Glyceraldehyde phosphate dehydrogenase oxidation during Cardiac ischemia and reperfusion

Objectives: Protein S-glutathiolation is a predicted mechanism by which protein thiol groups are oxidized during the oxidative stress of ischaemia and reperfusion. We measured protein S-thiolation during ischaemia and reperfusion and investigated the effect of this oxidative modification on the function of GAPDH. Methods: Glutathione was biotinylated (biotin-GSH) and used to probe for protein S-glutathiolation in isolated rat hearts using non-reducing Western blots and streptavidin-HRP. Streptavidin-agarose was used to purify S-glutathiolated proteins and these were identified using N-terminal sequencing and database searching.Results: Little protein S-glutathiolation occurred in control preparations, but this increased 15-fold during reperfusion. Protein S-glutathiolation was attenuated by the antioxidant mercaptopropionylglycine and was shown to occur only during the first minutes of reperfusion. Affinity purification of the S-glutathiolated proteins showed 20 dominant S-glutathiolation substrates. A dominant S-thiolated protein was N-terminally sequenced (VKVGVNGFG) and HPLC peptide mapping gave additional sequence nearer the site of oxidation (TGVFTTMEKA). The first sequence was the N-terminus of GAPDH, and the second a peptide from the same protein starting at residue 96. GAPDH was immunopurified from aerobic, ischemic or reperfused hearts. Maleimidofluorescein labeling of purified GAPDH provided an index of its reduced thiol status. In the absence of DTT, ischemia induced a reduction in the number of free thiols on GAPDH that was reversed on reperfusion. When treated with DTT, the free thiol status of GAPDH could be increased in ischemic but not reperfused samples. Ischemia induced a reduction in GAPDH activity that was partially restored by reperfusion. DTT-treatment reactivated ischemic GAPDH, but had little effect on the activity from reperfused tissue. Mass spectra acquired from aerobic GAPDH preparations were relatively simple whereas spectra from ischemic or reperfused preparations were highly complex, possibly indicative of oxidation by multiple oxidants.Conclusions: Many proteins, including GAPDH, are targets for S-glutathiolation during cardiac oxidative stress. GAPDH oxidation is associated with a loss in reduced cysteine status that correlates with the inactivation of this enzyme

Inhibition of human mesangial cell proliferation by targeting T-Type calcium channels

Background: Aberrant glomerular mesangial cell (MC) proliferation is a common finding in renal diseases. T-type calcium channels (T-CaCN) play an important role in the proliferation of a number of cell types, including vascular smooth muscle cells. The hypothesis that T-CaCN may play a role in the proliferation of human MC was investigated. Methods: The presence of T-CaCN in primary cultures of human MC was examined using voltage clamping and by RT-PCR. The effect of calcium channel inhibitors, and of siRNA directed against the Cav3.2 T-CaCN isoform, on MC proliferation was assessed using the microculture tetrazolium assay and nuclear BrdU incorporation. Results: Human MC express only the Cav3.2 T-CaCN isoform. Co-incubation of MC with a T-CaCN inhibitor (mibefradil, TH1177 or Ni2+) results in a concentration-dependent attenuation of proliferation. This effect cannot be attributed to direct drug-induced cytotoxicity or apoptosis and is not seen with verapamil, an L-type channel blocker. Transfection of MC with siRNA results in knockdown of T-CaCN Cav3.2 mRNA and a clear attenuation of MC proliferation. Conclusions: These results demonstrate for the first time an important role for T-CaCN in human MC proliferation. This could potentially lead to a novel therapy in the treatment of proliferative renal diseases

Leading change in higher education

This article considers the situation in the UK higher education system and investigates specifically the leadership practice in a cluster of UK institutions as they changed their status. The research goes further to advocate a form of contextualized leadership that is relevant to higher institutions under change

Rational design of HIV vaccines and microbicides: report of the Europrise network annual conference 2010

Novel, exciting intervention strategies to prevent infection with HIV have been tested in the past year, and the field is rapidly evolving. EUROPRISE is a network of excellence sponsored by the European Commission and concerned with a wide range of activities including integrated developmental research on HIV vaccines and microbicides from discovery to early clinical trials. A central and timely theme of the network is the development of the unique concept of co-usage of vaccines and microbicides. This review, prepared by the PhD students of the network captures much of the research ongoing between the partners. The network is in its 5th year and involves over 50 institutions from 13 European countries together with 3 industrial partners; GSK, Novartis and Sanofi-Pasteur. EUROPRISE is involved in 31 separate world-wide trials of Vaccines and Microbicides including 6 in African countries (Tanzania, Mozambique, South Africa, Kenya, Malawi, Rwanda), and is directly supporting clinical trials including MABGEL, a gp140-hsp70 conjugate trial and HIVIS, vaccine trials in Europe and Africa

Vaccine-Induced Linear Epitope-Specific Antibodies to Simian Immunodeficiency Virus SIVmac239 Envelope Are Distinct from Those Induced to the Human Immunodeficiency Virus Type 1 Envelope in Nonhuman Primates.

To evaluate antibody specificities induced by simian immunodeficiency virus (SIV) versus human immunodeficiency virus type 1 (HIV-1) envelope antigens in nonhuman primate (NHP), we profiled binding antibody responses to linear epitopes in NHP studies with HIV-1 or SIV immunogens. We found that, overall, HIV-1 Env IgG responses were dominated by V3, with the notable exception of the responses to the vaccine strain A244 Env that were dominated by V2, whereas the anti-SIVmac239 Env responses were dominated by V2 regardless of the vaccine regimen