Electron microscopy of alpha 2-macroglobulin with a thiol ester bound ligand.

International audienceIn order to covalently bind the hydrolyzed thiol ester groups of the human alpha 2-macroglobulin (alpha 2M) transformed by methylamine, the phospholipase A2 (PLA2), a small enzyme (M(r) = 13,000) from Naja nigricollis snake venom was activated by succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Average images determined from electron micrographs of the methylamine-transformed alpha 2M, with and without activated PLA2, were determined by image processing and compared. A localization of the PLA2 was achieved by subtracting the average image of alpha 2M transformed by methylamine from that containing PLA2. The results are consistent with previous work showing the central localization of chymotrypsin trapped in alpha 2M. They also suggest that the four thiol esters are located near the center of the alpha 2M molecule

Intravenous and oral itraconazole versus intravenous amphotericin B deoxycholate as empirical antifungal therapy for persistent fever in neutropenic patients with cancer who are receiving Broad-spectrum antibacterial therapy: A randomized, controlled trial

Prolonged neutropenia is a major risk factor for invasive fungal infection (1–6). The incidence among neutropenic patients with cancer who are receiving intensive cytotoxic therapy ranges from 2% to 47%, depending on other concomitant risk factors (7). Mortality rates range from 35% to 90% (8). Fever may be the only clinical sign of infection, and definitive diagnosis is often problematic. Empirical therapy with amphotericin B deoxycholate reduces the relative risk for documented infection by 50% to 80% and overall mortality rates by 23% to 45% (1–2, 9–10). This practice is now standard in neutropenic patients with cancer who have persistent fever that does not respond to 3 to 7 days of treatment with broad-spectrum antibiotics (11)

Comparison of crotoxin isoforms reveals that stability of the complex plays a major role in its pharmacological action

Crotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus is a potent neurotoxin consisting of a weakly toxic phospholipase-A2 subunit (CB) and a non-enzymic, non-toxic subunit (CA). Crotoxin complex (CACB) dissociates upon interaction with membranes: CB binds while CA does not. Moreover, CA enhances the toxicity of CB by preventing its non-specific adsorption. Several crotoxin isoforms have been identified. Multiple variants of each subunit give different crotoxin complexes that can be subdivided into two classes: those of high toxicity and low enzymic activity and those of moderate toxicity and a high phospholipase-A2 activity. In this study, we demonstrate that the more-toxic isoforms block neuromuscular transmission of chick biventer cervicis preparations more efficiently than weakly toxic isoforms. The less-toxic crotoxin complexes have the same Km and Vmax as CB alone. In contrast, the more-toxic isoforms are enzymically less active than CB. These differences correlate with the stability of the complexes: less-toxic isoforms are less stable (Kd = 25 nM) and dissociate rapidly (half-life about 1 min), whereas the more-toxic isoforms are more stable (Kd = 4.5 nM) and dissociate more slowly (half-life 10-20 min). The rate of interaction of crotoxin complexes with vesicles of negatively charged phospholipids paralleled the rate of dissociation of the complexes in the absence of vesicles. The differences of pharmacological and biochemical properties of crotoxin isoforms indicate that the stability of crotoxin complexes plays a major role in the synergistic action of crotoxin subunits: a stronger association between the two crotoxin subunits would account for their slower dissociation rate, a weaker enzymic activity, a slower interaction with phosphatidylglycerol vesicles, a faster blockade of neuromuscular transmission and a higher lethal potency