) in activated peripheral T-cell in vitro, and its relation to autonomous cell death by flow cytometry. Following the stimulation of lymph node T-cells with anti-CD3 and rIL2, the mRNA level of FasL increased more than four times during the first 2 days over the level before stimulation. The surface expression of FasL was observed on 27% of the population at day 2 after stimulation and increased to approximately 50% at day 3. Kinetic analysis by flow cytometry, however, indicated that all T-blasts transformed during activation did not express FasL. FasL expression became evident simultaneously with the termination of cell expansion. Since cells remained viable (> 90%) at day 3 as judged by trypan blue-exclusion, cell membranes expressing FasL were supposed to be still intact. Concomitantly with FasL-expression, spontaneous DNA fragmentation was observed. These observations support the idea that autonomous Fas/FasL interaction mediates apoptosis in activated peripheral T-cells as demonstrated in T-cell hybridoma or established T-cells.

Animals↗

PubMed1997

Relationship between a low toxicity of the mutant A subunit of enterotoxigenic Escherichia coli enterotoxin and its strong adjuvant action.

In the work described here it was determined if and how unnicking in the A subunit of Escherichia coli enterotoxin at Arg192 or nearby residues affected biological activities of the toxin. The mutant toxin was constructed to lack the nick site in the A subunit by deleting the tripeptide Arg192-Thr193-Ile194, which is essential for toxicity. The mutant toxin did not exhibit agmatine ADP-ribosyltransferase activity in the presence or absence of the ADP-ribosylation factor and had less diarrhoeal activity and lower induction of cyclic AMP than did LT. The mutant toxin exhibited a much stronger adjuvant action on antibody responses to measles virus, keyhole limpt haemocyanin, bovine immunoglobulin and ovalbumin compared with LT. The altered toxicity of the mutant toxin might be closely related to the potent adjuvant action on antibody responses to antigens. The relationship between two activities is discussed.

ADP Ribose Transferases↗

PubMed1997

The serum resistance of malaria-infected erythrocytes.

IgG and IgM antibodies were detected on non-parasitized as well as parasitized erythrocytes (E) from mice surviving over 15 days after infection with rodent malaria, Plasmodium berghei, whereas C3 was detected exclusively on parasitized E. Parasitized E, however, were quite resistant to the haemolytic activity of guinea pig complement and effectively inactivated human C3b to iC3b on their surface. Similarly, parasitized E were extremely resistant to homologous complement as assessed by haemolysis and C3 binding even when regulatory proteins (decay-accelerating factor, DAF; complement receptor related gene y, Crry; heat-stable antigen, HSA) were blocked with specific antibodies. DAF and Crry were equally expressed on both normal E and parasitized E from mice within a week post-infection; therefore, molecules that inhibit the haemolysis or C3 binding of parasitized E appear to be independent of DAF and Crry. Unexpectedly, the molecular forms of HSA and DAF in parasitized erythrocyte membranes were found to be different from those of normal erythrocyte membranes: DAF was detected as three bands (85,000, 64,000 and 30,000 MW) by immunoblotting. HSA was detected as more highly glycosylated forms than normal HSA. These alterations of DAF and HSA could be explained by the modification of membrane proteins and polysaccharides induced by parasitization, and we hypothesize that these changes of membranes or membrane proteins are involved in the resistance of parasitized E against homologous complement.

Animals↗

PubMed1996

Rapid killing of murine lymph node T blasts by intestinal intraepithelial lymphocytes in vitro.

We examined the effect of murine intestinal intraepithelial lymphocytes (IEL) on the proliferation of murine lymph node T cells (LN-T) in vitro. An IEL fraction prevented the proliferation of LN-T stimulated with antigen and X-irradiated spleen cells, or with anti-CD3 monoclonal antibodies (mAb). Concanavalin A-activated LN-T were less sensitive. Such an inhibitory activity was recovered from a CD8-depleted population by panning of bulk IEL using anti-CD8 alpha mAb. This population of BALB/c IEL showed less granzyme A activity, and its surface markers were positive for CD8 (4%), CD3 (80-90%), CD4 (2-6%), alpha-beta TcR (45-70%), and gamma-delta TcR (4-9%). Asialo-GM1 and Thy1.2 were variably expressed, but interleukin-2 (IL-2) receptor-alpha and Fc gamma receptor were not. By contrast, no cytotoxicity against YAC-1 was detected in a CD8-depleted IEL population by a 6-h 51Cr-release assay. Although IEL from severe-combined immunodeficient mice lacking CD4, CD8 and TcR, but expressing IL-2 receptor, showed cytotoxicity against YAC-1, their inhibitory activity against LN-T was almost the same as that by IEL from BALB/c mice. When LN-T blasts (greater than 75% CD4+) activated with anti-CD3 were treated with CD8-depleted IEL, intact cellular DNA of the T blasts disappeared within 1 h with increased amounts of small-sized DNA. These results suggest that CD8- IEL directly and nonspecifically kill lymph node CD4+ T blasts and possibly down-regulate TcR-mediated proliferation of peripheral T cells in the gut epithelium.

Animals↗

PubMed1996

The amino acids of Escherichia coli enterotoxin B subunit involved in binding to Bio-Gel A-5m or to the glycoprotein from mouse intestinal epithelial cells.

We determined whether Arg13, Met31, and Ser95 of the heat-labile enterotoxin B subunit (LT-B) might be involved in Lt-B binding to oligosaccharides, which did not bind to the B subunit of the cholera toxin (CT-B). Three LT-B mutants, R13H, M31L, and S95A were prepared by substituting three amino acid residues that differ in CT-B. These mutants formed a pentamer and exhibited the same binding ability to the GM1 ganglioside as native LT-B. Although these mutants did not bind to Bio-Gel A-5m, they did bind to the glycoprotein from mouse intestinal cells in the order R13H > M31L > S95A. These data suggest that Ser95, Met31, and Arg13 are important for LT-B binding to Bio-Gel A-5m, and that although Ser95 is also partially responsible for LT-B binding to the glycoprotein, Arg13 has no significant involvement in it.

Animals↗

PubMed1995

Natural killer (NK)-like cytotoxicity of murine intraepithelial lymphocytes in the small intestine (iIEL) and the effect of the serine proteases.

The natural killer (NK)-like cytotoxicity of murine intraepithelial lymphocytes in the small intestine (iIEL) and the participation of serine proteases in it were investigated. We monitored the cytotoxicity of iIEL with a sensitive cytotoxic assay using laser flow cytometry. iIEL exhibited NK-like cytotoxicity on YAC-1 target cells. Benzamidine, a serine protease inhibitor, inhibited significantly both Na-CBZ-L-lysine thiobenzyl ester (BLT)-specific serine protease activity and iIEL-mediated NK-like cytotoxicity. These results suggest that BLT-specific serine proteases may participate in NK-like cytotoxicity of murine iIEL.

Amino Acid Sequence↗

PubMed1995

Monomer of the B subunit of heat-labile enterotoxin from enterotoxigenic Escherichia coli has little ability to bind to GM1 ganglioside compared to its coligenoid.

Coligenoid, composed of the B subunit of heat-labile enterotoxin from enterotoxigenic Escherichia coli, was separated into monomers in the presence of 2% propionic acid containing 6 M urea (pH 3.8). Monomers equilibrated against 0.75% or 0.5% propionic acid containing 3 M urea (pH 3.8) did not reassemble into coligenoid. Complexes of GM1 ganglioside and coligenoid in these buffers were detected by SDS-polyacrylamide gel electrophoresis, but those of the GM1 ganglioside and monomers were not. The binding ability of monomer to GM1 ganglioside in these buffers was about 1% of that of normal coligenoid by GM1-enzyme-linked immunosorbent assay. Moreover, monomers in these buffers reassembled into coligenoid by buffering against original TEAN buffer, and the binding ability of the resulting coligenoid to GM1 ganglioside was identical to that of native coligenoid. These data suggest that although coligenoid formation is important for the receptor binding of the B subunit, little binding ability to GM1 ganglioside remains in monomer of the B subunit.

Bacterial Toxins↗

PubMed1995

Mutagenicity of bile and pancreatic juice from patients with pancreatico-biliary maljunction.

We attempted to detect mutagenic activity in bile and pancreatic juice from patients with biliary tract disease using the spore rec assay and wild (H17) and mutant (M45) strains. Three bile samples out of 5 obtained from patients with pancreatico-biliary maljunction showed positive reaction in the spore rec assay, and all contained a high level of amylase activity, while 300 microliters of bile samples obtained from 10 control patients without pancreatico-biliary maljunction did not show any positive reaction. Moreover, 300 microliters of the in vitro mixture of bile with an equal volume of pancreatic juice also showed a positive reaction after treatment for 12 days at 37 degrees C or for 10 min at 100 degrees C, suggesting that they were very stable and long-acting in vivo. These data suggest that possible mutagens might be formed by the mixing of bile with pancreatic juice regurgitated into the biliary tract, and that there might be a relationship to biliary tract cancer which often accompanies pancreatico-biliary maljunction.

Amylases↗

PubMed1994

Construction of plasmids useful for production of the B subunit of cholera toxin from Vibrio cholerae or a heat-labile enterotoxin from enterotoxigenic Escherichia coli.

A simple method to construct the plasmids producing the B subunit of porcine or human heatlabile enterotoxin or cholera toxin was developed, and the B subunits produced by the resulting plasmids were purified. The gene of LTp from pEWD 299 was ligated to pHSG 396 or pBluescript SK(+)-1 and the vector carrying one Xbal and EcoR1 site in the LTp-A gene was constructed. The Xbal-EcoR1 fragment of LTp-A gene was exchanged for the multicloning site of pHSG 396 containing Xbal, BamH1, Cla 1, Kpn1, Sac1 and EcoR1 sites. This plasmid (pTSU28) produced the LTp-B subunit. Moreover, the fragment of the LTp-B gene of pTSU 28 was exchanged by the EcoR1-HindIII fragment of LTh-B from E. coli H10407 strain (pTSU 35) or by the Cla 1-Hind III fragment of CT-B gene amplified by the PCR procedure with the chromosomal DNA of V. cholerae 86KT25 (pTSU 32). The DNA sequence of the CT-B subunit amplified by PCR procedure was compared and found identical to that cited in the literature [11]. After these plasmids were transformed into E. coli MV 1184 strain, the toxins produced by them were purified using a Bio-Gel A 5m affinity column for both LT-Bs and an immunobilized D-galactose affinity column for CT-B. Though both columns absorbed only the B subunit, the eluates contained a single protein corresponding to the B subunit, suggesting that each mutant produces only the B subunit.

Animals↗

PubMed1993

Production of heat-stable enterotoxin II by chicken clinical isolates of Escherichia coli.

Enterotoxigenic Escherichia coli isolated from diarrhea stools of chickens were examined for production of heat-stable enterotoxin II which is considered to be implicated only in diarrhea of pigs. Seven out of 38 strains examined were found to contain heat-stable enterotoxin II gene, determined by colony hybridization and the polymerase chain reaction. The culture supernatants of these strains caused fluid accumulation in the mouse intestinal loop test. This fluid accumulation activity was not lost by heating at 100 degrees C and was neutralized by anti-heat-stable enterotoxin II antiserum. Purified heat-stable enterotoxin II caused fluid accumulation in the chicken intestinal loop assay. These results indicate that STII-producing E. coli is implicated in chicken diarrhea.

Animals↗

PubMed1993

Amino acid sequence of heat-labile enterotoxin from chicken enterotoxigenic Escherichia coli is identical to that of human strain H 10407.

The DNA sequence of heat-labile enterotoxin from the chicken enterotoxigenic Escherichia coli 21d strain was determined by direct dideoxy sequencing of polymerase chain reaction (PCR)-amplified DNA and was compared with those of heat-labile enterotoxins from porcine and human enterotoxigenic E. coli strains EWD 299 and H 10407. The structural genes of the A and B subunits of chicken heat-labile enterotoxin were identical to those of human heat-labile enterotoxin from the human H 10407 strain. Moreover, 67 base pairs of the upstream and 60 base pairs of the downstream region of the chicken heat-labile enterotoxin gene were also identical to that of the human heat-labile enterotoxin from strain H 10407. However, the patterns of plasmids from the 21d and H 10407 strains were different. The 21d strain had no band corresponding to the 42-MDa plasmid of the H 10407 strain encoding the heat-labile enterotoxin gene but it had a smaller plasmid. These data suggest that although the DNA sequence of chicken heat-labile enterotoxin is identical to that of human heat-labile enterotoxin, the plasmid encoding the chicken heat-labile enterotoxin gene in the chicken might be different from that encoding the human heat-labile enterotoxin gene in the H 10407 strain.

Amino Acid Sequence↗

PubMed1993

Inhibitory effect of FUT-175 on complement activation and its application for glomerulonephritis with hypocomplementemia.

FUT-175 (6-amidino-2-naphthyl p-guanidinobenzoate dimethane-sulphonate), a potent serine protease inhibitor, has been reported to inhibit complement activity in vitro, and especially the classical complement pathway effectively. In the present study, we examined the inhibitory effect of FUT-175 on the classical complement pathway components by hemolytic assay using purified human complement components. As a result, 50% inhibition of the C1 protease activity for classical C3 convertase formation and for C2 was obtained with 3.0 x 10(-8) M and 7.0 x 10(-8) M of FUT-175, respectively. FUT-175 did not inhibit the C2 protease activity at all. We then administered FUT-175 to 5 glomerulonephritic patients with hypocomplementemia and proteinuria in order to assess the clinical effectiveness of this drug. When FUT-175 was administered intravenously and continuously at a rate of 0.1 to 0.2 mg/kg/hr for 2 weeks, the urinary protein excretion decreased significantly from 2.9 +/- 0.8 to 1.4 +/- 0.5 g/day (P < 0.025). In these patients, some of the serum complement markers (serum C3, C4 level and the hemolytic activity via the classical complement pathway (CH50)) were increased after FUT-175 administration. The above findings suggests that FUT-175 can exert beneficial effects on glomerulonephritis with hypocomplementemia by inhibiting complement activation.

Adult↗

PubMed1992

Enterotoxicity and immunological properties of two mutant forms of Escherichia coli STIp with lysine or arginine substituted for the asparagine residue at position 11.

Two variants of Escherichia coli heat-stable enterotoxin Ip, in which the amino acid residue at position 11 was substituted with lysine or arginine, were purified to near homogeneity from the culture supernatants of toxin-producing mutant strains. Neither the purified heat-stable enterotoxin Ip(Lys-11) nor the purified heat-stable enterotoxin Ip(Arg-11) showed a positive response in the suckling mouse assay or in the mouse intestinal loop assay. Furthermore, live bacteria producing these mutant heat-stable Ip enterotoxins did not cause fluid accumulation in mouse intestinal loops, in contrast to bacteria producing native heat-stable enterotoxin Ip. Nevertheless, antisera raised against both heat-stable enterotoxin Ip(Lys-11) and heat-stable enterotoxin Ip(Arg-11) neutralized the enterotoxic activity of native heat-stable enterotoxin Ip. These results demonstrate that heat-stable enterotoxin Ip(Lys-11) and heat-stable enterotoxin Ip(Arg-11) lose enterotoxicity but retain epitopes which are common to native heat-stable enterotoxin Ip.

Animals↗

PubMed1992

The protease from Vibrio cholerae nicks arginine at position 192 from the N-terminus of the heat-labile enterotoxin a subunit from enterotoxigenic Escherichia coli.

It was examined where a protease purified from Vibrio cholerae might nick the heat-labile enterotoxin (LT) A subunit from enterotoxigenic Escherichia coli. LT was digested by the protease and contained a fragment which had the same mobility on SDS-PAGE as that of the A1 fragment of LT digested by trypsin. The biological activity of LT by this protease was also identical to that of LT by trypsin. The amino acid sequence of the N-terminus of the A2-like fragment was Thr-Ser-Thr-Gly, which corresponded to the sequence from 193 to 196 of the A subunit. These data suggest that this protease, like trypsin, nicks arginine at position 192 from the N-terminus of the A subunit and that the biological activation of LT by this protease is similar to that by trypsin.

Amino Acid Sequence↗

PubMed1992

Heterogeneity among heat-labile enterotoxins produced by porcine enterotoxigenic Escherichia coli.

The heat-labile enterotoxins (LT) produced by various porcine strains (LTp) of enterotoxigenic Escherichia coli were purified to homogeneity and their molecular properties were compared with each other. By double gel diffusion and rabbit skin permeability test, LTps from WT-1 (LTp-WT-1) and BO-149 (LTp-BO-149) strains were antigenically and biologically identical. By polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS), the mobilities of A and B subunits of LTp (BO-149) were identical to those of LTp (WT-1). However, on polyacrylamide gel electrophoresis without SDS, LTp (BO-149) and LTp (WT-1) differed in mobility, suggesting their molecular differences. Though the pI value of the B subunit of LTp (BO-149) was identical to that of LTp (WT-1), the pI value of the A subunit of LTp (BO-149) was higher than that of LTp (WT-1). These data suggest that there is molecular heterogeneity among LTps produced by the two porcine enterotoxigenic Escherichia coli strains.

Animals↗

PubMed1992

Chinese hamster ovary cells produce an enzyme that nicks heat-labile enterotoxin from enterotoxigenic Escherichia coli.

Chinese hamster ovary (CHO) cells were examined for production of an enzyme that nicked the polypeptide chain of the heat-labile enterotoxin from enterotoxigenic Escherichia coli between the A1 and A2 fragments of its A subunit. Serum-free culture medium prepared each day after CHO cell inoculation was concentrated 100 times and its proteolytic activity for formation of the A1 fragment was examined by Western blotting with anti-LT A antibody. The A subunit was detected in culture medium on day 6 after cell inoculation, although not in media on day 1 or 3, indicating that CHO cells produced a nicking enzyme. This nicking enzyme had an optimal pH of about 7.5 and an apparent Mr. of 120,000, as seen by Superose 12 TM gel filtration with an FPLC system. The activity of this enzyme was strongly inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, but not by p-chloromercuribenzoic acid, EDTA or ethyleneglycol bis (beta-aminoethylether)-N,N-N',N'-tetraacetic acid, suggesting that this enzyme was a serine protease. The activity was not stimulated by plasminogen or fibrin. These findings suggest that the nicking enzyme was different from proteases such as elastase, collagenase and plasminogen activator, which are probably also secreted by fibroblast-like CHO cells.

Animals↗