Expression of recombinant human argininosuccinate synthetase in Escherichia coli

Escherichia coli strain BL21(DE3)/pET42a/ASS – an efficient producer of recombinant human argininosuccinate synthetase (rhASS) – was constructed, and preparations of purified rhASS were obtained using His-tag affinity chromatography. The effect of specific inhibitor, α-methyl-DL-aspartate, and nitric oxide donor, sodium nitroprusside, on the ASS specific activity was evaluated with purified rhASS protein and in mouse liver lysates. The developed expression platform is a useful tool in search for new ASS inhibitors efficient under in vitro and in vivo conditions

A potential role for T-type calcium channels in homocysteinemia-induced peripheral neuropathy

Homocysteinemia is a metabolic condition characterized by abnormally high level of homocysteine in the blood and is considered to be a risk factor for peripheral neuropathy. However, the cellular mechanisms underlying toxic effects of homocysteine on the processing of peripheral nociception have not yet been investigated comprehensively. Here, using a rodent model of experimental homocysteinemia, we report the causal association between homocysteine and the development of mechanical allodynia. Homocysteinemia-induced mechanical allodynia was reversed on pharmacological inhibition of T-type calcium channels. In addition, our in vitro studies indicate that homocysteine enhances recombinant T-type calcium currents by promoting the recycling of Cav3.2 channels back to the plasma membrane through a protein kinase C-dependent signaling pathway that requires the direct phosphorylation of Cav3.2 at specific loci. Altogether, these results reveal an unrecognized signaling pathway that modulates the expression of T-type calcium channels, and may potentially contribute to the development of peripheral neuropathy associated with homocysteinemia

A potential role for T-type calcium channels in homocysteinemia-induced peripheral neuropathy

Homocysteinemia is a metabolic condition characterized by abnormally high level of homocysteine in the blood and is considered to be a risk factor for peripheral neuropathy. However, the cellular mechanisms underlying toxic effects of homocysteine on the processing of peripheral nociception have not yet been investigated comprehensively. Here, using a rodent model of experimental homocysteinemia, we report the causal association between homocysteine and the development of mechanical allodynia. Homocysteinemia-induced mechanical allodynia was reversed on pharmacological inhibition of T-type calcium channels. In addition, our in vitro studies indicate that homocysteine enhances recombinant T-type calcium currents by promoting the recycling of Cav3.2 channels back to the plasma membrane through a protein kinase C-dependent signaling pathway that requires the direct phosphorylation of Cav3.2 at specific loci. Altogether, these results reveal an unrecognized signaling pathway that modulates the expression of T-type calcium channels, and may potentially contribute to the development of peripheral neuropathy associated with homocysteinemia