Until recently, hydrogen sulfide (H2S) was exclusively viewed a toxic gas and an environmental hazard, with its toxicity primarily attributed to the inhibition of mitochondrial Complex IV, resulting in a shutdown of mitochondrial electron transport and cellular ATP generation. Work over the last decade established multiple biological regulatory roles of H2S, as an endogenous gaseous transmitter. H2S is produced by cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST). In striking contrast to its inhibitory effect on Complex IV, recent studies showed that at lower concentrations, H2S serves as a stimulator of electron transport in mammalian cells, by acting as a mitochondrial electron donor. Endogenous H2S, produced by mitochondrially localized 3-MST, supports basal, physiological cellular bioenergetic functions; the activity of this metabolic support declines with physiological aging. In specialized conditions (calcium overload in vascular smooth muscle, colon cancer cells), CSE and CBS can also associate with the mitochondria; H2S produced by these enzymes, serves as an endogenous stimulator of cellular bioenergetics. The current article overviews the biochemical mechanisms underlying the stimulatory and inhibitory effects of H2S on mitochondrial function and cellular bioenergetics and discusses the implication of these processes for normal cellular physiology. The relevance of H2S biology is also discussed in the context of colonic epithelial cell physiology: colonocytes are exposed to high levels of sulfide produced by enteric bacteria, and serve as a metabolic barrier to limit their entry into the mammalian host, while, at the same time, utilizing it as a metabolic 'fuel'.

While searching for natural ligands for the peroxisome proliferator-activated receptor (PPAR) ␥, we identified a synthetic compound that binds to this receptor. Bisphenol A diglycidyl ether (BADGE) is a ligand for PPAR␥ with a K d(app) of 100 M. This compound has no apparent ability to activate the transcriptional activity of PPAR␥; however, BADGE can antagonize the ability of agonist ligands such as rosiglitazone to activate the transcriptional and adipogenic action of this receptor. BADGE also specifically blocks the ability of natural adipogenic cell lines such as 3T3-L1 and 3T3-F442A cells to undergo hormone-mediated cell differentiation. These results provide the first pharmacological evidence that PPAR␥ activity is required for the hormonally induced differentiation of adipogenic cells. Peroxisome proliferator-activated receptor (PPAR)1 ␥ is a nuclear hormone receptor that is expressed at highest levels in adipose tissue and lower levels in several other tissues. PPAR␥ is a major coordinator of adipocyte gene expression and differentiation (1). The expression of this receptor occurs early during the differentiation of preadipocytes, and it is expressed in a highly adipose-selective manner.PPAR␥ has been considered an orphan member of the nuclear hormone receptor superfamily, because no high affinity endogenous ligand has been identified for this receptor. However, a number of synthetic compounds have been shown to bind and activate PPAR␥ including a relatively new class of antidiabetic drugs, the thiazolidinediones (2). Thiazolidinediones (TZD) can ameliorate glucose metabolism and improve whole body insulin sensitivity in many animal models of obesity and diabetes. One TZD, troglitazone (Rezulin TM ), is currently used in the treatment of Type II diabetes in humans, and a second, rosiglitazone (Avandia TM ), was recently approved by the United States Food and Drug Administration. In addition to synthetic ligands, a number of natural ligands have been described for PPAR␥ that include primarily fatty acids and their metabolites (3-5). These ligands, however, have relatively low affinities with K d Ϸ 2-50 M, and hence it is possible that, analogous to other nuclear hormone receptors, a higher affinity ligand for PPAR␥ might exist.The evidence supporting a key role for PPAR␥ in adipogenesis is strong, but it is entirely based on "gain of function" experiments. For example, it has been shown that the ectopic expression and activation of PPAR␥ in undetermined fibroblasts are sufficient to induce an adipogenic response that includes morphological changes, lipid accumulation, and expression of most of the genes characteristic of this cell type (6). However, until now no experiments have addressed whether PPAR␥ function is required for adipocyte differentiation. During a screen for endogenous ligands of PPAR␥ we purified and characterized a compound that exhibited PPAR␥ binding activity. High pressure liquid and gas chromatography/mass spectrometry (LC/MS/MS and GC/MS, respectively)-based analyses identified this ...

The purpose of the current study was to investigate the effect of the recently synthesized mitochondrially-targeted H2S donor, AP39 [10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol-5yl)phenoxy)decyl) triphenylphosphonium bromide], on bioenergetics, viability, and mitochondrial DNA integrity in bEnd.3 murine microvascular endothelial cells in vitro, under normal conditions, and during oxidative stress. Intracellular H2S was assessed by the fluorescent dye 7-azido-4-methylcoumarin. For the measurement of bioenergetic function, the XF24 Extracellular Flux Analyzer was used. Cell viability was estimated by the combination of the MTT and LDH methods. Oxidative protein modifications were measured by the Oxyblot method. Reactive oxygen species production was monitored by the MitoSOX method. Mitochondrial and nuclear DNA integrity were assayed by the Long Amplicon PCR method. Oxidative stress was induced by addition of glucose oxidase. AP39 (30 – 300 nM) to bEnd.3 cells increased intracellular H2S levels, with a preferential response in the mitochondrial regions. AP39 exerted a concentration-dependent effect on mitochondrial activity, which consisted of a stimulation of mitochondrial electron transport and cellular bioenergetic function at lower concentrations (30–100 nM) and an inhibitory effect at the higher concentration of 300 nM. Under oxidative stress conditions induced by glucose oxidase, an increase in oxidative protein modification and an enhancement in MitoSOX oxidation was noted, coupled with an inhibition of cellular bioenergetic function and a reduction in cell viability. AP39 pretreatment attenuated these responses. Glucose oxidase induced a preferential damage to the mitochondrial DNA; AP39 (100 nM) pretreatment protected against it. In conclusion, the current paper documents antioxidant and cytoprotective effects of AP39 under oxidative stress conditions, including a protection against oxidative mitochondrial DNA damage.

Various titanium(IV) complexes of the type Cp′Ti(OAr)Cl2 (Cp′ ) cyclopentadienyl; OAr ) aryloxy) could be prepared in high yields from Cp′TiCl3. Cp*Ti(O-2,6-i Pr2C6H3)Me2 (Cp* ) C5Me5) could also be prepared from Cp*TiMe3 with 2,6-i Pr2C6H3OH in high yield (77%). These complexes showed notable catalytic activities for ethylene polymerization with MAO or Al i Bu3-Ph3CB(C6F5)4: Cp*Ti(O-2,6-i Pr2C6H3)X2 [X ) Cl (2b), Me (8b), CF3SO3 (9b)] showed the highest activities among these complexes. The effects of substituents on both cyclopentadienyl (pentamethylcyclopentadienyl) and aryloxy (2,6diisopropylphenoxy) groups are important for the remarkable activity. The crystallographic analyses of CpTi(O-2,6-i Pr2C6H3)Cl2 (1b), Cp*Ti(O-2,6-i Pr2C6H3)Cl2 (2b), and (1,3-t Bu2C5H3)Ti(O-2,6-i Pr2C6H3)Cl2 (6b) could be performed, and the bond angle of Ti-O-C (phenyl group) for 2b (173.0°) was found to be significantly different from those for other complexes (162.3-163.1°), although no significant differences are observed for other bond lengths and angles among these compounds. CpTi(O-2,4,6-Me3C6H2)2Cl (7a) and CpTi(O-2,6-i Pr2C6H3)2Cl (7b) could be prepared from CpTiCl3 with the corresponding phenol under the refluxing conditions of toluene, and the structure of 7a could be determined by X-ray crystallography. These complexes also exhibited moderate catalytic activities for ethylene polymerization in the presence of MAO, which was prepared by removing toluene and an excess amount of AlMe3, and the effect of the bulk of phenoxy ligand on the activity was demonstrated. 2b was also found to be an effective catalyst precursor for ethylene/1-butene copolymerization, and the smaller rErB values (0.25-0.36) compared to [Me2Si(C5Me4)(N t Bu)]TiCl2 (2.45) were observed by microanalysis of the resultant copolymers.

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Various titanium complexes of the type Cp′Ti-(OAr)Cl 2 (Cp′ ) cyclopentadienyl; OAr ) aryloxy) could be prepared in high yields from Cp′TiCl 3 . These complexes show remarkable catalytic activities for alkene polymerization with MAO or Al i Bu 3 -Ph 3 CB(C 6 F 5 ) 4 : (C 5 -Me 5 )Ti(O-2,6-i Pr 2 C 6 H 3 )X 2 (X ) Cl (2b), Me (8b), CF 3 -SO 3 (9b)) showed the highest activities. The bond angle Ti-O-C (phenyl group) in 2b (173.0°) is significantly different from those for other complexes (162.3-163.1°).

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