NO* (nitric oxide) is a pleiotropic signalling molecule, with many of its effects on cell function being elicited at the level of the mitochondrion. In addition to the well-characterized binding of NO* to the Cu(B)/haem-a3 site in mitochondrial complex IV, it has been proposed by several laboratories that complex I can be inhibited by S-nitrosation of a cysteine. However, direct molecular evidence for this is lacking. In this investigation we have combined separation techniques for complex I (blue-native gel electrophoresis, Superose 6 column chromatography) with sensitive detection methods for S-nitrosothiols (chemiluminescence, biotin-switch assay), to show that the 75 kDa subunit of complex I is S-nitrosated in mitochondria treated with S-nitrosoglutathione (10 microM-1 mM). The stoichiometry of S-nitrosation was 7:1 (i.e. 7 mol of S-nitrosothiols per mol of complex I) and this resulted in significant inhibition of the complex. Furthermore, S-nitrosothiols were detected in mitochondria isolated from hearts subjected to ischaemic preconditioning. The implications of these results for the physiological regulation of respiration, for reactive oxygen species generation and for a potential role of S-nitrosation in cardioprotection are discussed.

A key pathologic event in cardiac ischemia reperfusion (I-R) injury is mitochondrial energetic dysfunction, and several studies have attributed this to complex I (CxI) inhibition. In isolated perfused rat hearts, following I-R, we found that CxI-linked respiration was inhibited, but isolated CxI enzymatic activity was not. Using the mitochondrial thiol probe iodobutyl-triphenylphosphonium in conjunction with proteomic tools, thiol modifications were identified in several subunits of the matrix-facing 1alpha sub-complex of CxI. These thiol modifications were accompanied by enhanced ROS generation from CxI, but not complex III. Implications for the pathology of cardiac I-R injury are discussed.

The mechanisms of mitochondrial proton (H+) leak under various pathophysiological conditions are poorly understood. In the present study it was hypothesized that different mechanisms underlie H+ leak in cardiac IR (ischaemia/reperfusion) injury and IPC (ischaemic preconditioning). Potential H(+) leak mechanisms examined were UCPs (uncoupling proteins), allosteric activation of the ANT (adenine nucleotide translocase) by AMP, or the PT (permeability transition) pore. Mitochondria isolated from perfused rat hearts that were subjected to IPC exhibited a greater H+ leak than did controls (202+/-27%, P<0.005), and this increased leakage was completely abolished by the UCP inhibitor, GDP, or the ANT inhibitor, CAT (carboxyattractyloside). Mitochondria from hearts subjected to IR injury exhibited a much greater amount of H+ leak than did controls (411+/-28%, P<0.001). The increased leakage after IR was weakly inhibited by GDP, but was inhibited, >50%, by carboxyattractyloside. In addition, it was inhibited by cardioprotective treatment strategies including pre-IR perfusion with the PT pore inhibitors cyclosporin A or sanglifehrin A, the adenylate kinase inhibitor, AP5A (diadenosine pentaphosphate), or IPC. Together these data suggest that the small increase in H+ leak in IPC is mediated by UCPs, while the large increase in H+ leak in IR is mediated by the ANT. Furthermore, under all conditions studied, in situ myocardial O2 efficiency was correlated with isolated mitochondrial H+ leak (r2=0.71). In conclusion, these data suggest that the modulation of H+ leak may have important implications for the outcome of IR injury.

Corpus cavernosum abscesses are uncommon with only 23 prior reports in the literature. Several precipitating factors for cavernosal infections have been described including injection therapy for erectile dysfunction, trauma, and priapism. Common causal organisms include Staphylococcus aureus, Streptococci, and Bacteroides. We report a unique case of a corpus cavernosum abscess due to proctitis with hematological seeding and review the literature on cavernosal abscesses.

The intracellular signaling network triggered by binding of growth factors and cytokines to cellular surface receptors is rigorously regulated in cells and often involves recruitment of adapter molecules to transmembrane receptors (1, 2). Downstream of kinase (Dok) 1 proteins are a recently discovered family of adapter molecules (including Dok-1, -2, and -3), which have emerged as an expanding group of insulin receptor substrates (IRSs)-related signaling molecules, consisting of an NH 2 -terminal tandem of PH and PTB domains. The previously identified Dok members, p62 Dok (Dok-1), Dok-2 (Dok-R), and Dok-3, are predominantly expressed in hematopoietic cells (3). Although these three Doks have similar domains to IRSs, they can be distinguished from the IRS family based on sequence homology and functional interactions. Hematopoietic Dok molecules are prominent substrates of the Src and Abl tyrosine kinases. Upon tyrosine phosphorylation, they acquire the ability to bind SH2 domain-containing molecules such as RasGAP, Csk,. More recently, Grimm et al. (6) identified two novel Dok-like molecules, Dok-4 and Dok-5, as partners and substrates for the receptor tyrosine kinase Ret. Whereas Dok-5 appears concentrated in the central nervous system, Dok-4 seems most highly expressed in epithelial and endothelial cells (4, 6). We have reported (4) that a significant proportion of Dok-4 is constitutively associated with the cell membrane and that it can serve as a substrate for tyrosine kinases of the Src family. However, because Dok-4 lacks consensus binding sites for known SH2 domains, its exact function has been difficult to define. We found that Dok-4 inhibited activation of the Erk substrate Elk-1 by Ret and by the Src kinase Fyn in epithelial cells. On the other hand, by using different approaches and cellular systems, Grimm et al. (6) and Cai et al.