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1.  Inhibitors of ROS production by the ubiquinone-binding site of mitochondrial complex I identified by chemical screening 
Free radical biology & medicine  2013;65:1047-1059.
Mitochondrial production of reactive oxygen species is often considered an unavoidable consequence of aerobic metabolism and currently cannot be manipulated without perturbing oxidative phosphorylation. Antioxidants are widely used to suppress effects of reactive oxygen species after formation, but they can never fully prevent immediate effects at the sites of production. To identify site-selective inhibitors of mitochondrial superoxide/H2O2 production that do not interfere with mitochondrial energy metabolism, we developed a robust small-molecule screen and secondary profiling strategy. We describe the discovery and characterization of a compound (N-cyclohexyl-4-(4-nitrophenoxy)benzenesulfonamide; CN-POBS) that selectively inhibits superoxide/H2O2 production from the ubiquinone-binding site of complex I (site IQ) with no effects on superoxide/H2O2 production from other sites or on oxidative phosphorylation. Structure/activity studies identified a core structure that is important for potency and selectivity for site IQ. By employing CN-POBS in mitochondria respiring on NADH-generating substrates, we show that site IQ does not produce significant amounts of superoxide/H2O2 during forward electron transport on glutamate plus malate. Our screening platform promises to facilitate further discovery of direct modulators of mitochondrially-derived oxidative damage and advance our ability to understand and manipulate mitochondrial reactive oxygen species production in both normal and pathological conditions.
PMCID: PMC4321955  PMID: 23994103
superoxide; hydrogen peroxide; antioxidant; glycerol 3-phosphate dehydrogenase; NADH:Q oxidoreductase; complex II; complex III; energy metabolism; respiratory complexes
2.  Impaired Mitochondrial Dynamics and Bioenergetics in Diabetic Skeletal Muscle 
PLoS ONE  2014;9(3):e92810.
In most cells, mitochondria are highly dynamic organelles that constantly fuse, divide and move. These processes allow mitochondria to redistribute in a cell and exchange contents among the mitochondrial population, and subsequently repair damaged mitochondria. However, most studies on mitochondrial dynamics have been performed on cultured cell lines and neurons, and little is known about whether mitochondria are dynamic organelles in vivo, especially in the highly specialized and differentiated adult skeletal muscle cells. Using mitochondrial matrix-targeted photoactivatable green fluorescent protein (mtPAGFP) and electroporation methods combined with confocal microscopy, we found that mitochondria are dynamic in skeletal muscle in vivo, which enables mitochondria exchange contents within the whole mitochondrial population through nanotunneling-mediated mitochondrial fusion. Mitochondrial network promotes rapid transfer of mtPAGFP within the cell. More importantly, the dynamic behavior was impaired in high-fat diet (HFD)-induced obese mice, accompanying with disturbed mitochondrial respiratory function and decreased ATP content in skeletal muscle. We further found that proteins controlling mitochondrial fusion MFN1 and MFN2 but not Opa1 were decreased and proteins governing mitochondrial fission Fis1 and Drp1 were increased in skeletal muscle of HFD-induced mice when compared to normal diet-fed mice. Altogether, we conclude that mitochondria are dynamic organelles in vivo in skeletal muscle, and it is essential in maintaining mitochondrial respiration and bioenergetics.
PMCID: PMC3962456  PMID: 24658162
3.  Novel Inhibitors of Mitochondrial sn-Glycerol 3-phosphate Dehydrogenase 
PLoS ONE  2014;9(2):e89938.
Mitochondrial sn-glycerol 3-phosphate dehydrogenase (mGPDH) is a ubiquinone-linked enzyme in the mitochondrial inner membrane best characterized as part of the glycerol phosphate shuttle that transfers reducing equivalents from cytosolic NADH into the mitochondrial electron transport chain. Despite the widespread expression of mGPDH and the availability of mGPDH-null mice, the physiological role of this enzyme remains poorly defined in many tissues, likely because of compensatory pathways for cytosolic regeneration of NAD+ and mechanisms for glycerol phosphate metabolism. Here we describe a novel class of cell-permeant small-molecule inhibitors of mGPDH (iGP) discovered through small-molecule screening. Structure-activity analysis identified a core benzimidazole-phenyl-succinamide structure as being essential to inhibition of mGPDH while modifications to the benzimidazole ring system modulated both potency and off-target effects. Live-cell imaging provided evidence that iGPs penetrate cellular membranes. Two compounds (iGP-1 and iGP-5) were characterized further to determine potency and selectivity and found to be mixed inhibitors with IC50 and Ki values between ∼1–15 µM. These novel mGPDH inhibitors are unique tools to investigate the role of glycerol 3-phosphate metabolism in both isolated and intact systems.
PMCID: PMC3933693  PMID: 24587137
4.  Identification Of Small Molecule TRABID Deubiquitinase Inhibitors By Computation-Based Virtual Screen 
BMC Chemical Biology  2012;12:4.
Wnt/β-catenin-mediated gene transcription plays important roles in a wide range of biological and pathophysiological processes including tumorigenesis where β-catenin-mediated transcription activity frequently elevates. TRABID, a deubiquitinase, was shown to have a positive Wnt/β-catenin-mediated gene transcription and hence holds a promise as a putative anti-cancer target.
In this study, we used a combination of structure based virtual screening and an in vitro deubiquitinase (DUB) assay to identify several small molecules that inhibit TRABID DUB activity. However, these inhibitors failed to show inhibitory effects on β-catenin-mediated gene transcription. In addition, expression of TRABID shRNAs, wildtype TRABID, or the DUB activity-deficient mutant showed little effects on β-catenin-mediated gene transcription.
TRABID may not be a critical component in canonical Wnt/β-catenin signal transduction or that a minute amount of this protein is sufficient for its role in regulating Wnt activity.
PMCID: PMC3475094  PMID: 22584113
5.  The inflammatory response following an epidermal burn depends on the activities of mouse mast cell proteases 4 and 5 
A second degree epidermal scald burn in mice elicits an inflammatory response mediated by natural IgM directed to non-muscle myosin with complement activation that results in ulceration and scarring. We find that such burn injury is associated with early mast cell (MC) degranulation and is absent in WBB6F1-KitW/KitWv mice which lack MCs in a context of other defects due to a mutation of the KIT receptor. To further address a MC role, we used transgenic strains with normal lineage development and a deficiency in a specific secretory granule component. Mouse strains lacking the MC-restricted chymase, mouse MC protease (mMCP)-4, or elastase, mMCP-5, show decreased injury following a second degree scald burn while mice lacking the MC-restricted tryptases, mMCP-6 and mMCP-7, or the MC-specific carboxypeptidase A3 activity are not protected. Histologic sections showed some disruption of the epidermis at the scald site in the protected strains suggesting the possibility of topical reconstitution of full injury. Topical application of recombinant mMCP-5 or human neutrophil elastase to the scalded area increases epidermal injury with subsequent ulceration and scarring, both clinically and morphologically, in mMCP-5-deficent mice. Restoration of injury requires that topical administration of recombinant mMCP-5 occurs within the first h post burn. Importantly, topical application of human MC chymase restores burn injury to scalded mMCP-4-deficient mice but not to mMCP-5-deficient mice revealing non-redundant actions for these two MC proteases in a model of innate inflammatory injury with remodeling.
PMCID: PMC3058366  PMID: 21076070
6.  Cyclodextrin-erythromycin complexes as a drug delivery device for orthopedic application 
Erythromycin, a hydrophobic antibiotic used to treat infectious diseases, is now gaining attention because of its anti-inflammatory effects and ability to inhibit osteoclasts formation. The aim of this study was to explore a cyclodextrin-erythromycin (CD-EM) complex for sustained treatment of orthopedic inflammation.
Methods and results
Erythromycin was reacted with β-cyclodextrin to form a nonhost-guest CD-EM complex using both kneading and stirring approaches. Physiochemical measurement data indicated that erythromycin and cyclodextrin formed a packing complex driven by intermolecular forces instead of a host-guest structure due to the limited space in the inner cavity of β-cyclodextrin. The CD-EM complex improved the stability of erythromycin in aqueous solution and had a longer duration of bactericidal activity than free erythromycin. Cytotoxicity and cell differentiation were evaluated in both murine MC3T3 preosteoblast cells and RAW 264.7 murine macrophage cells. The CD-EM complex was noncytotoxic and showed significant inhibition of osteoclast formation but had little effect on osteoblast viability and differentiation.
These attributes are especially important for the delivery of an adequate amount of erythromycin to the site of periprosthetic inflammation and reducing local inflammation in a sustained manner.
PMCID: PMC3252670  PMID: 22228990
erythromycin; cyclodextrin; drug stability; bactericidal activity; osteoclastogenesis
7.  Magnesium sulfate enhances non-depolarizing muscle relaxant vecuronium action at adult muscle-type nicotinic acetylcholine receptor in vitro 
Acta Pharmacologica Sinica  2011;32(12):1454-1459.
To investigate the effect of magnesium sulfate and its interaction with the non-depolarizing muscle relaxant vecuronium at adult muscle-type acetylcholine receptors in vitro.
Adult muscle-type acetylcholine receptors were expressed in HEK293 cells. Drug-containing solution was applied via a gravity-driven perfusion system. The inward currents were activated by brief application of acetylcholine (ACh), and recorded using whole-cell voltage-clamp technique.
Magnesium sulfate (1–100 mmol/L) inhibited the inward currents induced ACh (10 μmol/L) in a concentration-dependent manner (IC50=29.2 mmol/L). The inhibition of magnesium sulfate was non-competitive. In contrast, vecuronium produced a potent inhibition on the adult muscle-type acetylcholine receptor (IC50=8.7 nmol/L) by competitive antagonism. Magnesium sulfate at the concentrations of 1, 3, and 6 mmol/L markedly enhanced the inhibition of vecuronium (10 nmol/L) on adult muscle-type acetylcholine receptors.
Clinical enhancement of vecuronium-induced muscle relaxation by magnesium sulfate can be attributed partly to synergism between magnesium sulfate and non-depolarizing muscle relaxants at adult muscle-type acetylcholine receptors.
PMCID: PMC4010211  PMID: 21986576
magnesium sulfate; non-depolarizing muscle relaxant; vecuronium; adult muscle-type acetylcholine receptor; synergism
8.  Potency of nondepolarizing muscle relaxants on muscle-type acetylcholine receptors in denervated mouse skeletal muscle 
Acta Pharmacologica Sinica  2010;31(12):1541-1546.
To investigate the changing resistance to nondepolarizing muscle relaxants (NDMRs) during the first month after denervation.
The denervated and innervated skeletal muscle cells were examined on days 1, 4, 7, 14, 21, and 28 after denervation. Individual denervated and innervated cells were prepared from the flexor digitorum brevis of the surgically denervated and contralateral hind feet, respectively. Nicotinic acetylcholine receptors (nAChRs) in the cells were activated with 30 μmol/L acetylcholine, either alone or in combination with various concentrations of vecuronium. Currents were recorded using a whole-cell patch-clamp technique.
The concentrations of vecuronium resulting in half-maximal inhibitory responses (IC50) increased 1.2- (P>0.05), 1.7-, 3.7-, 2.5-, 1.9-, and 1.8-fold (P<0.05) at Days 1, 4, 7, 14, 21, and 28 after denervation, respectively, compared to the innervated control. Resistance to vecuronium appeared at Day 4, peaked at Day 7, and declined at Day 14 after denervation. Nevertheless, IC50 values at Day 28 remained significantly higher than those for the innervated control, suggesting that the resistance to vecuronium had not disappeared at Day 28.
The NDMR doses required to achieve satisfactory clinical effects differ at different times after muscle denervation.
PMCID: PMC4002946  PMID: 21102480
denervation; nondepolarizing muscle relaxants; nicotinic acetylcholine receptors; skeletal muscle cells; vecuronium
9.  A Locomotor Innovation Enables Water-Land Transition in a Marine Fish 
PLoS ONE  2010;5(6):e11197.
Morphological innovations that significantly enhance performance capacity may enable exploitation of new resources and invasion of new ecological niches. The invasion of land from the aquatic realm requires dramatic structural and physiological modifications to permit survival in a gravity-dominated, aerial environment. Most fishes are obligatorily aquatic, with amphibious fishes typically making slow-moving and short forays on to land.
Methodology/Principal Findings
Here I describe the behaviors and movements of a little known marine fish that moves extraordinarily rapidly on land. I found that the Pacific leaping blenny, Alticus arnoldorum, employs a tail-twisting movement on land, previously unreported in fishes. Focal point behavioral observations of Alticus show that they have largely abandoned the marine realm, feed and reproduce on land, and even defend terrestrial territories. Comparisons of these blennies' terrestrial kinematic and kinetic (i.e., force) measurements with those of less terrestrial sister genera show A. arnoldorum move with greater stability and locomotor control, and can move away more rapidly from impending threats.
My results demonstrate that axial tail twisting serves as a key innovation enabling invasion of a novel marine niche. This paper highlights the potential of using this system to address general evolutionary questions about water-land transitions and niche invasions.
PMCID: PMC2887833  PMID: 20585564
10.  Different magnitude of resistance to nondepolarizing muscle relaxants in the denervated mouse skeletal muscle 
Acta Pharmacologica Sinica  2010;31(4):399-404.
To test the hypothesis that different magnitude of resistance of denervated skeletal muscle to nondepolarizing muscle relaxants (NDMRs) is related to their varying potencies at ɛ-AChR and γ-AChR.
Both innervated and denervated mouse muscle cells, and human embryonic kidney 293 (HEK293) cells expressing ɛ-AChR or γ-AChR were used. The effects of NDMRs on nAChR were explored using whole-cell patch clamp technique.
NDMRs vecuronium (VEC), atracurium (ATR) and rocuronium (ROC) produced reversible, dose-dependent inhibition on the currents induced by 30 μmol/L acetylcholine both in innervated and denervated skeletal muscle cells. Compared to those obtained in innervated skeletal muscle cells, denervation shifted the concentration-response curves rightward and significantly increased the 50% inhibitory concentration (IC50) values (VEC: from 11.2 to 39.2 nmol/L, P<0.01; ATR: from 24.4 to 129.0 nmol/L, P<0.01; ROC: from 37.9 to 101.4 nmol/L, P<0.01). In HEK293 cell expression system, ATR was less potent at γ-AChR than ɛ-AChR (IC50 values: 35.9 vs 22.3 nmol/L, P<0.01), VEC was equipotent at both receptor subtypes (IC50 values: 9.9 vs 10.2 nmol/L, P>0.05), while ROC was more potent at γ-AChR than ɛ-AChR (IC50 values: 22.3 vs 33.5 nmol/L, P<0.05).
Magnitude differences of resistance to different NDMRs caused by denervation are associated with distinct potencies of NDMRs at nAChR subtypes.
PMCID: PMC4007661  PMID: 20305678
acetylcholine; atracurium; denervation; nicotinic acetylcholine receptor; rocuronium; nondepolarizing muscle relaxants; vecuronium
11.  Enzootic Angiostrongyliasis in Shenzhen, China 
Emerging Infectious Diseases  2008;14(12):1955-1956.
PMCID: PMC2634632  PMID: 19046534
Angiostrongylus cantonensis; eosinopilic meningitis; Shenzhen; human; rates; snails; letter

Results 1-11 (11)