Proteomics of the nucleus ovoidalis and field L brain regions of zebra finch

The purpose of present study is to analyze the brain proteome of the nucleus ovoidalis (OV) and Field L regions of the zebra finch (Taeniopygia guttata). The OV and Field L are important brain nuclei in song learning in zebra finches; their analyses identified a total of 79 proteins. The zebra finch brain proteome analyses are poised to provide clues about cell and circuit layout as well as possible circuit function

Proteomic analyses of songbird (Zebra finch; Taeniopygia guttata) retina

Proteomic analyses of male songbird (Zebra finch; Taeniopygia guttata; ZF) retina were performed resulting in identification of 129 proteins. Comparison of T. guttata retinal proteome with that of chicken found proteins detected in both retinas. Immunohistochemical analyses of T. guttata retinal sections and Western analyses of total retinal protein extract were performed confirming presence of select bona fide retinal proteins. Results demonstrate the utility of one-dimensional gel fractionation for mass spectrometry and will be useful for future proteomic comparison of songbird retina and brain tissues in different behavioral and pharmacological studies

Neurotoxicity of Organomercurial Compounds

ReviewMercury is a ubiquitous contaminant, and a range of chemical species is generated by human activity and natural environmental change. Elemental mercury and its inorganic and organic compounds have different toxic properties, but all them are considered hazardous in human exposure. In an equimolecular exposure basis, organomercurials with a short aliphatic chain are the most harmful compounds and they may cause irreversible damage to the nervous system. Methylmercury (CH3Hg+) is the most studied following the neurotoxic outbreaks identified as Minamata disease and the Iraq poisoning. The first description of the CNS pathology dates from 1954. Since then, the clinical neurology, the neuropathology and the mechanisms of neurotoxicity of organomercurials have been widely studied. The high thiol reactivity of CH3Hg+, as well as all mercury compounds, has been suggested to be the basis of their harmful biological effects. However, there is clear selectivity of CH3Hg+ for specific cell types and brain structures, which is not yet fully understood. The main mechanisms involved are inhibition of protein synthesis, microtubule disruption, increase of intracellular Ca2+ with disturbance of neurotransmitter function, oxidative stress and triggering of excitotoxicity mechanisms. The effects are more damaging during CNS development, leading to alterations of the structure and functionality of the nervous system. The major source of CH3Hg+ exposure is the consumption of fish and, therefore, its intake is practically unavoidable. The present concern is on the study of the effects of low level exposure to CH3Hg+ on human neurodevelopment, with a view to establishing a safe daily intake. Recommendations are 0.4 µg/kg body weight/day by the WHO and US FDA and, recently, 0.1 µg/kg body weight/day by the US EPA. Unfortunately, these levels are easily attained with few meals of fish per week,depending on the source of the fish and its position in the food chain.This work was supported by grants FIS 00-1094 and CIRIT SGR00047. J.S. is a recipient of an IDIBAPS fellowship. We thank Dr. Santiago Barambio, Yolanda Trejo and Irene Gallego from Tutor Médica Clinics for providing the human tissues used for the cell culture experiments illustrating this review. Permission to use human tissue was obtained from the Ethics Committee of the CSIC.Peer reviewe

A cell based screening approach for identifying protein degradation regulators

<p>Cellular transitions are achieved by the concerted actions of regulated degradation pathways. In the case of the cell cycle, ubiquitin mediated degradation ensures unidirectional transition from one phase to another. For instance, turnover of the cell cycle regulator cyclin B1 occurs after metaphase to induce mitotic exit. To better understand pathways controlling cyclin B1 turnover, the N-terminal domain of cyclin B1 was fused to luciferase to generate an N-cyclin B1-luciferase protein that can be used as a reporter for protein turnover. Prior studies demonstrated that cell-based screens using this reporter identified small molecules inhibiting the ubiquitin ligase controlling cyclin B1-turnover. Our group adapted this approach for the G2-M regulator Wee1 where a Wee1-luciferase construct was used to identify selective small molecules inhibiting an upstream kinase that controls Wee1 turnover. In the present study we present a screening approach where cell cycle regulators are fused to luciferase and overexpressed with cDNAs to identify specific regulators of protein turnover. We overexpressed approximately 14,000 cDNAs with the N-cyclin B1-luciferase fusion protein and determined its steady-state level relative to other luciferase fusion proteins. We identified the known APC/C regulator Cdh1 and the F-box protein Fbxl15 as specific modulators of N-cyclin B1-luciferase steady-state levels and turnover. Collectively, our studies suggest that analyzing the steady-state levels of luciferase fusion proteins in parallel facilitates identification of specific regulators of protein turnover.</p

Activation Domain-dependent Degradation of Somatic Wee1 Kinase*

Cell cycle progression is dependent upon coordinate regulation of kinase and proteolytic pathways. Inhibitors of cell cycle transitions are degraded to allow progression into the subsequent cell cycle phase. For example, the tyrosine kinase and Cdk1 inhibitor Wee1 is degraded during G2 and mitosis to allow mitotic progression. Previous studies suggested that the N terminus of Wee1 directs Wee1 destruction. Using a chemical mutagenesis strategy, we report that multiple regions of Wee1 control its destruction. Most notably, we find that the activation domain of the Wee1 kinase is also required for its degradation. Mutations in this domain inhibit Wee1 degradation in somatic cell extracts and in cells without affecting the overall Wee1 structure or kinase activity. More broadly, these findings suggest that kinase activation domains may be previously unappreciated sites of recognition by the ubiquitin proteasome pathway