SelK is a novel ER stress-regulated protein and protects HepG2 cells from ER stress agent-induced apoptosis

Selenoprotein K (SelK), an endoplasmic reticulum (ER) resident protein, its biological function has been less-well studied. To investigate the role of SelK in the ER stress response, effects of SelK gene silence and ER stress agents on expression of SelK and cell apoptosis in HepG2 cells were studied. The results showed that SelK was regulated by ER stress agents, Tunicamycin (Tm) and β-Mercaptoethanol (β-ME), in HepG2 cells. Moreover, the SelK gene silence by RNA interference could significantly aggravate HepG2 cell death and apoptosis induced by the ER stress agents. These results suggest that SelK is an ER stress-regulated protein and plays an important role in protecting HepG2 cells from ER stress agent-induced apoptosis.     

RESPONSES OF LEAF SPECTRAL REFLECTANCE TO PLANT STRESS

Leaf spectral reflectances were measured to determine whether leaf reflectance responses to plant stress may differ according to the agent of stress and species. As a result of decreased absorption by pigments, reflectance at visible wavelengths increased consistently in stressed leaves for eight stress agents and among six vascular plant species. Visible reflectance was most sensitive to stress in the 535–640-nm and 685–700-nm wavelength ranges. A sensitivity minimum occurred consistently near 670 nm. Infrared reflectance was comparatively unresponsive to stress, but increased at 1,400–2,500 nm with severe leaf dehydration and the accompanying decreased absorption by water. Thus, visible rather than infrared reflectance was the most reliable indicator of plant stress. Visible reflectance responses to stress were spectrally similar among agents of stress and species.     

Enhancement of glutathione levels in mouse peritoneal macrophages by sodium arsenite, cadmium chloride and glucose/glucose oxidase

Glutathione content of mouse peritoneal macrophages markedly increased when they were exposed to insulting agents like sodium arsenite, cadmium chloride, and glucose/glucose oxidase which generates hydrogen peroxide. This increase was attributed to the induction of the cystine transport activity by these agents. The transport activity for other amino acids was not induced, but rather diminished by these agents. Heat shock treatment did not induce the cystine transport activity, nor did it augment glutathione. Since glutathione protects cells against the cytotoxic effects of these agents, the induction of the cystine transport activity constitutes a protective mechanism related to the stress caused by the agents. The protein component(s) for cystine transport may fall into the category of the stress protein.     

A comparative study of stress-mediated immunological functions with the adjuvanticity of alum

The efficacy of a vaccine is generally dependent on an adjuvant, which enhances the immune functions and alum has been widely used in human immunization. Alum activates the intracellular stress sensors inflammasomes, but whether these are responsible for the adjuvanticity is controversial. The objectives of this investigation were to examine the hypothesis that alum-mediated adjuvanticity is a function of stress and conversely that stress agents will elicit adjuvanticity. The investigation was carried out in BALB/c mice by SC immunization with ovalbumin (OVA) mixed with alum. This elicited inflammasomes, with significant activation of caspase 1, production of IL-1β, and adjuvanticity, demonstrated by enhancing OVA-specific serum IgG antibodies, CD4(+) T cells, and proliferation. The novel finding that alum induced HSP70 suggests that stress is involved in the mechanism of adjuvanticity. This was confirmed by inhibition studies with PES (phenylethynesulfonamide), which disrupts inducible HSP70 function, and inhibited both inflammasomes and the adjuvant function. Parallel studies were pursued with an oxidative agent (sodium arsenite), K-releasing agent (Gramicidin) and a metal ionophore (dithiocarbamate). All 3 stress agents induced HSP70, inflammasomes, and the adjuvant functions. Furthermore, up-regulation of membrane associated IL-15 on DC and CD40L on T cells in the animals treated with alum or the stress agents mediate the interactions between splenic CD11c DC and CD4(+) or CD8(+) T cells. The results suggest that the three stress agents elicit HSP70, a hallmark of stress, as well as inflammasomes and adjuvanticity, commensurate with those of alum, which may provide an alternative strategy in developing novel adjuvants.     

Monocyte chemotactic protein-induced protein (MCPIP) promotes inflammatory angiogenesis via sequential induction of oxidative stress, endoplasmic reticulum stress and autophagy

Major diseases such as cardiovascular diseases, rheumatoid arthritis, diabetes, obesity and tumor growth are known to involve inflammation. Inflammatory molecules such as MCP-1, TNF-α, IL-1β and IL-8 are known to promote angiogenesis. MCP-induced protein (MCPIP), originally discovered as a novel zinc finger protein induced by MCP-1, is also induced by other inflammatory agents. MCPIP was shown to mediate MCP-1-induced angiogenesis. Whether angiogenesis induced by other inflammatory agents is mediated via MCPIP is unknown and the molecular mechanisms involved in angiogenesis induced by MCPIP have not been elucidated. The aim of this study was to bridge this gap and delineate the sequential processes involved in angiogenesis mediated via MCPIP. siRNA knockdown of MCPIP was used to determine whether different inflammatory agents, MCP-1, TNF-α, IL-1β and IL-8, mediate angiogenesis via MCPIP in human umbilical vein endothelial cells (HUVECs). Chemical inhibitors and specific gene knockdown approach were used to inhibit each process postulated. Oxidative stress was inhibited by apocynin or cerium oxide nanoparticles or knockdown of NADPH oxidase subunit, phox47. Endoplasmic reticulum (ER) stress was blocked by tauroursodeoxycholate or knockdown of ER stress signaling protein IRE-1 and autophagy was inhibited by the use of 3'methyl adenine, or LY 294002 or by specific knockdown of beclin1. Matrigel assay was used as a tool to study angiogenic differentiation induced by inflammatory agents or MCPIP overexpression in HUVECs. Tube formation induced by inflammatory agents, TNF-α, IL-1β, IL-8 and MCP-1 was inhibited by knockdown of MCPIP. Forced MCPIP-expression induced oxidative stress, ER stress, autophagy and angiogenic differentiation in HUVECs. Inhibition of each step caused inhibition of each subsequent step postulated. The results reveal that angiogenesis induced by inflammatory agents is mediated via induction of MCPIP that causes oxidative and nitrosative stress resulting in ER stress leading to autophagy required for angiogenesis. The sequence of events suggested to be involved in inflammatory angiogenesis by MCPIP could serve as possible targets for therapeutic intervention of angiogenesis-related disorders.     

Effects of oxidative stress and antioxidant treatments on eicosanoid synthesis and lipid peroxidation in long term human umbilical vein endothelial cells culture.

We analyzed the influence of oxidative stress and agents that modify its effect in human umbilical vein endothelial cell cultures (HUVEC). The parameters analyzed were PGI2, TXA2, PGI2/TXA2 ratio, lipid peroxidation and cell viability. Oxidative stress was induced by H2O2. The agents (treatments) that were tested are: antioxidant enzymes (superoxide dismutase and catalase), oxygen free radical scavenger (vitamin E) and eicosanoids of the series 2 and 3 (Arachidonic acid, Eicosapentanoic acid). In this study we show, in long term endothelial cell cultures, the effects of different levels of oxidative stress alone or in combination with the different treatment agents, over the analyzed parameters. With induced oxidative stress alone the results obtained indicate that it has a harmful effect over cell function and viability, and that this effect is dose and time dependent. In absence of oxidative stress in basal situation, none of the treatments assayed showed significant differences compared to control cultures in the different analyzed parameters. When oxidative stress increased, antioxidant enzymes reduced cell damage and had a protective function, whereas Eicosapentanoic acid and vitamin E presented a lower level of protection. No beneficial effect was observed with arachidonic acid treatments. A significant increase in cell survival was observed in culture cells with oxidative stress when they were treated with antioxidant enzymes.     

Use of chemosensitization to overcome fludioxonil resistance in Penicillium expansum

Aim: To overcome fludioxonil resistance of Penicillium expansum, a mycotoxigenic fungal pathogen causing postharvest decay in apple, by using natural phenolic chemosensitizing agents. Methods and Results: Fludioxonil‐resistant mutants of P. expansum were co‐treated with different oxidising and natural phenolic agents. Resistance was overcome by natural phenolic chemosensitizing agents targeting the oxidative stress–response pathway. These agents also augmented effectiveness of the fungicide, kresoxim‐methyl. Results indicated that alkyl gallates target mitochondrial respiration and/or its antioxidation system. Fungal mitochondrial superoxide dismutase (Mn‐SOD) plays a protective role against alkyl gallates. Conclusions: Natural chemosensitizing agents targeting the oxidative stress–response system, such as Mn‐SOD, can synergize commercial fungicides. Significance and Impact of the Study: Redox‐active compounds can serve as potent chemosensitizing agents to overcome resistance and lower effective dosages of fungicides. This can reduce costs with coincidental lowering of environmental and health risks.     

Novel role of VMP1 as modifier of the pancreatic tumor cell response to chemotherapeutic drugs

We hypothesized that inhibiting molecules that mediate the adaptation response to cellular stress can antagonize the resistance of pancreatic cancer cells to chemotherapeutic drugs. Toward this end, here, we investigated how VMP1, a stress‐induced autophagy‐associated protein, modulate stress responses triggered by chemotherapeutic agents in PDAC. We find that VMP1 is particularly over‐expressed in poorly differentiated human pancreatic cancer. Pharmacological studies show that drugs that work, in part, via the endoplasmic reticulum stress response, induce VMP1 expression. Similarly, VMP1 is induced by known endoplasmic reticulum stress activators. Genetic inactivation of VMP1 using RNAi‐based antagonize the pancreatic cancer stress response to antitumoral agents. Functionally, we find that VMP1 regulates both autophagy and chemotherapeutic resistance even in the presence of chloroquin, ATG5 or Beclin 1 siRNAs, or a Beclin 1‐binding VMP1 mutant. In addition, VMP1 modulates endoplasmic reticulum stress independently of its coupling to the molecular and cellular autophagy machinery. Preclinical studies demonstrate that xenografts expressing an inducible and tractable form of VMP1 show increased resistance to the gemcitabine treatment. These results underscore a novel role for VMP1 as a potential therapeutic target for combinatorial therapies aimed at sensitizing pancreatic cancer cells to chemotherapeutic agents as well as provide novel molecular mechanisms to better understand this phenomenon. J. Cell. Physiol. 228: 1834–1843, 2013. © 2013 Wiley Periodicals, Inc.     

Comparative analysis of mutants in the mycothiol biosynthesis pathway in Mycobacterium smegmatis

The role of mycothiol in mycobacteria was examined by comparative analysis of mutants disrupted in the four known genes encoding the protein machinery needed for mycothiol biosynthesis. These mutants were sensitive to acid stress, antibiotic stress, alkylating stress, and oxidative stress indicating that mycothiol and mycothiol-dependent enzymes protect the mycobacterial cell against attack from various different types of stresses and toxic agents.     

Protective peptides derived from novel glial proteins

In studying the mediators of VIP neurotrophism in the central nervous system, two glial proteins have been discovered. Both of these proteins contain short peptides that exhibit femtomolar potency in preventing neuronal cell death from a wide variety of neurotoxic substances. Extension of these peptides to models of oxidative stress or neurodegeneration in vivo have indicated significant efficacy in protection. These peptides, both as individual agents and in combination, have promise as possible protective agents in the treatment of human neurodegenerative disease and in pathologies involving oxidative stress.