Chip electrophoretic separation of highly homologous ammodytoxin isoforms: three neurotoxic phospholipases A2 of Vipera ammodytes ammodytes venom.

Ammodytoxins (Atxs), a group of Ca(2+) -dependent neurotoxic phospholipases A2 of Vipera ammodytes ammodytes venom, are mainly responsible for venom toxicity. Within the Atx group, LD50 values between three isoforms, A, B, and C are differing with AtxA exhibiting an LD50 value by an order of magnitude lower (more toxic) than the other two isoforms. This difference in toxicity justifies the necessity to prepare suitable antibodies and thus isoform separation to characterize the Atx content of Vipera ammodytes ammodytes venom is of importance. However, a high homology between the three Atx isoforms (differences in only two, respectively, three residues within the last 18 amino acids at the C-terminus, total length 122 residues) hindered the successful separation of isoforms to date. As the investigated phospholipases A2 were reported to exhibit differences in pI values, we concentrate with the current work on the separation of Atx isoforms after fluorescence labeling via chip electrophoresis on a commercially available instrument to build the basis for a fast and easy to handle screening method. In the course of our work, we were able to show that samples of AtxA, AtxB, and AtxC declared to be homogenous by standard analytical techniques consisted indeed of more than one isoform of which the relative amounts were calculated by using the newly developed method.

Degeneration and atherosclerosis inducing increased deposition of type IIA secretory phospholipase A2, C-reactive protein and complement in aortic valves cause neutrophilic granulocyte influx.

BACKGROUND AND AIM OF THE STUDY: Recent studies have indicated that atherosclerosis-like changes are involved in the pathogenesis of aortic valve stenosis. Increased blood and valve tissue levels of C-reactive protein (CRP) have been reported in patients with aortic valve disease, although the different pathological conditions involved were not analyzed. The study aim was to monitor the deposition of CRP, its activator sPLA2-IIA and its effector complement, and the subsequent influx of neutrophilic granulocytes in degenerative and atherosclerotic aortic valves. METHODS: Human tricuspid aortic valves (n = 57) obtained at autopsy included five control valves, 36 aortic valves with atherosclerotic changes, and 16 with degenerative changes. All valves were analyzed immunohistochemically for the presence of sPLA2-IIA, CRP, C3d and MPO (to detect neutrophilic granulocytes), and subsequently quantified using computer-assisted morphometry. RESULTS: In aortic valves with degeneration, the areas of sPLA2-IIA, CRP and complement deposition were all significantly increased compared to control valves. These mediators were even more extensively deposited in atherosclerotically changed aortic valves. The increased deposition of these mediators coincided with a significant increase of neutrophilic granulocytes in atherosclerotic and degenerated aortic valves, compared to control valves. CONCLUSION: The study results indicate that sPLA2-IIA, CRP, and C3d are significantly more activated in atherosclerotic aortic valves compared to degeneratively changed aortic valves. A significant increase was also identified in neutrophilic granulocytes in non-infectious, diseased valves (atherosclerosis and degeneration).

Secretory type II phospholipase A2 is produced and secreted by epicardial adipose tissue and overexpressed in patients with coronary artery disease.

CONTEXT: Epicardial adipose tissue (EAT) is a visceral adipose tissue in close contact with coronary vessels, the excess of which is associated with coronary artery disease (CAD). OBJECTIVE: Our goal was to identify candidate molecule(s) characterizing EAT that could intervene in the pathogenesis of CAD. DESIGN: An approach combining microarrays and bioinformatic sequence analysis tools for predicting secreted proteins (TargetP) was applied to paired biopsies of sc adipose tissue (SAT) and EAT, obtained from patients with or without CAD (NCAD). RESULTS were validated in three independent groups of subjects by quantitative RT-PCR, Western blot, immunohistochemistry, and explant secretion. RESULTS: Secretory type II phospholipase A2 (sPLA2-IIA) ranked as the highest gene coding for potentially secreted proteins with the highest overexpression in EAT in both CAD and NCAD. Quantitative RT-PCR confirmed its increased expression in EAT (P < 0.01) as well as EAT from CAD as compared with NCAD (49.3 +/- 13 vs. 17.4 +/- 9.7 P < 0.01). sPLA2-IIA protein levels were higher in EAT than SAT (P < 0.001). EAT explants also showed significantly higher sPLA2-IIA secretion levels than SAT ones (4.37 +/- 2.7 vs. 0.67 +/- 0.28 ng/ml to 1 per gram tissue per 24 h, P < 0.03). sPLA2-IIA labeling was seen in the stroma vascular fraction between adipocytes and in connective capsules in EAT, with no immunostaining of the adipocytes. SAT was weakly labeled following the same process. CONCLUSION: We have shown for the first time an increased expression of sPLA2-IIA in EAT in patients with CAD. sPLA2-IIA is a phospholipase, which has been shown to be an independent risk factor for CAD. These findings suggest that EAT has a potentially pathophysiological role in CAD.

Crucial role of group IIA phospholipase A2 in pancreatitis-associated adrenal injury in acute necrotizing pancreatitis.

This study investigated the mechanistic role of group IIA phospholipase A2 (sPLA2-IIA) in the process of pancreatitis-associated adrenal injury in acute necrotizing pancreatitis. One hundred and sixty male Wistar rats were subdivided into a sham-operated group, a sodium taurocholate-induced pancreatitis group, and a pancreatitis group pretreated with sPLA2 inhibitor (pku-mdl-101). The sPLA2 inhibitor was administered by intravenous injection 30 min before induction of pancreatitis. The severity of pancreatitis was evaluated by serum amylase and pancreatic histological score. The serum corticosterone level was measured. Adrenal injury was evaluated by histological evaluation, as well as by sPLA2 activity and sPLA2-IIA protein analysis. Pancreatitis resulted in a time-related increase in serum amylase and in the pancreatic histological score. At first, serum corticosterone increased after 3h and decreased rapidly after 6h in pancreatitis. Adrenal injury aggravated during the observation period. The sPLA2 activity in the serum and adrenal glands, as well as the expression of sPLA2-IIA protein in adrenal glands increased and peaked 6h after the induction of pancreatitis. Pretreatment of sPLA2 inhibitor significantly reduced the severity of pancreatitis and adrenal histological injury, improved the 24-h serum corticosterone levels, and effectively inhibited sPLA2 activity and sPLA2-IIA expression in adrenal glands. The sPLA2 inhibitor attenuated the severity of pancreatitis and pancreatitis-associated adrenal injury. The observations indicate that sPLA2-IIA plays a crucial role in pancreatitis-associated adrenal injury in acute necrotizing pancreatitis.