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Biomedical subjects

A Miyama

Publications and source records attributed to A Miyama.

At least 19 recordsLinked to original sources
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↗
PubMed1992

Plasmodium berghei: sporozoites are sensitive to human serum but not susceptible host serum.

Human complement was activated by rodent malaria, Plasmodium berghei, sporozoites through the alternative pathway, as revealed by C3 deposition on sporozoites using the fluorescent antibody technique. Sporozoites exposed to fresh human serum decreased in infectivity to HepG2 cells, but those exposed to heated or C3-deficient human serum showed normal infectivity to HepG2 cells. In contrast, C3 deposition was not observed on the sporozoites treated with mouse or rat serum even in the presence of specific polyclonal anti-sporozoite antibody. However, following treatment with trypsin (250 micrograms/ml), 81% of salivary gland sporozoites and 49% of oocyst sporozoites became reactive with mouse serum, and reactive sporozoites deposited mouse C3 on their surface in the presence of 30 mM EGTA and 1 mM Mg2+ without antibody. Concomitantly some sporozoites lost reactivity to anti-circumsporozoite protein monoclonal antibody. These results suggest that P. berghei sporozoites possibly express surface molecules that regulate the complement activation pathway of susceptible hosts but not of nonhosts, and that the putative structures consist of protease-sensitive molecule(s) which are closely associated with the circumsporozoite protein.

Animals↗
PubMed1992

Fluid-phase activation of the alternative pathway of complement by excess factor D in regularly dialyzed patients.

We examined the effect of excess factor D on the alternative pathway of complement (APC). First, we demonstrated that the production of C3a is accelerated in the fluid-phase with the addition of purified factor D. Analysis by sodium dodecylsulfate polyacrylamide gel electrophoresis under reducing conditions showed that the serum iC3b level was elevated when incubated with excess factor D. Secondly, we demonstrated, by measuring the C5a-des-Arg level, that the generation of C5a was promoted in the fluid-phase with the addition of purified factor D. We then studied whether activation of APC is elevated in the blood of patients on maintenance hemodialysis whose sera contained a high concentration of factor D. First, we detected, by fluorescence activated cell sorter analysis, greater amounts of C3d on erythrocytes from the patients (mean fluorescence intensity +/- SD: 7.7 +/- 1.7 arbitrary units) than those from healthy individuals (5.4 +/- 0.5 arbitrary units; p less than 0.001). Secondly, serum C3 level was significantly lower (p less than 0.001) in patients (mean +/- SD: 63.3 +/- 8.2 mg/dl) than in healthy individuals (84.8 +/- 9.5 mg/dl), whereas there was no difference in serum C4 level between patients (32.4 +/- 6.9 mg/dl) and healthy individuals (33.0 +/- 7.4 mg/dl). Serum C5 level was almost the same in patients (10.5 +/- 1.5 mg/dl) and in healthy individuals (11.2 +/- 1.3 mg/dl). These results provide supportive evidence of elevated APC activation in patients with high serum factor D.

Blood Circulation↗
PubMed1991

Glutamic acid-112 of the A subunit of heat-labile enterotoxin from enterotoxigenic Escherichia coli is important for ADP-ribosyltransferase activity.

A mutant strain of enterotoxigenic Escherichia coli (E. coli pTUH 6A) produced an abnormal heat-labile enterotoxin (LT), the A subunit of which has a single amino acid substitution at position 112 (Glu-112 to Lys-112). As already reported, this mutant LT had no ileal loop and vascular permeability activities [(1990) J. Biol. Chem. 265, 22520-22525]. In this paper we report that the mutant LT showed no CHO cell elongation activity and did not activate adenylate cyclase of target cells. Moreover, no ADP-ribosyltransferase activity was detected in the mutant LT. It is concluded that the amino acid substitution at position 112 abolished the ADP-ribosyltransferase activity of the A subunit and this leads to the loss of toxic activities of LT.

Amino Acid Sequence↗
PubMed1991

Detection and purification of the free A subunit of heat-labile enterotoxin produced by enterotoxigenic Escherichia coli.

After removal of total B subunit and heat-labile enterotoxin (LT) from crude cell extracts of enterotoxigenic Escherichia coli (HB 101-EWD 299) by Bio-gel A 5 m column chromatography, the crude cell extract was shown to contain a free A subunit (A' subunit) that did not bind to the coligenoid of the B subunits. The A' subunit was found to be immunologically identical to the A subunit of holo-LT and was purified to show only one band in SDS-poly-acrylamide gel electrophoresis (PAGE). The mobility of the A' subunit was identical to that of the A subunit of holo-LT. The pI value of the A' subunit was also the same as that of the A subunit of holo-LT. These data suggest that in enterotoxigenic E. coli there is free A subunit which may be involved in formation of holo-LT, analogously to free B subunit (coligenoid), and that the free A subunit is physicochemically and immunologically identical to the A subunit of holo-LT.

Bacterial Toxins↗
PubMed1991

FUT-175 as a potent inhibitor of C5/C3 convertase activity for production of C5a and C3a.

We examined the inhibitory effect of FUT-175 on the C3/C5 convertase activity of the cobra venom factor-derived enzyme CVF,Bb by measuring C5b6-mediated reactive lysis of unsensitized guinea pig erythrocytes and by measuring directly the released fragments C3-des-Arg and C5a-des-Arg. In this study, we showed that the concentration of 4.5 X 10(-6) M of FUT-175 caused 50% inhibition of C5 convertase activity of CVF,Bb in reactive hemolysis assays, and that 4.0 X 10(-6) M FUT-175 caused 50% inhibition of the production of C3a and C5a generated by the C3/C5 convertase activity of CVF,Bb.

Animals↗
PubMed1991

Deterioration of immune complex solubilization activity of serum by increased concentration of factor D.

We investigated the effect of excess complement factor D on the immune complex solubilization activity (ICSA) of serum. First, we estimated the concentration of factor D, ICSA and the hemolytic activity via the classical complement pathway (CH50) in the sera of 16 healthy individuals and 36 patients on hemodialysis for end-stage renal failure. The serum concentration of factor D in these patients (mean +/- SD: 12.12 +/- 2.38 micrograms/ml) was significantly higher (p less than 0.001) than that in the healthy subjects (1.02 +/- 0.11 micrograms/ml). In this study, peroxidase and antiperoxidase rabbit IgG were used as immune precipitates for ICSA. The ICSA in patients (45.8 +/- 7.4 normal human serum %, NHS%) was significantly lower (p less than 0.001) than that in the healthy subjects (100.2 +/- 12.5 NHS%). There was no difference in CH50 between the sera of the patients (31.8 +/- 2.5) and that of the healthy group (32.4 +/- 2.6). Second, we determined that increasing amounts of purified factor D added to fresh serum resulted in a decrease in ICSA. This occurred in the serum of a healthy individual as well as in that of a patient. CH50 did not change regardless of the concentration of factor D used. There was an increase in C3 conversion in the sera to which purified factor D had been added, as observed by crossed immunoelectrophoresis. It is suggested that ICSA had deteriorated due to the excess of factor D, which had activated the alternative pathway of complement.

Antigen-Antibody Complex↗
PubMed1990

A single amino acid substitution in the A subunit of Escherichia coli enterotoxin results in a loss of its toxic activity.

A plasmid encoding a mutant gene of heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, was induced by treatment of plasmid EWD 299 with hydroxylamine. A mutant strain of E. coli HB 101 carrying the mutant plasmid pTUH 6A produced a low toxic LT analogue (mutant LT), which was cross-reactive with anti-LT antibody. The mutant LT activity was less than 0.15 and 0.006% of the normal LT in the rabbit ileal loop test and in the rabbit skin permeability test, respectively. The amino acid composition of the mutant LT-B subunit was the same as that of the normal B subunit. Though the A2 fragment of the mutant LT was identical to normal LT by DNA analysis, the A1 fragment of the mutant LT differed from the normal A1 fragment in one amino acid at position 112; namely it had lysine instead of glutamic acid from the N terminus. These data suggest that glutamic acid at position 112 from the N terminus of the A1 fragment is important for the A subunit to express its biological activity.

Amino Acid Sequence↗
PubMed1990

Comparison of coligenoid formation by B subunits of porcine and human Escherichia coli heat-labile enterotoxins.

A hybrid B subunit (coligenoid) of heat-labile enterotoxin could not be made from human heat-labile enterotoxin B subunit(LTh-B) and porcine LTp-B subunit(LTp-B). LTp-B monomer was able to form coligenoid by reassociation with homologous LTp-B monomer, but not with heterogeneous LTh-B monomer and vice versa. The dissociation of both coligenoids into monomers by SDS treatment occurred in a time-dependent manner, but the dissociation of LTh-B colligenoid was faster than that of LTp-B coligenoid. The association of LTp-B monomer is tighter than that of LTh-B monomer. The pI values of LTp-B coligenoid, LTp-B monomer and denatured LTp-B monomer were similar at 9.6-9.8, while the pI values of LTh-B coligenoid, LTh-B monomer and denatured LTh-B monomer were determined as 5.6-5.8, 9.2-9.6 and 9.2-9.6, respectively. All the ionic amino acids of LTp-B exist on the coligenoid surface. The difference in pI values between LTh-B coligenoid and LTh-B monomer suggests that some basic amino acids are located within the LTh-B coligenoid complex, but are exposed in the LTh-B monomer. These data suggest that the 4 amino acid substitutions between LTh-B and LTp-B result in a three dimensional structure difference and a less stable formation of LTh-B coligenoid compared to LTp-B coligenoid.

Animals↗
PubMed1990

Identification of hemolytic granules isolated from human myocardial cells.

Human myocardial cells from fresh autopsy material contained granules which possessed hemolytic activity against guinea pig and rabbit erythrocytes. The hemolytic granules, which had a density of 1.02 and a diameter of 200-300 nm, were recovered as a microsome fraction from subcellular homogenates of human myocardial cells by differential centrifugation in 300 mM sucrose containing 0.1 mM PMSF and 10 mM EDTA. The membrane lesions caused by the granules were ring-like structures with an internal diameter of about 10-17 nm, analogous to that caused by perforin- and complement-induced lysis. However, the requirement for divalent cation differed from that for perforin-induced lysis, since the microsome-mediated lysis occurred in the presence of EDTA.

Animals↗
PubMed1990

Molecular and functional identification and purification of complement component factor D from urine of patients with chronic renal failure.

Urine proteins of normal subject and patients with impaired renal function were analyzed by two-dimensional polyacrylamide gel electrophoresis. As a result, a clear spot was detected specifically in urine from patients with obvious renal dysfunction. The isoelectrical point of this unique spot was pH 7.1-7.2 and the flow-rate (Rf) was 0.50-0.55 as that of albumin was 1.0. Partial amino acid sequence analysis revealed that the NH2-terminal to 22nd amino acid sequence was identical with that of complement factor D. We purified 22 mg of this protein (factor D) from 5000 ml of urine from a patient on hemodialysis by three chromatographic steps using DEAE-Sephadex A-50 and Sephacryl S-200. The purified urine factor D gave a single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis at the position of 23 kD, and displayed normal factor D hemolytic activity. The concentrations of factor D estimated by hemolytic assay were 1.9 micrograms/ml of normal serum, less than 0.1 microgram/ml of normal urine, 15 micrograms/ml of patient serum and 50 micrograms/ml of patient urine.

Amino Acid Sequence↗
PubMed1979

Complement proteins and macrophages. 1. Quantitative estimation of factor B produced by mouse peritoneal macrophages.

Cobra venom factor was used for the detection of factor B synthesized by mouse peritoneal macrophages. This method was shown to be specific for factor B assay by neutralization by antimouse factor B antibody. The amount of factor B in the culture supernatant, assessed by this method, was found to be dependent on the medium used for cultivation of macrophages. The addition of 25% L cell-conditioned medium to minimal essential medium (LCM-MEM) enhanced the production of factor B and also of lysozyme. Kinetic analysis in LCM-MEM showed that factor B produced by 6 x 10(4) cells/cm2 increased up to 72 hr and reached a plateau at 96 hr. The amounts of factor B and lysozyme produced in LCM-MEM depended upon the number of macrophages. Production of factor B was completely inhibited by 1 microgram of cycloheximide per ml and was restored by its removal.

Animals↗