Insights into the local pathogenesis induced by fish toxins: role of natterins and nattectin in the disruption of cell-cell and cell-extracellular matrix interactions and modulation of cell migration.

Combined proteomic and transcriptomic approaches to study the composition of the venom of Thalassophryne nattereri venomous fish revealed the primary structures of the major toxins as a family of proteases natterins, never described on venoms and a C-type lectin nattectin. To gain new insights into the mechanisms of venom pathogenesis and to further elucidate the role of its major toxins, the natterins and nattectin, we undertook in vitro investigations using these isolated toxins. Here we demonstrated the specific ability of the nattectin to bind types I and V collagen and natterins to bind and cleave type I collagen as well as type IV collagen, disrupting cell attachment and HeLa cells survival. Natterins have cytotoxic effect on both adherent cells or at in suspension, showing direct induction of necrosis that is followed by cell detachment. Nattectin improves integrin-mediated HeLa cell adhesion and resistance to apoptosis by its binding to RGD-dependent integrins, especially the ß1 subunit. Based on our studies we now report that extracellular matrix (ECM) components as well as the integrin ß1 subunit are targets for the natterins and nattectin. The ECM degradation or remodeling activities exerted by these toxins affect cell-cell and cell-ECM adhesion and survival and impair inflammatory cell migration into inflamed tissues.

[Long-term and acute toxicity of kallikrein from the venom of Agkistrodon hlays Pallas].

A novel serine protease with high purity was extracted from the venom of Agkistrodon hlays Pallas using monoclonal antibody affinity chromatography. This protease releases bradykinin and has arginine esterase activity without being activated. After purification, its hydrolytic activity exceeded 800 U/mg, far higher than its counterparts from mammalian sources. The purity of the kininogenase could exceed 95%. The acute toxicity and the long-term toxicity of this kallikrein was studied for its potential clinical application. The maximum tolerance dose in adult was 150,000 times greater than the maximum applied dose, and long-term administration at the dose 50 times of allowed clinical dose did not obviously after the animals' body weight, survival condition, liver function, renal function, or blood routines, suggesting the extremely low toxicity of the kallidrein.

Early activation of bradykinin B2 receptor aggravates reactive oxygen species generation and renal damage in ischemia/reperfusion injury.

The kallikrein/kinin system is beneficial in ischemia/reperfusion injury in heart, controversial in brain, but detrimental in lung, liver, and intestine. We examined the role of the kallikrein/kinin system in acute ischemia/reperfusion renal injury induced by 40 min occlusion of the renal artery followed by reperfusion. Rats were infused with tissue kallikrein protein 5 days before (pretreated group) or after (treated group) ischemia. Two days later, the pretreated group exhibited the worst renal dysfunction, followed by the treated group, then the control group. Kallikrein increased tubular necrosis and inflammatory cell infiltration with generation of more tumor necrosis factor-alpha and monocyte chemoattractant protein-1. Reactive oxygen species (ROS), malondialdehyde, and reduced/oxidized glutathione measurement revealed that the oxidative stress was augmented by kallikrein administration in both ischemic and reperfusion phases. The groups with more ROS generation also had more apoptotic renal cells. The deleterious effects of kallikrein on ischemia/reperfusion injury were reversed by cotreatment with bradykinin B2 receptor (B2R) antagonist, but not B1 receptor antagonist, and were not associated with hemodynamic changes. We conclude that early activation of B2R augmented ROS generation in ischemia/reperfusion renal injury, resulting in subsequent apoptosis, inflammation, and tissue damage. This finding suggests the potential application of B2R antagonists in acute ischemic renal disease associated with bradykinin activation.

Highly selective synthetic inhibitors with regard to plasma-kallikrein activities.

The synthetic inhibitors of plasma kallikrein (PK) were found, which are called PKSI-1007, PKSI-0180 and PKSI-0527 in our laboratories. (1) The inhibitors inhibited PK competitively with D-Pro-Phe-Arg-pNA and the Ki values obtained were considerably small, 10(-6) M-10(-7) M. However, the Ki values for glandular kallikrein (GK), plasmin (PL), thrombin (TH) and factor Xa (FXa) were larger. In particular, a selectivity of PKSI-0527 towards PK was very high and the toxicity was weak (i.v. LD50 for mice is over 100 mg/kg). (2) The inhibitors were effective (a) to prevent the bradykinin formation in the kaolin-activated human plasma and the acid-treated ascites taken from the mice bearing Sarcoma 180, (b) to prolong the coagulation time by contact activation, and (c) to inhibit the enhancement of ADP-platelet aggregation by PK. The results indicated that the some PKSI-inhibitors will be much useful for the basic studies, furthermore they deem to be even promising towards the clinical application.