Enhanced calmodulin binding concurrent with increased kinase-dependent phosphorylation of cytoskeletal proteins following a single subcutaneous injection of diisopropyl phosphorofluoridate in hens.

Diisopropyl phosphorofluoridate (DFP) produces Type I organophosphorus compound-induced delayed neurotoxicity (OPIDN) in adult female chickens. We have proposed that calcium/calmodulin protein kinase II (CaM kinase II) plays a role in the development of OPIDN by increasing the phosphorylation of cytoskeletal proteins. We investigated in vivo the effects of treatment of DFP on CaM kinase II-dependent phosphorylation. In isolated brain supernatants from DFP-treated hens, calmodulin binding increased concurrent with increases in CaM kinase II-dependent autophosphorylation and phosphorylation of cytoskeleton proteins. There were no changes in the relative amounts of the enzyme based on immunobinding studies of antibodies to the CaM kinase II. In the absence of any exogenously added substrate. CaM kinase II and microtubule associated protein-2 (MAP-2) exhibited substantially increased phosphorylation, 833 and 275%, respectively, over brain supernatants from untreated hens. Moreover, isolated brain supernatants from treated hens with exogenously added cytoskeletal proteins and myelin basic protein (MBP) exhibited significant increases in phosphorylation over control, 233, 332 and 60%, for MAP-2, tubulin, and MBP, respectively. 125I-Calmodulin binding studies revealed a 136% increase in calmodulin binding to CaM kinase II in treated hens when compared to control groups. The data suggest that in vivo DFP treatment increases the percentage of unphosphorylated, active CaM kinase II resulting in increased calmodulin binding and subsequent enhanced phosphorylation of cytoskeletal proteins that leads to their aggregation and the production of axonal degeneration.

Carbon disulfide inhalation increases Ca2+/calmodulin-dependent kinase phosphorylation of cytoskeletal proteins in the rat central nervous system.

The Ca2+/calmodulin-dependent phosphorylation of neuronal cytoskeletal proteins was studied in brain supernatants prepared from rats exposed via inhalation to 600 to 800 ppm carbon disulfide (CS2) for 14 days. Exposure to CS2 resulted in increased phosphorylation of endogenous MAP-2 and exogenously added neurofilament triplet proteins. There also was an observed increase in the autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). Slight increases in the binding of a monoclonal antibody to the alpha subunit of CaM kinase II were seen, while large increases in the binding of [125I]calmodulin to the alpha subunit of CaM kinase II also were observed. The finding of large increases in the autophosphorylation and calmodulin-binding to CaM kinase II with only slight increases in the amount of antibody-binding suggests that CS2 exposure results in increased Ca2+/calmodulin-dependent phosphorylation of proteins by inducing an increase in kinase activity.

Genotoxic effects of stannous chloride (SnCl2) in K562 cell line.

The toxic effects of SnCl2 in K562 cells were analyzed in this study. This cell line is resistant to reactive oxygen species (ROS) making it suitable to evaluate the impact of SnCl2 in culture either through ROS or by direct toxicity using Trypan blue dye exclusion, comet and flow cytometry assays. An important loss of viability induced by SnCl2 in a dose-response manner was observed in cells treated in Tris-buffered saline (TBS). This necrotic cell death was further confirmed by flow cytometry. On the other hand, there was no loss of viability when cells were treated in rich medium (RPMI). DNA damage was visualized in SnCl2-treated K562 cells in both tested conditions. The data indicate that SnCl2 induces DNA damage and reduces K562 viability. Both actions seem to be correlated with ROS formation and direct linkage to DNA.

An international perspective on the treatment of pediatric shock: the Brazilian experience.

Shock in the pediatric population has many preventable causes. Treatment of children in shock will depend on access to health services, training of health personnel, availability of diagnostic procedures, monitoring, and therapeutic measures. Countries will differ among themselves and within themselves in the care provided to children developing shock. In Brazil, the majority of children are cared for in public hospitals, which often lack resources for basic care. Many children in shock do not even reach healthcare services. Investment in training healthcare personnel in a simplified and systematic approach to shock and access to equipped health services are basic to improved outcomes in the treatment of pediatric shock. The Brazilian experience in the treatment of children in shock outside hospital facilities, in the emergency department, and in the ICU is described.