A Biparatopic Intrabody Renders Vero Cells Impervious to Ricin Intoxication.

Expression of camelid-derived, single-domain antibodies (VHHs) within the cytoplasm of mammalian cells as "intrabodies" has opened-up novel avenues for medical countermeasures against fast-acting biothreat agents. In this report, we describe a heterodimeric intrabody that renders Vero cells virtually impervious to ricin toxin (RT), a potent Category B ribosome-inactivating protein (RIP). The intrabody consists of two structurally defined VHHs that target distinct epitopes on RT's enzymatic subunit (RTA): V9E1 targets RTA's P-stalk recruitment site, and V2A11 targets RTA's active site. Resistance to RT conferred by the biparatopic VHH construct far exceeded that of either of the VHHs alone and effectively inhibited all measurable RT-induced cytotoxicty in vitro. We propose that targeted delivery of bispecific intrabodies to lung tissues may represent a novel means to shield the airways from the effects of inhalational RT exposure.

Ortho-substituted but not coplanar PCBs rapidly kill cerebellar granule cells.

Several PCB congeners were assessed for their cytotoxicity on cerebellar granule cells in an attempt to compare their structure-activity relationship as potential neurotoxicants and to assess the mechanisms associated with their toxicity. Flow cytometry was used to monitor the changes of a number of biochemical endpoints: membrane integrity, intracellular free calcium concentration ([Ca(2+)](i)), reactive oxygen species (ROS) production, mitochondrial membrane potential (Delta psi(m)), and cell size. The non-coplanar, ortho-substituted congeners, PCB 8 (2,4'-dichlorobiphenyl), PCB 28 (2,4,4'-trichlorobiphenyl), PCB 47 (2,4,2',4'-tetrachlorobiphenyl), and PCB 52 (2,5,2',5'-tetrachlorobiphenyl) (10 microM) killed neurons to different degrees within 30 min. Loss of viability was accompanied by increased [Ca(2+)](i) and decreased Delta psi(m). No significant changes of ROS level were observed during exposure. The coplanar congeners, PCB 77 (3,4,3',4'-tetrachlorobiphenyl), PCB 80 (3,5,3',5'-tetrachlorobiphenyl), and PCB 81 (3,4,5,4'-tetrachlorobiphenyl) (10 microM), had no effects on membrane integrity, [Ca(2+)](i) or Delta psi(m) in this time period of exposure. In Ca(2+)-free Tyrode's medium, there was no [Ca(2+)](i) increase after exposure to the ortho-substituted congeners, but also no reduction in loss of membrane integrity, suggesting Ca(2+) influx was not the cause of viability loss. The mitochondrial uncoupler, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (1-2 microM), caused a large decrease of Delta psi(m), but only a slight loss of viability, which suggested that Delta psi(m) is not the primary cause of PCB 52-induced cell death. These studies show that ortho-substituted PCBs are toxic to cerebellar granule cells; however, their toxic action is not secondary to elevation of intracellular calcium, a change in mitochondrial membrane potential, or free radical generation.