Masahiko Akita

Purification and characterization of enterotoxin produced by Aeromonas sobria.

We purified the toxin of Aeromonas sobria capable of inducing a positive response in the mouse intestinal loop assay. The purified toxin showed a positive response not only in the loop assay but also in a hemolytic assay. Subsequently, we cloned the toxin gene and demonstrated that the product of this gene possessed both hemolytic and enterotoxic activities. These results showed that the enterotoxin of A. sobria possesses hemolytic activity. Nucleotide sequence determination of the toxin gene and amino acid sequence analysis of the purified toxin revealed that it is synthesized as a precursor composed of 488 amino acid residues, and that the 24 amino‐terminal amino acid residues of the precursor is removed in the mature toxin. As antiserum against the purified toxin neutralized the fluid accumulation induced by living cells not only of A. sobria but also of A. hydrophila, this and antigenically related toxin(s) are thought to play an essential role in the induction of diarrhea by these organisms. The toxin‐injured Chinese hamster ovary (CHO) cells induced the release of intracellular lactose dehydrogenase (LDH). The release of LDH from CHO cells and the lysis of erythrocytes by the toxin were repressed by the addition of dextran to the reaction solution, indicating that the toxin forms pores in the membranes and that the cells were injured by the osmotic gradient developed due to pore formation. However, the histopathological examination of intestinal cells exposed to the toxin showed that it caused fluid accumulation in the mouse intestinal loop without causing cellular damage.

Involvement of tachykinin receptors in Clostridium perfringens beta-toxin-induced plasma extravasation

Clostridium perfringens beta‐toxin causes dermonecrosis and oedema in the dorsal skin of animals. In the present study, we investigated the mechanisms of oedema induced by the toxin. The toxin induced plasma extravasation in the dorsal skin of Balb/c mice. The extravasation was significantly inhibited by diphenhydramine, a histamine 1 receptor antagonist. However, the toxin did not cause the release of histamine from mouse mastocytoma cells. Tachykinin NK1 receptor antagonists, [D‐Pro2, D‐Trp7,9]‐SP, [D‐Pro4, D‐Trp7,9]‐SP and spantide, inhibited the toxin‐induced leakage in a dose‐dependent manner. Furthermore, the non‐peptide tachykinin NK1 receptor antagonist, SR140333, markedly inhibited the toxin‐induced leakage. The leakage induced by the toxin was markedly reduced in capsaicin‐pretreated mouse skin but the leakage was not affected by systemic pretreatment with a calcitonin gene‐related peptide receptor antagonist (CGRP8‐37). The toxin‐induced leakage was significantly inhibited by the N‐type Ca2+ channel blocker, ω‐conotoxin MVIIA, and the bradykinin B2 receptor antagonist, HOE140 (D‐Arg‐[Hyp3, Thi5, D‐Tic7, Oic8]‐bradykinin), but was not affected by the selective L‐type Ca2+ channel blocker, verapamil, the P‐type Ca2+ channel blocker, ω‐agatoxin IVA, tetrodotoxin (TTX), the TTX‐resistant Na+ channel blocker, carbamazepine, or the sensory nerve conduction blocker, lignocaine. These results suggest that plasma extravasation induced by beta‐toxin in mouse skin is mediated via a mechanism involving tachykinin NK1 receptors.

Physicochemical and biological properties of an extracellular serine protease of Aeromonas sobria.

Previously, we cloned a protease gene of Aeromonas sobria, determined its nucleotide sequence and established a method of purifying its product. In this study, we examined the properties of the purified protease. The protease was temperature‐labile and had an optimal pH of 7.5. Metallo‐protease inhibitors and a cysteine protease inhibitor did not block the proteolytic activity of the enzyme. The treatment with reagents to modify sulfhydryl group did not reduce the activity. But, serine protease inhibitors did, showing that it was a serine protease. Subsequently, we examined the ability of the protease to enhance vascular permeability in dorsal skin. The protease showed activity and the reaction was inhibited by a simultaneously injected antihistaminic agent. Histopathological examination showed that mast cells appeared around the site where the protease was injected. These findings show that the vascular permeability‐enhancing effect of the protease is due to histamine released at the site. Furthermore, we found that a soybean trypsin inhibitor (Kunitz) did not block the proteolytic action of the protease in vitro, but inhibited its vascular permeability‐enhancing activity in skin. This suggests that a trypsin‐like protease from skin mediates the activity of the protease to enhance its vascular permeability.