The mitochondrion is at the core of cellular energy metabolism, being the site of most ATP generation. Calcium is a key regulator of mitochondrial function and acts at several levels within the organelle to stimulate ATP synthesis. However, the dysregulation of mitochondrial Ca(2+) homeostasis is now recognized to play a key role in several pathologies. For example, mitochondrial matrix Ca(2+) overload can lead to enhanced generation of reactive oxygen species, triggering of the permeability transition pore, and cytochrome c release, leading to apoptosis. Despite progress regarding the independent roles of both Ca(2+) and mitochondrial dysfunction in disease, the molecular mechanisms by which Ca(2+) can elicit mitochondrial dysfunction remain elusive. This review highlights the delicate balance between the positive and negative effects of Ca(2+) and the signaling events that perturb this balance. Overall, a "two-hit" hypothesis is developed, in which Ca(2+) plus another pathological stimulus can bring about mitochondrial dysfunction.

Mitochondria play an important role in controlling the life and death of a cell. Consequently, mitochondrial dysfunction leads to a range of human diseases such as ischemia-reperfusion injury, sepsis, and diabetes. Although the molecular mechanisms responsible for mitochondria-mediated disease processes are not fully elucidated yet, the oxidative stress appears to be critical. Accordingly, strategies are being developed for the targeted delivery of antioxidants to mitochondria. In this review, we shall briefly discuss cellular reactive oxygen species metabolism and its role in pathophysiology; the currently existing antioxidants and possible reasons why they are not effective in ameliorating oxidative stress-mediated diseases; and recent developments in mitochondrially targeted antioxidants and their future promise for disease treatment.

SummaryCullins are central scaffolding subunits in eukaryotic E3 ligases that facilitate the ubiquitination of target proteins. Arabidopsis contains at least 11 cullin proteins but only a few of them have been assigned biological roles. In this work Arabidopsis cullin 4 is shown to assemble with DDB1, RBX1, DET1 and DDB2 in vitro and in planta. In addition, by using T-DNA insertion and CUL4 antisense lines we demonstrate that corresponding mutants are severely affected in different aspects of development. Reduced CUL4 expression leads to a reduced number of lateral roots, and to abnormal vascular tissue and stomatal development. Furthermore, cul4 mutants display a weak constitutive photomorphogenic phenotype. These results therefore assign an important function to CUL4 during plant development and provide strong evidence that CUL4 assembles together with RBX1 and DDB1 proteins to form a functional E3 ligase in Arabidopsis.

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A B S T R A C T Methane (CH4) production from the anti-inflammatory agent, dimethyl sulfoxide (DMSO), was used to measure *OH from chemical reactions or human phagocytes. Reactions producing * OH (xanthine/xanthine oxidase or Fe++/EDTA/H202) generated CH4 from DMSO, whereas reactions yielding primarily 0r or H202 failed to produce CH4. Neutrophils (PMN), monocytes, and alveolar macrophages also produced CH4 from DMSO. Mass spectroscopy using cJ-DMSO showed formnation of d3-CH, indicating that CH4 was derived from DMSO. Methane generation by normal but not chronic granulomatous disease or heat-killed phagocytes increased after stimulation with opsonized zymosan particles or the chemical, phorbol myristate acetate. Methane production from DMSO increased as the number of stimulated PMN was increased and the kinetics of CH4 production approximated other metabolic activities of stimulated PMN. Methane production from stimulated phagocytes and DMSO was markedly decreased by purportedly potent -OH scavengers (thiourea or tryptophane) and diminished to lesser degrees by weaker -OH scavengers (mannitol, ethanol, or Indeed, stimulated neutrophils (PMN), monocytes (MN) or alveolar marcophages (AM) do produce C2H4 from methional or KMB (6-10). However, the strict dependence of C2H4 production upon -OH is in doubt (8,(10)(11)(12)(13)(14). Methional spontaneously forms C2H4, especially during prolonged incubations with tissues (8), and can also react with H202 to form C2H4 (10). The specificity of KMB as a detector of -OH is also in question because C2H4 production from KMB may reflect, at least in part, reactions with 02 or hypochlorous acid (10).In Ca++ and Mg++ (26). Contaminating erythrocytes were lysed by adding 6 ml of ice-cold sterile distilled water and mixing gently for 35 s. Tonicity was rapidly restored with 2 ml of hypertonic (4x) Ca++ and Mg++ free HBSS. The mixture was then centrifuged at 170 g for 10 min. Cells in the pellet were then washed once more, resuspended in HBSS with Ca++ and Mg++, counted, and used immediately. PMN preparations contained >98% PMN with only a few lymphocytes and rare erythrocytes, platelets or MN. Suspensions of MN were similarly prepared. The percentages of MN were determined by differential counting of 600 cells on Wright's stained smears and cytochemically confirmed by esterase stain. MN preparations contained -30% MN, <1% PMN, and the remainder lymphocytes. AM were obtained by bronchoscopic sterile saline lavage of the unaffected subsegments of the lungs of patients undergoing evaluations for localized pulmonary disease or from healthy volunteers (9). AM were recovered from lavage fluids, washed once, and counted. The final preparations contained >90% AM and <2% PMN. More than 85% of the AM excluded trypan blue. Concentrations of lymphocytes or platelets that were comparable to those remaining in phagocyte preparations did not produce significant amounts of CH4 from DMSO.Preparation of pooled human serum, opsonized zymosan, or PMA. Pooled human serum was prepared from clotted ...