Heat and chemical shock potentiation of glucocorticoid receptor transactivation requires heat shock factor (HSF) activity. Modulation of HSF by vanadate and wortmannin

Heat shock and other forms of stress increase glucocorticoid receptor (GR) activity in cells, suggesting cross-talk between the heat shock and GR signal pathways. An unresolved question concerning this cross-talk is whether heat shock factor (HSF1) activity is required for this response. We addressed this issue by modulating HSF1 activity with compounds acting by distinct mechanisms: sodium vanadate (SV), an inhibitor of protein phosphatases; and wortmannin, an inhibitor of DNA-dependent protein kinase. Using HSF1- and GR-responsive CAT reporters, we demonstrate that SV inhibits both HSF1 activity and the stress potentiation of GR, while having no effect on the hormone-free GR or HSF1. Paradoxically, SV increased hormone-induced GR activity in the absence of stress. In contrast, wortmannin increased HSF1 activity in stressed cells and had no effect on HSF1 in the absence of stress. Using the pMMTV-CAT reporter containing the negative regulatory element 1 site for DNA-dependent protein kinase, wortmannin was found to increase the GR response. However, in cells expressing a minimal promoter lacking negative regulatory element 1 sites, wortmannin had no effect on the GR in the absence of stress but increased the stress potentiation of GR. Our results show that the mechanism by which GR activity is increased in stressed cells requires intrinsic HSF1 activity.     

Anticancer activity of sclerotiorin, isolated from an endophytic fungus Cephalotheca faveolata Yaguchi, Nishim. & Udagawa

Biodiversity provides critical support for drug discovery. A significant proportion of drugs are derived, directly or indirectly, from biological sources. Through high throughput screening (HTS) and bioassay-guided isolation, bioactive compound sclerotiorin has been isolated from an endophytic fungus Cephalotheca faveolata. Sclerotiorin was found to be potent anti-proliferative against different cancer cells. In this study sclerotiorin has been found to induce apoptosis in colon cancer (HCT-116) cells through the activation of BAX, and down-regulation of BCL-2, those further activated cleaved caspase-3 causing apoptosis of cancer cells.     

Molecular modeling of cornulin (CRNN) for docking with phytocompounds from Justicia adhatoda L.

Cornulin (CRNN) is linked with tumour progression. Therefore, it is of interest to document data on the molecular modeling of cornulin (CRNN) for docking with phytocompounds (Pyrazinamide, Anisotine, Vasicinone, Vasicoline) from Justicia adhatoda L. Thus, we document the optimal binding features of these compounds with the cornulin model for further consideration.     

Lupeol and its ester ameliorate the cyclophosphamide provoked cardiac lysosomal damage studied in rat

Cyclophosphamide (CP), an alkylating agent widely used in cancer chemotherapy causes fatal cardiotoxicity. Lupeol, a pentacyclic triterpene, isolated from Crataeva nurvala stem bark and its ester, lupeol linoleate possess a wide range of medicinal properties. The effect of lupeol and its ester was evaluated in CP-induced myocardial toxicity in rats. Male albino rats of Wistar strain were categorized into six groups. Group I served as control. Rats in groups II, V and VI animals were injected intraperitoneally with a single dose of CP (200 mg/kg body weight) dissolved in saline. CP-treated groups V and VI received lupeol and lupeol linoleate (50 mg/kg body weight), respectively, dissolved in olive oil for 10 days by oral gavage. CP-administered rats showed a significant increase (p < 0.001) in the activities of lysosomal hydrolases in serum and heart, a decrease (p < 0.001) in the levels of cellular thiols and myofibres were swollen with loss of myofilaments in electron microscopical analysis in heart. Lupeol and its ester showed reversal of the above alterations induced by CP. These findings demonstrate that the supplementation with lupeol and its ester could preserve lysosomal integrity, improve thiol levels, highlighting their protective effect against CP-induced cardiotoxicity.     

Ouabain decreases sarco(endo)plasmic reticulum calcium ATPase activity in rat hearts by a process involving protein oxidation

The effect of cardiac glycosides to increase cardiac inotropy by altering Ca(2+) cycling is well known but still poorly understood. The studies described in this report focus on defining the effects of ouabain signaling on sarcoplasmic reticulum Ca(2+)-ATPase function. Rat cardiac myocytes treated with 50 microM ouabain demonstrated substantial increases in systolic and diastolic Ca(2+) concentrations. The recovery time constant for the Ca(2+) transient, tau(Ca(2+)), was significantly prolonged by ouabain. Exposure to 10 microM H(2)O(2), which causes an increase in intracellular reactive oxygen species similar to that of 50 microM ouabain, caused a similar increase in tau(Ca(2+)). Concurrent exposure to 10 mM N-acetylcysteine or an aqueous extract from green tea (50 mg/ml) both prevented the increases in tau(Ca(2+)) as well as the changes in systolic or diastolic Ca(2+) concentrations. We also observed that 50 microM ouabain induced increases in developed pressure in addition to diastolic dysfunction in the isolated perfused rat heart. Coadministration of ouabain with N-acetylcysteine prevented these increases. Analysis of sarcoplasmic reticulum Ca(2+)-ATPase protein revealed increases in both the oxidation and nitrotyrosine content in the ouabain-treated hearts. Liquid chromatography-mass spectrometric analysis confirmed that the sarcoplasmic reticulum Ca(2+)-ATPase protein from ouabain-treated hearts had modifications consistent with oxidative and nitrosative stress. These data suggest that ouabain induces oxidative changes of the sarcoplasmic reticulum Ca(2+)-ATPase structure and function that may, in turn, produce some of the associated changes in Ca(2+) cycling and physiological function.     

Activation of IKK/NF-κB provokes renal inflammatory responses in guanylyl cyclase/natriuretic peptide receptor-A gene-knockout mice

The present study was aimed at determining the consequences of the disruption of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene (Npr1) on proinflammatory responses of nuclear factor kappa B, inhibitory kappa B kinase, and inhibitory kappa B alpha (NF-κB, IKK, IκBα) in the kidneys of mutant mice. The results showed that the disruption of Npr1 enhanced the renal NF-κB binding activity by 3.8-fold in 0-copy (-/-) mice compared with 2-copy (+/+) mice. In parallel, IKK activity and IκBα protein phosphorylation were increased by 8- and 11-fold, respectively, in the kidneys of 0-copy mice compared with wild-type mice. Interestingly, IκBα was reduced by 80% and the expression of proinflammatory cytokines and renal fibrosis were significantly enhanced in 0-copy mice than 2-copy mice. Treatment of 0-copy mice with NF-κB inhibitors andrographolide, pyrrolidine dithiocarbamate, and etanercept showed a substantial reduction in renal fibrosis, attenuation of proinflammatory cytokines gene expression, and significantly reduced IKK activity and IkBα phosphorylation. These findings indicate that the systemic disruption of Npr1 activates the renal NF-κB pathways in 0-copy mice, which transactivates the expression of various proinflammatory cytokines to initiate renal remodeling; however, inhibition of NF-κB pathway repairs the abnormal renal pathology in mutant mice.     

Sediment Heavy Metal Contents,Ostracod Toxicity and Risk Assessment in Tropical Freshwater Lakes,Tamil Nadu,India

ABSTRACT

Coimbatore is one of the industrial cities in Tamil Nadu, India, which has been experiencing rapid urbanization and population growth. Coimbatore is also known for its unique freshwater lakes environment and serves as a rich ecosystem. However, the assessment of heavy metal levels in aquatic environments is limited. This study was aimed to investigate physicochemical parameters, heavy metals level and sources, and ecotoxicity in sediments collected from five different lakes in Coimbatore. The concentrations of heavy metals (Cr, Cu, Zn, As, Cd, and Pb) in sediments were determined by Inductive Coupled Plasma-Mass Spectroscopy (ICP-MS). Hg level was measured using Advanced Mercury Analyzer (AMA). The determined heavy metal concentrations in sediments varied significantly according to the lake location and consistent with local human linked anthropogenic activities. The metal concentrations in urban lakes were exceeding both the Sediment Quality Guidelines (SQGs) and the probable effect levels” (PELs) mostly; e.g., in sediments from the Lake Ukkadam, the values of 5.08 and 203.32 mg kg-1 dry weight were observed for Hg and Cu, respectively. The ecotoxicity test with ostracods exposed to the sediment samples revealed that mortality ranged between 6 and 23% for countryside lakes and 28 and 88% for the lakes within urban Zone. We used Spearman rank-order correlation and Principal components analysis (PCA) to assess the sources of pollutants and if they related to anthropogenic pressure and eutrophication of lakes. The main sources of heavy metals from studied lakes differed significantly. Urban and industrial effluents were dominant sources in urban lakes. Agricultural runoff, domestic wastes, and natural weathering were responsible for the metal sources in country lakes. This study provides baseline information on the heavy metal pollution status of sediments in the freshwater lakes in Coimbatore, which will be useful for pollution control measures to prevent possible metal sources on these lakes and impose appropriate management practices and continuous monitoring by relevant authorities.     

Mitigation of cocaine-mediated mitochondrial damage,defective mitophagy and microglial activation by superoxide dismutase mimetics

ABSTRACT

Although cocaine exposure has been shown to potentiate neuroinflammation by upregulating glial activation in the brain, the role of mitophagy in this process remains an enigma. In the present study, we sought to examine the role of impaired mitophagy in cocaine-mediated activation of microglia and to determine the ameliorative potential of superoxide dismutase mimetics in this context. Our findings demonstrated that exposure of mouse primary microglial cells (mPMs) to cocaine resulted in decreased mitochondrial membrane potential, that was accompanied by increased expression of mitophagy markers, PINK1 and PRKN. Exposure of microglia to cocaine also resulted in increased expression of DNM1L and OPTN with a concomitant decrease in the rate of mitochondrial oxygen consumption as well as impaired mitochondrial functioning. Additionally, in the presence of cocaine, microglia also exhibited upregulated expression of autophagosome markers, BECN1, MAP1LC3B-II, and SQSTM1. Taken together, these findings suggested diminished mitophagy flux and accumulation of mitophagosomes in the presence of cocaine. These findings were further confirmed by imaging techniques such as transmission electron microscopy and confocal microscopy. Cocaine-mediated activation of microglia was further monitored by assessing the expression of the microglial marker (ITGAM) and the inflammatory cytokine (Tnf, Il1b, and Il6) mRNAs. Pharmacological, as well as gene-silencing approaches aimed at blocking both the autophagy/mitophagy and SIGMAR1 expression, underscored the role of impaired mitophagy in cocaine-mediated activation of microglia. Furthermore, superoxide dismutase mimetics such as TEMPOL and MitoTEMPO were shown to alleviate cocaine-mediated impaired mitophagy as well as microglial activation.

Abbreviations: 3-MA: 3-methyladenine; Δψm: mitochondrial membrane potential; ACTB: actin, beta; AIF1: allograft inflammatory factor 1; ATP: adenosine triphosphate; BAF: bafilomycin A₁; BECN1: beclin 1, autophagy related; CNS: central nervous system; DNM1L: dynamin 1 like; DMEM: Dulbecco modified Eagle medium; DAPI: 4,6-Diamidino-2-phenylindole; DRD2: dopamine receptor D2; ECAR: extracellular acidification rate; FBS: fetal bovine serum; FCCP: Trifluoromethoxy carbonylcyanide phenylhydrazone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IL1B: interleukin 1, beta; IL6: interleukin 6; ITGAM: integrin subunit alpha M; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; mPMs: mouse primary microglial cells; MRC: maximal respiratory capacity; NFKB: nuclear factor kappa B; NLRP3: NLR family pyrin domain containing 3; NTRK2: neurotrophic receptor tyrosine kinase 2; OCR: oxygen consumption rate; OPTN: optineurin; PBS: phosphate buffered saline; PINK1: PTEN induced putative kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; ROS: reactive oxygen species; siRNA: small interfering RNA; SQSTM1: sequestosome 1; TNF: tumor necrosis factor