John C. Robinson

Proteomic Analysis of Pharmacological Preconditioning: Novel Protein Targets Converge to Mitochondrial Metabolism Pathways

Ischemic preconditioning is characterized by resistance to ischemia reperfusion injury in response to previous short ischemic episodes, a protective effect that can be mimicked pharmacologically. The underlying mechanism of protection remains controversial and requires greater understanding before it can be fully exploited therapeutically. To investigate the overall effect of preconditioning on the myocardial proteome, isolated rabbit ventricular myocytes were treated with drugs known to induce preconditioning, adenosine or diazoxide (each at 100 &mgr;mol/L for 60 minutes). Their protein profiles were then compared with vehicle-treated controls (n=4 animals per treatment) using a multitiered 2D gel electrophoresis approach. Of 28 significantly altered protein spots, 19 nonredundant proteins were identified (5 spots remained unidentified). The majority of these proteins are involved in mitochondrial energetics, including subunits of tricarboxylic acid cycle enzymes and oxidative phosphorylation complexes. These changes were not indiscriminate, with only a small number of enzymes or complex subunits altered, indicating a very specific and targeted affect of these 2 preconditioning mimetics. Among the changes were shifts in the extent of posttranslational modification of 4 proteins. One of these, the adenosine-induced phosphorylation of the ATP synthase β subunit, was fully characterized with the identification of 5 novel phosphorylation sites. This proteomics approach provides an overall assessment of the cellular response to pharmacological treatment with adenosine and diazoxide and identifies a distinct subset of enzymes and protein complex subunit that may underlie the preconditioned phenotype.

STK33 Kinase Activity Is Nonessential in KRAS-Dependent Cancer Cells

Despite the prevalence of KRAS mutations in human cancers, there remain no targeted therapies for treatment. The serine-threonine kinase STK33 has been proposed to be required for the survival of mutant KRAS-dependent cell lines, suggesting that small molecule kinase inhibitors of STK33 may be useful to treat KRAS-dependent tumors. In this study, we investigated the role of STK33 in mutant KRAS human cancer cells using RNA interference, dominant mutant overexpression, and small molecule inhibitors. As expected, KRAS downregulation decreased the survival of KRAS-dependent cells. In contrast, STK33 downregulation or dominant mutant overexpression had no effect on KRAS signaling or survival of these cells. Similarly, a synthetic lethal siRNA screen conducted in a broad panel of KRAS wild-type or mutant cells identified KRAS but not STK33 as essential for survival. We also obtained similar negative results using small molecule inhibitors of the STK33 kinase identified by high-throughput screening. Taken together, our findings refute earlier proposals that STK33 inhibition may be a useful therapeutic approach to target human KRAS mutant tumors.

A Critical Method of Evaluating Tests for Male Infertility

Considerable uncertainty surrounds the selection of test values that separate infertile from fertile men in the evaluation of male infertility. We herein describe an objective method of determining these values, referred to as threshold values, for different infertility tests. Using test results from fertile men threshold values were chosen such that 96 per cent of the semen samples from the fertile men were scored as fertile. These threshold values then were used to evaluate 100 semen samples from 74 men presenting for evaluation of infertility. Using this method we constructed infertility profiles on each of the 100 semen samples presented for infertility evaluation and found that the zona pellucida-free hamster egg penetration test (a measure of a spermatozoons ability to undergo capacitation and penetrate an egg) identified 66 per cent of these samples as infertile, while multiple exposure photomicrography (a quantitative measure of sperm motility) identified 54 per cent of these samples as infertile. This compares with results from routine semen analyses using the same method, which identified none of the samples as infertile by sperm motility grade, 1 per cent by semen pH, 4 per cent by the percentage of motile sperm, 7 per cent by the total count of motile sperm, 10 per cent by the total sperm count, 11 per cent by the semen leukocyte concentration, 12 per cent by the concentration of motile sperm, 13 per cent by ejaculate volume, 16 per cent by sperm concentration and 27 per cent by sperm morphology. This method of analyzing infertility test results provides insight into the potential causes of male infertility and offers a critical approach towards understanding the complex problem of male fertility dysfunction.