Sparing effects of selenium and ascorbic acid on vitamin C and E in guinea pig tissues

BACKGROUND: Selenium (Se), vitamin C and vitamin E function as antioxidants within the body. In this study, we investigated the effects of reduced dietary Se and L-ascorbic acid (AA) on vitamin C and α-tocopherol (AT) status in guinea pig tissues. METHODS: Male Hartley guinea pigs were orally dosed with a marginal amount of AA and fed a diet deficient (Se-D/MC), marginal (Se-M/MC) or normal (Se-N/MC) in Se. An additional diet group (Se-N/NC) was fed normal Se and dosed with a normal amount of AA. Guinea pigs were killed after 5 or 12 weeks on the experimental diets at 24 and 48 hours post AA dosing. RESULTS: Liver Se-dependent glutathione peroxidase activity was decreased (P < 0.05) in guinea pigs fed Se or AA restricted diets. Plasma total glutathione concentrations were unaffected (P > 0.05) by reduction in dietary Se or AA. All tissues examined showed a decrease (P < 0.05) in AA content in Se-N/MC compared to Se-N/NC guinea pigs. Kidney, testis, muscle and spleen showed a decreasing trend (P < 0.05) in AA content with decreasing Se in the diet. Dehydroascorbic acid concentrations were decreased (P < 0.05) in several tissues with reduction in dietary Se (heart and spleen) or AA (liver, heart, kidney, muscle and spleen). At week 12, combined dietary restriction of Se and AA decreased AT concentrations in most tissues. In addition, restriction of Se (liver, heart and spleen) and AA (liver, kidney and spleen) separately also reduced AT in tissues. CONCLUSION: Together, these data demonstrate sparing effects of Se and AA on vitamin C and AT in guinea pig tissues

Effects of Helicobacter suis γ-glutamyl transpeptidase on lymphocytes: modulation by glutamine and glutathione supplementation and outer membrane vesicles as a putative delivery route of the enzyme

Helicobacter (H.) suis colonizes the stomach of the majority of pigs as well as a minority of humans worldwide. Infection causes chronic inflammation in the stomach of the host, however without an effective clearance of the bacteria. Currently, no information is available about possible mechanisms H. suis utilizes to interfere with the host immune response. This study describes the effect on various lymphocytes of the γ-glutamyl transpeptidase (GGT) from H. suis. Compared to whole cell lysate from wild-type H. suis, lysate from a H. suis ggt mutant strain showed a decrease of the capacity to inhibit Jurkat T cell proliferation. Incubation of Jurkat T cells with recombinantly expressed H. suis GGT resulted in an impaired proliferation, and cell death was shown to be involved. A similar but more pronounced inhibitory effect was also seen on primary murine CD4+ T cells, CD8+ T cells, and CD19+ B cells. Supplementation with known GGT substrates was able to modulate the observed effects. Glutamine restored normal proliferation of the cells, whereas supplementation with reduced glutathione strengthened the H. suis GGT-mediated inhibition of proliferation. H. suis GGT treatment abolished secretion of IL-4 and IL-17 by CD4+ T cells, without affecting secretion of IFN-γ. Finally, H. suis outer membrane vesicles (OMV) were identified as a possible delivery route of H. suis GGT to lymphocytes residing in the deeper mucosal layers. Thus far, this study is the first to report that the effects on lymphocytes of this enzyme, not only important for H. suis metabolism but also for that of other Helicobacter species, depend on the degradation of two specific substrates: glutamine and reduced glutatione. This will provide new insights into the pathogenic mechanisms of H. suis infection in particular and infection with gastric helicobacters in general

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Protection by ascorbate against apoptosis of thymocytes: implications of ascorbate-induced nonlethal oxidative stress and poly(ADP-ribosyl)ation

Apoptosis can be triggered in thymocytes with stimuli (6α- methylprednisolone, thapsigargin, and etoposide) acting by different mechanisms. In each of these instances cell death is extensively prevented until 5 h of incubation when cells are preincubated with 250 μM ascorbic acid (AA) for 1 h, then washed, and incubated in fresh medium containing the above mentioned apoptotic stimuli. In addition, the degree of spontaneous apoptosis of untreated thymocytes is somewhat lower in the AA-preincubated cells. The protection against apoptosis does not seem to be dependent on the intracellular enrichment of AA, as measured at the end of the preincubation period. On the contrary, such a protection is strictly related to a partial loss of ascorbate in the medium (possibly due to its autooxidation), is catalase-inhibitable, and is reproduced by a preincubation of the cells with nontoxic concentrations of hydrogen peroxide. The AA-supplemented cells show a remarkable decrease in NAD+ levels and a significant increase of poly(ADP- ribose) polymerase (PARP) activity. Consistently with these results, the addition of PARP inhibitors, such as thymidine and 3-aminobenzamide, during the preincubation with AA, prevents NAD+ depletion and abolishes the protective effect of AA against apoptosis. The possibility is discussed that an early activation of PARP by stimuli which are nontoxic per se makes the cells able to withstand subsequent apoptotic stimuli which are otherwise lethal

Histochemical detection of lipid peroxidation in the liver of bromobenzene-poisoned mice.

The possibility of detecting lipid peroxidation histochemically was investigated in liver tissue in vivo, in conditions in which the process has been demonstrated by biochemical methods. The technique was based on the detection of aldehyde functions with the use of the Schiff's reagent. The study was carried out on bromobenzene-intoxicated mice, which generally exhibit levels of lipid peroxidation considerably higher than those observed in the case of other hepatotoxins. Liver sections from control animals were unstainable by the reagent, while sections from bromobenzene-poisoned mice showed a purple stain of various intensity, unhomogeneously distributed, sometimes with a mediolobular localization. Microphotometric measurements were performed at 565 nm by means of a computer-controlled microscope photometer. The ratios of Schiff-positive area relative to total section area, as well as the total extinctions referred to 100 sq mu of the sections, showed a high correlation with the corresponding hepatic contents of malonic dialdehyde, chosen as biochemical index of lipid peroxidation. In vitro studies in which liver sections were incubated in the presence of NADPH-Fe2+, showed a Schiff positivity which increased with the incubation time, confirming the reliability of the histochemical method. Another procedure, based on the use of 2-OH-3-naphtoic acid hydrazide coupled with fast blue B, was also developed and proved to be possibly more sensitive than Schiff's reagent in the detection of lipid peroxidation in liver tissue

Calpains and cancer: friends or enemies?

Calpains are a complex family of ubiquitous or tissue-specific cysteine proteases that proteolyze a variety of substrates (leading to their degradation or functional modulation) and are implicated in several pathophysiological phenomena. In tumor cell biology, calpains are implicated in a triple way: they are involved in different processes crucial for tumor progression, including cell proliferation, apoptotic cell death, survival mechanisms, migration and invasiveness; they have aberrant expression in several human cancers; a variety of anticancer drugs induce cytotoxicity through activation of calpains or the latter can influence response to therapy. This review covers established and recent literature showing these diverse aspects in tumor cells

The dark side of gamma-glutamyltransferase (GGT): Pathogenic effects of an ‘antioxidant’ enzyme

Having long been regarded as just a member in the cellular antioxidant systems, as well as a clinical biomarker of hepatobiliary diseases and alcohol abuse, gamma-glutamyltransferase (GGT) enzyme activity has been highlighted by more recent research as a critical factor in modulation of redox equilibria within the cell and in its surroundings. Moreover, due to the prooxidant reactions which can originate during its metabolic function in selected conditions, experimental and clinical studies are increasingly involving GGT in the pathogenesis of several important disease conditions, such as atherosclerosis, cardiovascular diseases, cancer, lung inflammation, neuroinflammation and bone disorders. The present article is an overview of the laboratory findings that have prompted an evolution in interpretation of the significance of GGT in human pathophysiology