Calcium Export from Neurons and Multi-Kinase Signaling Cascades Contribute to Ouabain Neuroprotection in Hyperhomocysteinemia.

Pathological homocysteine (HCY) accumulation in the human plasma, known as hyperhomocysteinemia, exacerbates neurodegenerative diseases because, in the brain, this amino acid acts as a persistent N-methyl-d-aspartate receptor agonist. We studied the effects of 0.1-1 nM ouabain on intracellular Ca2+ signaling, mitochondrial inner membrane voltage (φmit), and cell viability in primary cultures of rat cortical neurons in glutamate and HCY neurotoxic insults. In addition, apoptosis-related protein expression and the involvement of some kinases in ouabain-mediated effects were evaluated. In short insults, HCY was less potent than glutamate as a neurotoxic agent and induced a 20% loss of φmit, whereas glutamate caused a 70% decrease of this value. Subnanomolar ouabain exhibited immediate and postponed neuroprotective effects on neurons. (1) Ouabain rapidly reduced the Ca2+ overload of neurons and loss of φmit evoked by glutamate and HCY that rescued neurons in short insults. (2) In prolonged 24 h excitotoxic insults, ouabain prevented neuronal apoptosis, triggering proteinkinase A and proteinkinase C dependent intracellular neuroprotective cascades for HCY, but not for glutamate. We, therefore, demonstrated here the role of PKC and PKA involving pathways in neuronal survival caused by ouabain in hyperhomocysteinemia, which suggests existence of different appropriate pharmacological treatment for hyperhomocysteinemia and glutamate excitotoxicity.

Crystal structure and location of gp131 in the bacteriophage phiKZ virion.

Pseudomonas phage ϕKZ and its two close relatives ϕPA3 and 201ϕ2-1 are very large bacteriophages that form a separate branch in phage classification because their genomes are very different from the rest of GenBank sequence data. The contractile tail of ϕKZ is built from at least 32 different proteins, but a definitive structural function is assigned to only one of them-the tail sheath protein. Here, we report the crystal structure of the C-terminal domain of another phiKZ tail protein, gene product 131 (gp131C). We show that gp131 is located at the periphery of the baseplate and possibly associates with fibers that emanate from the baseplate. Gp131C is a seven-bladed ß-propeller that has a shape of a skewed toroid. A small but highly conserved and negatively charged patch on the surface of gp131C might be important for substrate binding or for interaction with a different tail protein.