Reactive Oxygen Species Quencher Research Articles

Abstract 1237: Down-regulation of chemokine receptor CXCR4 by gambogic acid suppresses metastasis of pancreatic cancer in an orthotopic mouse model

Abstract More than 90% of patients die of pancreatic cancer due to metastasis. Among the cytokines, CXCL12 and its CXC chemokine receptor 4 (CXCR4) are known to play a role in invasion and metastasis in many types of cancer. Thus, agents that can interrupt the CXCR4/CXCL12 signaling cascade have the potential to suppress cancer metastasis. In the present study, we demonstrate that gambogic acid (GA), a xanthone from Gamboge hanburyi, down-regulated CXCR4 expression in pancreatic cancer cells in a dose- and time-dependent manner. We found that the decrease in CXCR4 by GA was not specific to cell type since its expression was abrogated in multiple myeloma cell lines and in colorectal, head and neck, prostate, and breast cancer cell lines. Furthermore, down-regulation of CXCR4 was not due to proteolytic degradation since proteasome inhibitors had no effect. Instead, the down-regulation of CXCR4 was due to transcriptional regulation, as indicated by the corresponding down-regulation of mRNA expression. This xanthone also inhibited the invasion of pancreatic tumor cells in vitro. Down-regulation of CXCR4 and prevention of cell invasion by the xanthone were both found to be mediated through the reactive oxygen species (ROS) as N-acetylcysteine, a ROS quencher, abolished these effects. When examined in an orthotopic nude mouse model of human pancreatic cancer, intraperitoneal administration of GA suppressed both tumor growth and metastasis. Immunohistochemical analysis of tumor tissue showed that besides decreasing the level of CXCR4, GA also reduced the expression of VEGF and MMP-9, which are also linked to invasion and metastasis. Thus, our results demonstrate gambogic acid’s potential in suppressing tumor invasion and metastasis through down-modulation of CXCR4/CXCL12 signaling pathways. Citation Format: Sahdeo Prasad, Sanjay Srivastava. Down-regulation of chemokine receptor CXCR4 by gambogic acid suppresses metastasis of pancreatic cancer in an orthotopic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1237.

Study of pyridoxamine against glycation and reactive oxygen species production in human serum albumin as model protein: An in vitro & ex vivo approach

Hyperglycaemia is considered to be a driving factor for advanced glycated end products (AGEs). Inhibiting the process of glycation play an important role in reducing the diabetes related complications. We have explored the glucose mediated glycation and antiglycation activity of pyridoxamine using human serum albumin (HSA). Protein was incubated with glucose for 28 days at physiological temperature to achieve glycation. Antiglycation activity was assessed by the estimation of carbonyl content, free lysine and AGE specific fluorescence. Molecular docking was used to study the interaction of pyridoxamine with HSA and to get a detailed understanding of binding sites and binding energy. Glycation was reduced by pyridoxamine to commendable levels which was evident by the quantification of free lysine and carbonyl content. Pyridoxamine treatment also prevented the loss in secondary structure induced by glycation. It has also emerged as the quencher of reactive oxygen species which lead to the protection of DNA from oxidative damage. Pyridoxamine was found to be located at subdomain IIA of HSA with binding energy of −5.6 kcal/mol. These results are high points in the antiglycation activity of pyridoxamine. Its antioxidant nature and antiglycation activity are proof of its potential in preventing disease progression in diabetes.

Selenium amelioration of arsenic toxicity in rice shows genotypic variation: A transcriptomic and biochemical analysis

The toxic metalloid arsenic (As) is consumed mostly through contaminated rice. Therefore, reducing its accumulation and maintaining nutrient homeostasis in crop plants are imperative to ensure food safety. However, there is a dearth of information on the interrelationship between nutrient homeostasis and the regulatory mechanisms of arsenic-selenium (As-Se) interactive pathways responsible for stress tolerance. In the present study, experiments were conducted in hydroponically grown 12-day-old seedlings of rice (Oryza sativa L.) varieties (Pusa Basmati1 and IR64) treated with arsenite (AsIII) (150 μM), selenium (SeVI) (20 μM), and As + Se. It was observed that selenium supplementation ameliorated As toxicity by reducing its accumulation and retrieving As-induced nutrient deficiency. Significant decrease in As accumulation, H2O2 content, and fluorescent intensity of nitric oxide (NO), reactive oxygen species (ROS), and superoxide radical (O2.−) along with cell death with Se supplementation in both rice varieties demonstrated the protective role of Se as a probable ROS quencher. Addition of Se increased the enzyme activities of thiol metabolism and induced differential transcript accumulation patterns of sulfur-related genes. Nutrient level positively correlated with the differential expression pattern of NPK-related genes that play roles in metabolism and nutrient availability in both varieties. Though Pusa Basmati1 (PB1) showed higher tolerance to As, IR64 overcomes As toxicity more efficiently than PB1 in the presence of Se, which highlights that IR64 is a better performer in the presence of Se. Overall, this study provides novel insight into the role of Se in As-stressed rice genotypes through alteration of nutrient transporters and thiol-related genes.

Differential transcriptomic analysis reveals hidden light response in Streptomyces lividans.

Recently, a comprehensive screening workflow for the filamentous bacterium Streptomyces lividans, a highly performant source for pharmaceutically active agents was introduced. This framework used parallelized cultivation in microtiter plates to efficiently accelerate early upstream process development. Focusing on growth performance, cultivation was successfully scaled-up to 1 L stirred tank reactors. However, metabolic adaptation was observed on the transcriptomic level as among others, several genes incorporated in light response were upregulated during bioreactor cultivation. Despite it was assumed that this was attributed to the fact that reactor cultivations were performed in glass vessels exposed to daylight and artificial room light, this setup did not allow distinguishing exclusively between light and other effects. Upon that, the present study directly investigates the influence of light by defined illumination of microtiter plate cultures. Almost identical growth performance was observed for cultures grown in the dark or with illumination. Transcriptomics revealed the upregulation of seven genes of which 6 have previously been described to be relevant for carotenoid synthesis and its regulation. These pigments are effective quenchers of reactive oxygen species. The seventh transcript coded for a photo-lyase incorporated in UV-damage repair of DNA further confirming induced light response. However, this was fully compensated by metabolic adaptation on the transcriptomic level and overall process performance was maintained. Consequently, environmental conditions need extremely careful control and evaluation during in-depth omics analysis of bioprocesses. Otherwise metabolic adaptation induced by such issues can easily be misinterpreted, especially during studies addressing cultivation system comparisons. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:287-292, 2018.

Carnosic Acid and Carnosol, Two Major Antioxidants of Rosemary, Act through Different Mechanisms.

Carnosic acid, a phenolic diterpene specific to the Lamiaceae family, is highly abundant in rosemary (Rosmarinus officinalis). Despite numerous industrial and medicinal/pharmaceutical applications of its antioxidative features, this compound in planta and its antioxidant mechanism have received little attention, except a few studies of rosemary plants under natural conditions. In vitro analyses, using high-performance liquid chromatography-ultraviolet and luminescence imaging, revealed that carnosic acid and its major oxidized derivative, carnosol, protect lipids from oxidation. Both compounds preserved linolenic acid and monogalactosyldiacylglycerol from singlet oxygen and from hydroxyl radical. When applied exogenously, they were both able to protect thylakoid membranes prepared from Arabidopsis (Arabidopsis thaliana) leaves against lipid peroxidation. Different levels of carnosic acid and carnosol in two contrasting rosemary varieties correlated with tolerance to lipid peroxidation. Upon reactive oxygen species (ROS) oxidation of lipids, carnosic acid was consumed and oxidized into various derivatives, including into carnosol, while carnosol resisted, suggesting that carnosic acid is a chemical quencher of ROS. The antioxidative function of carnosol relies on another mechanism, occurring directly in the lipid oxidation process. Under oxidative conditions that did not involve ROS generation, carnosol inhibited lipid peroxidation, contrary to carnosic acid. Using spin probes and electron paramagnetic resonance detection, we confirmed that carnosic acid, rather than carnosol, is a ROS quencher. Various oxidized derivatives of carnosic acid were detected in rosemary leaves in low light, indicating chronic oxidation of this compound, and accumulated in plants exposed to stress conditions, in parallel with a loss of carnosic acid, confirming that chemical quenching of ROS by carnosic acid takes place in planta.

Graphene as Metal‐Free Catalyst for Aqueous Phase Reforming of Ethylene Glycol

AbstractGraphene obtained by pyrolysis of alginate shows in the absence of any metal, catalytic activity towards the decomposition of ethylene glycol into hydrogen and carbon dioxide at 250 °C due to the presence of hydrogenating/dehydrogenating sites. This reaction has interest in the context of valorization of waste waters from cellulose depolymerization and it is typically catalyzed by platinum and other transition metals. In this regard, the use of graphene as metal‐free catalyst may have a considerable advantage from the point of sustainability of the catalyst. Based on the influence of the presence of acids and bases and the lack of influence of quenchers of reactive oxygen species, it is proposed that the active sites on the graphene could be frustrated acid‐base Lewis pairs acting as dehydrogenating centers. Controls with oxalic acid and glyoxal have shown that if ethylene glycol is converted into these α‐dicarbonylic compounds, spontaneous decarboxylation would occur totally (oxalic acid) or in some extent (glyoxal. 30%). In the last case, decarboxylation is also assisted by graphene.

Role of A-Kinase anchor protein (AKAP4) in growth and survival of ovarian cancer cells.

Ovarian cancer represents one of the most common malignancies among women with very high mortality rate worldwide. A-kinase anchor protein 4 (AKAP4), a unique cancer testis (CT) antigen has been shown to be associated with various malignant properties of cancer cells. However, its involvement in various molecular pathways in ovarian cancer remains unknown. In present investigation, employing gene silencing approach, we examined the role of AKAP4 in cell cycle, apoptosis and epithelial-mesenchymal transition (EMT). Further, we also investigated the effect of ablation of AKAP4 on tumor growth in SCID mice ovarian cancer xenograft mouse model. Our results showed that ablation of AKAP4 resulted in increased reactive oxygen species (ROS) generation, DNA damage, cell cycle arrest and apoptosis in ovarian cancer cells. AKAP4 knockdown lead to degradation of protien kinase A (PKA) which was rescued by proteosome inhibitor MG-132. ROS quencher N-acetyl cysteine (NAC) treatment rescued cell cycle arrest and resumed cell division. Subsequently, increased expression of pro-apoptotic molecules and decreased expression of pro-survival/anti-apoptotic factors was observed. As a result of AKAP4 depletion, DNA damage response proteins p-γH2AX, p-ATM and p21 were upregulated. Also, knockdown of CREB resulted in similar findings. Further, PKA inhibitor (H89) and oxidative stress resulted in similar phenotype of ovarian cancer cells as observed in AKAP4 ablated cells. Collectively, for the first time our data showed the involvement of AKAP4 in PKA degradation and perturbed signaling through PKA-CREB axis in AKAP4 ablated ovarian cancer cells.

Roles of Reactive Oxygen Species in Modulation of Thymocyte Subpopulations during Thymic Atrophy

Abstract The thymus is the site of T cell development, selection and maturation. Precursor cells migrate from the bone marrow to the thymus, where they undergo sequential development followed by selection to give rise to mature and functional T cells. The cell surface expressions of various markers define distinct stages of development and maturation of thymocytes. With the help of these markers and multicolour flow cytometry, we have standardized staining procedures to study different thymocyte subsets during atrophy. We have also standardized an acute and lethal model of oral Salmonella Typhimurium infection in BALB/c mice, wherein drastic reduction of thymic cellularity is observed. Using multicolour flow cytometry, we investigated the changes occurring in thymocyte subsets in this model of infection-induced thymic atrophy. In addition, thymic atrophy was also induced by administration of LPS, the chemotherapeutic drug etoposide and the synthetic glucocorticoid, dexamethasone. Extensive thymic atrophy occurred across the different groups and thymocyte subsets were studied in a comprehensive manner. CD4 and CD8 profile on total thymocytes revealed CD4+CD8+ cells to be the most sensitive across all the modes of thymic atrophy studied. Subsequent detailed analysis of the thymic subpopulations revealed distinct changes during thymic atrophy. The roles of reactive oxygen species (ROS) in modulation of thymocyte subsets in the above mentioned conditions were studied using the ROS quencher, N-acetyl cysteine. We further intend to understand the mechanisms involved in changes occurring in the thymocyte subsets that may aid in development of candidate therapeutic molecules with thymopoietic potential.

Bacterial persistence induced by salicylate via reactive oxygen species

Persisters are phenotypic variants of regular cells that exist in a dormant state with low metabolic activity, allowing them to exhibit high tolerance to antibiotics. Despite increasing recognition of their role in chronic and recalcitrant infections, the mechanisms that induce persister formation are not fully understood. In this study, we find that salicylate can induce persister formation in Escherichia coli via generation of reactive oxygen species (ROS). Salicylate-induced ROS cause a decrease in the membrane potential, reduce metabolism and lead to an increase in persistence. These effects can be recovered by culturing cells in the presence of a ROS quencher or in an anaerobic environment. Our findings reveal that salicylate-induced oxidative stress can lead to persistence, suggesting that ROS, and their subsequent impact on membrane potential and metabolism, may play a broad role in persister formation.

Cold atmospheric-pressure air plasma treatment of C6 glioma cells: effects of reactive oxygen species in the medium produced by the plasma on cell death

An atmospheric-pressure air plasma is employed to treat C6 glioma cells in vitro. To elucidate on the mechanism causing cell death and role of reactive species (RS) in the medium produced by the plasma, the concentration of the long-lived RS such as hydrogen peroxide, nitrate, and ozone in the plasma-treated liquid (phosphate-buffered saline solution) is measured. When vitamin C is added to the medium as a ROS quencher, the viability of C6 glioma cells after the plasma treatment is different from that without vitamin C. The results demonstrate that reactive oxygen species (ROS) such as H2O2, and O3 constitute the main factors for inactivation of C6 glioma cells and the reactive nitrogen species (RNS) may only play an auxiliary role in cell death.

Auranofin, an inhibitor of thioredoxin reductase, induces apoptosis in hepatocellular carcinoma Hep3B cells by generation of reactive oxygen species.

Mammalian thioredoxin reductase (TrxR) plays a vital role in restoring cellular redox balance disrupted by reactive oxygen species (ROS) generation and oxidative damage. Here, we evaluated whether auranofin, a selective inhibitor of TrxR, could serve as a potential anti-cancer agent through its selective targeting of TrxR activity in Hep3B hepatocellular carcinoma cells. Auranofin treatment reduced the TrxR activity of these cells and induced apoptosis, which were accompanied by up-regulation of death receptors (DRs) and activation of caspases, as well as promotion of proteolytic degradation of poly(ADP-ribose)-polymerase. Treatment with a pan-caspase inhibitor reversed the auranofin-induced apoptosis and growth suppression, indicating that auranofin may induce apoptosis through a caspase-dependent mechanism involving both the intrinsic and extrinsic apoptotic pathways. Auranofin also significantly altered mitochondrial function, promoting mitochondrial membrane permeabilization and cytochrome c release by regulating Bcl-2 family proteins; these events were accompanied by an accumulation of ROS. Inhibition of ROS generation with the ROS quencher significantly attenuated the inactivation of TrxR in auranofin-treated cells and almost completely suppressed the auranofin-induced up-regulation of DRs and activation of caspases, thereby preventing auranofin-induced apoptosis and loss of cell viability. Taken together, these findings indicate that auranofin inhibition of TrxR activity in Hep3B cells activates ROS- and caspase-dependent apoptotic signaling pathways and triggers cancer cell death.

Hormetic shifting of redox environment by pro-oxidative resveratrol protects cells against stress

Resveratrol has gained tremendous interest owing to multiple reported health-beneficial effects. However, the underlying key mechanism of action of this natural product remained largely controversial. Here, we demonstrate that under physiologically relevant conditions major biological effects of resveratrol can be attributed to its generation of oxidation products such as reactive oxygen species (ROS). At low nontoxic concentrations (in general <50µM), treatment with resveratrol increased viability in a set of representative cell models, whereas application of quenchers of ROS completely truncated these beneficial effects. Notably, resveratrol treatment led to mild, Nrf2-specific gene expression reprogramming. For example, in primary epidermal keratinocytes derived from human skin this coordinated process resulted in a 1.3-fold increase of endogenously generated glutathione (GSH) and subsequently in a quantitative reduction of the cellular redox environment by 2.61mVmmol GSH per g protein. After induction of oxidative stress by using 0.78% (v/v) ethanol, endogenous generation of ROS was consequently reduced by 24% in resveratrol pre-treated cells. In contrast to the common perception that resveratrol acts mainly as a chemical antioxidant or as a target protein-specific ligand, we propose that the cellular response to resveratrol treatment is essentially based on oxidative triggering. In physiological microenvironments this molecular training can lead to hormetic shifting of cellular defense towards a more reductive state to improve physiological resilience to oxidative stress.

Mannitol metabolism during pathogenic fungal–host interactions under stressed conditions

Numerous plants and fungi produce mannitol, which may serve as an osmolyte or metabolic store; furthermore, mannitol also acts as a powerful quencher of reactive oxygen species (ROS). Some phytopathogenic fungi use mannitol to stifle ROS-mediated plant resistance. Mannitol is essential in pathogenesis to balance cell reinforcements produced by both plants and animals. Mannitol likewise serves as a source of reducing power, managing coenzymes, and controlling cytoplasmic pH by going about as a sink or hotspot for protons. The metabolic pathways for mannitol biosynthesis and catabolism have been characterized in filamentous fungi by direct diminishment of fructose-6-phosphate into mannitol-1-phosphate including a mannitol-1-phosphate phosphatase catalyst. In plants mannitol is integrated from mannose-6-phosphate to mannitol-1-phosphate, which then dephosphorylates to mannitol. The enzyme mannitol dehydrogenase plays a key role in host–pathogen interactions and must be co-localized with pathogen-secreted mannitol to resist the infection.

HMGB1 in the pathogenesis of ultraviolet-induced ocular surface inflammation.

High-mobility group box 1 (HMGB1) functions as a transcription-enhancing nuclear protein as well as a crucial cytokine that regulates inflammation. This study demonstrated that secretion of HMGB1 due to ultraviolet (UV) radiation inducing ocular surface inflammation-mediated reactive oxygen species (ROS) production. After treating conjunctival epithelial cells with UV radiation, HMGB1 was translocated from the nucleus to the cytoplasm and then eventually to the extracellular space. HMGB1 played a crucial role in UV-induced conjunctival neutrophil infiltration, which subsided when mice were pretreated with the HMGB1 inhibitors soluble receptor for advanced glycation endproducts (sRAGEs) and HMGB1 A box protein. In case of using ROS quencher, there was decrease in UV-induced HMGB1 secretion in conjunctival epithelial cells and mice. Considering that UV-induced chronic inflammation causes ocular surface change as pterygium, we have confirmed high HMGB1 translocation and ROS expression in human pterygium. Our findings therefore revealed a previously unknown mechanism of UV-induced ocular inflammation related to ROS and HMGB1 suggesting a new medical therapeutic target.

Syndecan-4 ectodomain evokes mobilization of podocyte TRPC6 channels and their associated pathways: An essential role for integrin signaling

PodocyteTRPC6 channels have been implicated in glomerular diseases. Syndecan-4 (Sdc4) is a membrane proteoglycan that can be cleaved to release a soluble ectodomain capable of paracrine and autocrine signaling. We have confirmed that overexpression of Sdc4 core protein increases surface abundance of TRPC6 channels in cultured podocytes, whereas Sdc4 knockdown has the opposite effect. Exposure to soluble Sdc4 ectodomain also increased the surface abundance of TRPC6, and increased cationic currents evoked by a diacylglycerol analog in podocytes. Sdc4 ectodomain increased generation of reactive oxygen species (ROS), reduced activation of RhoA, increased activation of Rac1, increased nuclear abundance of NFATc1, and increased total β3-integrin. The effects of Sdc4 ectodomain on cell-surface TRPC6 were blocked by the ROS quencher TEMPOL, and by the Rac1 inhibitor NSC-23766, but were not blocked by inhibition of calcineurin–NFATc1 signaling. The Sdc4 core protein co-immunoprecipitates with β3-integrin in cultured podocytes. Moreover, effects of Sdc4 ectodomain on TRPC6, ROS generation, Rac1 and RhoA modulation, and NFATc1 activation were blocked by cilengitide, a selective inhibitor of outside-in signaling through αv-containing integrins. Exposure to TNF, or serum from three patients with recurrent FSGS in relapse, increased shedding of podocyte Sdc4 ectodomains into the surrounding medium. This was also observed after treating podocytes with the metalloproteinase ADAM17 or after overexpression of the Sdc4 core protein. Increased concentrations of Sdc4 ectodomain were detected in urine of rats during acute puromycin aminonucleoside nephrosis. Locally generated Sdc4 may play a role in regulating TRPC6 channels, and may contribute to glomerular pathology.

Induction of reactive oxygen species–mediated apoptosis by purified Schisandrae semen essential oil in human leukemia U937 cells through activation of the caspase cascades and nuclear relocation of mitochondrial apoptogenic factors

The aim of this study was to evaluate the beneficial effects of Schisandrae semen essential oil (SSeo) on apoptosis events and the mechanisms associated with these effects in human leukemia U937 cells. The treatment of U937 cells with SSeo significantly inhibited survival and induced apoptosis. Schisandrae semen essential oil treatment increased the levels of death receptors and Fas, and activated caspases accompanied by proteolytic degradation of poly(ADP-ribose)-polymerase, which was associated with the downregulation of members of the inhibitor of apoptosis protein family protein expression; however, a pan-caspase inhibitor reversed SSeo-induced apoptosis. Treating the cells with SSeo also caused truncation of Bid, translocation of proapoptotic Bax to the mitochondria, and loss of mitochondrial membrane permeabilization, thereby inducing the release of cytochrome c into the cytosol. Subsequently, SSeo upregulated the translocation of mitochondrial apoptogenic factors, such as endonuclease G and apoptosis-inducing factor, into the nucleus during the apoptotic process. Notably, SSeo immediately increased the generation of intracellular reactive oxygen species (ROS); however, pretreatment with N-acetylcysteine, a common ROS quencher, almost completely blocked SSeo-induced apoptosis. Taken together, these findings indicate that SSeo caused ROS- and caspase-dependent cell death involving mitochondrial dysfunction and nuclear translocation of mitochondrial proapoptosis proteins. Based on our data, the consumption of Schisandrae semen or its essential oil is a good natural therapeutic agent for anticancer activity and regression.

ATP acting through P2Y receptors causes activation of podocyte TRPC6 channels: role of podocin and reactive oxygen species

Extracellular ATP may contribute to Ca(2+) signaling in podocytes during tubuloglomerular feedback (TGF) and possibly as a result of local tissue damage. TRPC6 channels are Ca(2+)-permeable cationic channels that have been implicated in the pathophysiology of podocyte diseases. Here we show using whole cell recordings that ATP evokes robust activation of TRPC6 channels in mouse podocyte cell lines and in rat podocytes attached to glomerular capillaries in ex vivo glomerular explants. The EC50 for ATP is ~10 μM and is maximal at 100 μM, and currents were blocked by the P2 antagonist suramin. In terms of maximal currents that can be evoked, ATP is the strongest activator of podocyte TRPC6 that we have characterized to date. Smaller currents were observed in response to ADP, UTP, and UDP. ATP-evoked currents in podocytes were abolished by TRPC6 knockdown and by pretreatment with 10 μM SKF-96365 or 50 μM La(3+). ATP effects were also abolished by inhibiting G protein signaling and by the PLC/PLA2 inhibitor D-609. ATP effects on TRPC6 were also suppressed by knockdown of the slit diaphragm scaffolding protein podocin, and also by tempol, a membrane-permeable quencher of reactive oxygen species. Modulation of podocyte TRPC6 channels, especially in foot processes, could provide a mechanism for regulation of glomerular function by extracellular nucleotides, possibly leading to changes in permeation through slit diaphragms. These results raise the possibility that sustained ATP signaling could contribute to foot process effacement, Ca(2+)-dependent changes in gene expression, and/or detachment of podocytes.

Angiotensin II Activation of TRPC6 Channels in Rat Podocytes Requires Generation of Reactive Oxygen Species

Angiotensin II (AII) plays a major role in the progression of chronic kidney diseases. Podocytes are essential components of the ultrafiltration apparatus, and are targets for AII signaling. AII has been shown to increase generation of reactive oxygen species (ROS) in podocytes. Canonical transient receptor potential-6 (TRPC6) channels stimulate Ca(2+) influx in podocytes, and have been implicated in glomerular disease. We observed that AII increased cationic currents in rat podocytes in an isolated glomerulus preparation in which podocytes are still attached to the underlying capillary. This effect was completely blocked by SKF-96365, by micromolar La(3+) , and by siRNA knockdown of TRPC6, indicating that TRPC6 is the primary source of Ca(2+) influx mobilized by endogenously expressed angiotensin II receptors in these cells. These responses were also blocked by the AT1R antagonist losartan, the phospholipase C inhibitor D-609, and by inhibition of G protein signaling. The pan-protein kinase C inhibitor chelerythrine had no effect. Importantly, pretreating podocytes with the ROS quencher manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) eliminated AII activation of TRPC6. Significant reductions of AII effects on podocyte TRPC6 were also observed after pretreatment with NADPH oxidase inhibitors apocynin or diphenylene iodonium (DPI). These data suggest that ROS production permits activation of TRPC6 channels by G protein and PLC-dependent cascades initiated by AII acting on AT1Rs in podocytes. This pathway also provides a basis whereby two forms of cellular stress-oxidative stress and Ca(2+) overload-converge on common pathways relevant to disease.

In vitro Antioxidant and Free Radical Scavenging Activity of Isolongifolene

Heavy metals can be included in the main category of pollutants. Nickel is a heavy metal used extensively and can contaminate the soil mainly through sewage sludge, industrial compost and atmospheric fallout, especially near processing plants (Poulik, 1997).Nickel is an essential micronutrient for plant growth and it is also a component of enzyme urease which is required for nitrogen metabolism in higher plants (Dixon et al., 2004). However, excess nickel is known to be toxic and many studies have been conducted concerning Ni toxicity in various plant species (Pandolfini et al., 1992; Gajewska and Skodowska, 2008; Ahmad, et al., 2009; Khan and Khan, 2010; Gajewska and Skodowska, 2010; Singh and Pandey, 2011). The most common symptoms of nickel toxicity in plants are growth inhibition, photosynthesis, mineral nutrition, sugar transport and water relations (Seregin and Kozhevnikova, 2006).Over production of reactive oxygen species (ROS) is a common response of plants to heavy metal stress especially nickel stress (Blokhina et al., 2003). It is well established that the overproduction of ROS induces oxidative damage to various cellular constituents, such as lipid, proteins and nucleic acids (Shah et al., 2001). One of the most damaging oxidative effects is the peroxidation of membrane lipids, which results in concomitant production of melondialdehyde (MDA) (Hodges et al., 1999). Plants have evolved antioxidative mechanism to detoxify and eliminate these harmful ROS (Chri, et al., 2008). The antioxidative defense system includes antioxidant molecules like proline and antioxidant enzymes such as superoxide dismutase (SOD, EC 1.15.1.1) peroxidase (POD, EC 1.11.1.7) as well as catatlase (CAT, EC 1.11.1.6). SOD is the first enzyme in detoxifying process, converts O 2 to H 2 O 2 and POD and CAT catalyse the breakdown of H 2 O 2 (Asada, 1992, 1999). Proline also acts as an effective ROS quencher and accumulates heavily in plants under metal stress (Alia and Pardha Saradhi, 1991). The role of nitrogen fixing cyanobacteria in enhancing soil fertility has been long known and is well documented (De, 1939; Venkataraman, 1981; Sinha and Hader, 1996). Cyanobacteria contributes to overall soil health not only by its ability to perform biological nitrogen fixation but also because of its ability to produce polysaccharides and other bioactive compounds which has a growth stimulating effect on plants, as well as ensuring maintenance of soil quality and preventing erosion (Singh, 1950). The toxic effect of nickel on cyanobacteria especially on their growth, carbon fixation, nitrogen metabolism (Rai and Raizada, 1985, 1986), phosphorus metabolism (Asthana et al., 1992) and bioremediation (Shukla et al., 2009) are studied earlier Effect of nickel stress on growth and antioxidants in cyanobacterium Cylindrospermum sp.

Inactivation of Norovirus on Dry Copper Alloy Surfaces

Noroviruses (family Caliciviridae) are the primary cause of viral gastroenteritis worldwide. The virus is highly infectious and touching contaminated surfaces can contribute to infection spread. Although the virus was identified over 40 years ago the lack of methods to assess infectivity has hampered the study of the human pathogen. Recently the murine virus, MNV-1, has successfully been used as a close surrogate. Copper alloys have previously been shown to be effective antimicrobial surfaces against a range of bacteria and fungi. We now report rapid inactivation of murine norovirus on alloys, containing over 60% copper, at room temperature but no reduction of infectivity on stainless steel dry surfaces in simulated wet fomite and dry touch contamination. The rate of inactivation was initially very rapid and proportional to copper content of alloy tested. Viral inactivation was not as rapid on brass as previously observed for bacteria but copper-nickel alloy was very effective. The use of chelators and quenchers of reactive oxygen species (ROS) determined that Cu(II) and especially Cu(I) ions are still the primary effectors of toxicity but quenching superoxide and hydroxyl radicals did not confer protection. This suggests Fenton generation of ROS is not important for the inactivation mechanism. One of the targets of copper toxicity was the viral genome and a reduced copy number of the gene for a viral encoded protein, VPg (viral-protein-genome-linked), which is essential for infectivity, was observed following contact with copper and brass dry surfaces. The use of antimicrobial surfaces containing copper in high risk closed environments such as cruise ships and care facilities could help to reduce the spread of this highly infectious and costly pathogen.