Akio Miyama

Primary structure of heat-stable enterotoxin produced by Yersinia enterocolitica

A heat-stable enterotoxin was isolated and purified from the culture supernatant of Yersinia enterocolitica by reversed-phase high-performance liquid chromatography. The amino acid sequence of the purified toxin was determined to be as follows: (sequence; see text) (X: not determined). The C-terminal sequence containing 6 half-cystine residues was highly homologous to that of heat-stable enterotoxin of enterotoxigenic Escherichia coli.

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.

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.

Effect of Yersinia enterocolitica ST on Cyclic Guanosine 3′,5′-Monophosphate Levels in Mouse Intestines and Cultured Cells

We determined the effect of heat‐stable enterotoxin produced by Yersinia enterocolitica (Y. enterocolitica ST) on cyclic nucleotide levels in the intestines of 6‐day‐old mice and in cultured cell line cells. The concentration of cyclic guanosine 3′,5′‐monophosphate (cyclic GMP) in homogenates of the intestines increased four‐ to fivefold by 3 min after intragastric administration of 10 units of purified Y. enterocolitica ST. This increase continued for 60 min, and then the concentration of cyclic GMP fell toward the levels of the controls. On the other hand, fluid accumulation in the intestines was not evident until 60 min after administration of the toxin. Thus, the increase in intestinal cyclic GMP concentration preceded measurable fluid accumulation. The effect on both cyclic GMP levels and fluid accumulation was abolished by treatment of the ST with either alkali solution (pH 10.7) or 2‐mercaptoethanol. Likewise, cyclic GMP levels in cultured cells (CCL‐6, HeLa, L, and Mm‐1 cells) increased dose‐dependently by 10 min after incubation of the cells with the ST. Cyclic adenosine 3′,5′‐monophosphate levels in both intestines and cultured cells were not affected by the toxin.

Adherence of Yersinia enterocolitica to Mammalian Epithelial Cell Lines

Yersinia enterocolitica RIMD 2501003 grown at 25 C avidly adhered to various kinds of cultured epithelial cell lines (HeLa, FL, Y‐1 adrenal, human intestine, human conjunctiva) but the bacteria grown at 37 C did not adhere. This phenomenon paralleled the temperature‐dependent motility of the bacteria. To clarify the adherence mechanism, we obtained two kinds of mutants, an immobile mutant and a nonadherent mutant, by treatment with A‐methyl‐A‐nitro‐A‐nitrosoguanidine. The immobile mutant did not move on soft agar but retained the capacity to adhere to cultured epithelial cells when grown at 25 C. The nonadherent mutant did not adhere to cultured epithelial cells but retained the ability to move on soft agar when grown at 25 C.

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.

Complement proteins and macrophages. II. The secretion of factor B by lipopolysaccharide-stimulated macrophages.

The secretion of factor B by mouse peritoneal macrophages was found to be enhanced following in vivo or in vitro stimulation with lipopolysaccharide (LPS). The intravenous administration of LPS to mice of various strains caused an increased release of factor B but not the release of acid phosphatase by the peritoneal macrophages obtained from the stimulated mice. In vitro stimulation of cultured macrophages with LPS resulted in an enhanced secretion of both factor B and acid phosphatase. The dose‐dependent augmentation of factor B secretion by LPS was found in the macrophages from LPS‐responsive C3H/HeN mice, whereas the macrophages from LPS‐unresponsive C3H/HeJ mice did not respond to either phenol‐extracted LPS or butanol‐extracted LPS. The ability of LPS to cause the enhancement of factor B secretion by macrophages was abolished by alkali or acid treatment of LPS, indicating that its lipid A part was responsible for the observed effect.

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 enterotoxigenicEscherichia coli.LT was digested by the protease and contained a fragment which had the same mobility on SDS-PAGE as that of the Al 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.

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.

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.