The invertebrate Caenorhabditis elegans biosynthesizes ascorbate.

l-Ascorbate, commonly known as vitamin C, serves as an antioxidant and cofactor essential for many biological processes. Distinct ascorbate biosynthetic pathways have been established for animals and plants, but little is known about the presence or synthesis of this molecule in invertebrate species. We have investigated ascorbate metabolism in the nematode Caenorhabditis elegans, where this molecule would be expected to play roles in oxidative stress resistance and as cofactor in collagen and neurotransmitter synthesis. Using high-performance liquid chromatography and gas-chromatography mass spectrometry, we determined that ascorbate is present at low amounts in the egg stage, L1 larvae, and mixed animal populations, with the egg stage containing the highest concentrations. Incubating C. elegans with precursor molecules necessary for ascorbate synthesis in plants and animals did not significantly alter ascorbate levels. Furthermore, bioinformatic analyses did not support the presence in C. elegans of either the plant or the animal biosynthetic pathway. However, we observed the complete (13)C-labeling of ascorbate when C. elegans was grown with (13)C-labeled Escherichia coli as a food source. These results support the hypothesis that ascorbate biosynthesis in invertebrates may proceed by a novel pathway and lay the foundation for a broader understanding of its biological role

The mutualistic fungus Piriformospora indica protects barley roots from a loss of antioxidant capacity caused by the necrotrophic pathogen Fusarium culmorum

Fusarium culmorum causes root rot in barley (Hordeum vulgare), resulting in severely reduced plant growth and yield. Pretreatment of roots with chlamydospores of the mutualistic root-colonizing basidiomycete Piriformospora indica (Agaricomycotina) prevented necrotization of root tissues and plant growth retardation commonly associated with Fusarium root rot. Quantification of Fusarium infections with a real-time PCR assay revealed a correlation between root rot symptoms and the relative amount of fungal DNA. Fusarium-infected roots showed reduced levels of ascorbate and glutathione (GSH), along with reduced activities of antioxidant enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR), and monodehydroascorbate reductase (MDHAR). Consistent with this, Fusarium-infected roots showed elevated levels of lipid hydroperoxides and decreased ratios of reduced to oxidized forms of ascorbate and glutathione. In clear contrast, roots treated with P. indica prior to inoculation with F. culmorum showed levels of ascorbate and GSH that were similar to controls. Likewise, lipid peroxidation and the overall reduction in antioxidant enzyme activities were largely attenuated by P. indica in roots challenged by F. culmorum. These results suggest that P. indica protects roots from necrotrophic pathogens at least partly, through activating the plant’s antioxidant capacity

Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum.

In most aerobic organisms hemoperoxidases play a major role in H(2)O(2)-detoxification, but trypanosomatids have been reported to lack this activity. Here we describe the properties of an ascorbate-dependent hemoperoxidase (TcAPX) from the American trypanosome Trypanosoma cruzi. The activity of this plant-like enzyme can be linked to the reduction of the parasite-specific thiol trypanothione by ascorbate in a process that involves nonenzymatic interaction. The role of heme in peroxidase activity was demonstrated by spectral and inhibition studies. Ascorbate could saturate TcAPX activity indicating that the enzyme obeys Michaelis-Menten kinetics. Parasites that overexpressed TcAPX activity were found to have increased resistance to exogenous H(2)O(2). To determine subcellular location an epitope-tagged form of TcAPX was expressed in T. cruzi, which was observed to colocalize with endoplasmic reticulum resident chaperone protein BiP. These findings identify an arm of the oxidative defense system of this medically important parasite. The absence of this redox pathway in the human host may be therapeutically exploitable

Existing antioxidant levels are more important in acclimation to supplemental UV-B irradiation than inducible ones: Studies with high light pretreated tobacco leaves

Greenhouse grown tobacco plants were exposed to supplemental ultraviolet irradiation (280-400 nm, UV-B centered) for 6 days and changes in their photosynthesis (gas exchange and electron transport) and general and specific antioxidant activities were measured. UV irradiation corresponded to 8.95 kJ m-2 d-1 biologically effective dose and was supplemented to below ambient (200 μmol m-2 s-1 photon flux density) photosynthetic photon flux density (PPFD, 400-700 nm). Two groups of plants, which were different in their leaf antioxidant capacities due to one of them having been acclimated to high irradiance (1000 μmol m-2 s-1 PPFD) before the UV treatment, responded differently. High light pretreated leaves lost approximately 25% of photosynthetic activity during the UV exposure and showed no change either in the amounts of UV-absorbing pigments or antioxidant levels. On the other hand, leaves which were exposed to UV irradiation without the preceding high light acclimation had 60% lower photosynthesis by the end of the treatment, and increased antioxidant activities. Our results emphasize the importance of base antioxidant levels over inducible pools in leaf responses to low doses of UV irradiation and may also contribute to hypotheses on acclimation under field conditions

Vitamin C alleviates acute enterocolitis in Campylobacter jejuni infected mice

Human foodborne infections with the zoonotic pathogen Campylobacter jejuni are on the rise and constitute a significant socioeconomic burden worldwide. The health-beneficial, particularly anti-inflammatory effects of vitamin C (ascorbate) are well known. In our preclinical intervention study, we assessed potential anti-pathogenic and immunomodulatory effects of ascorbate in C. jejuni-infected secondary abiotic IL-10-/- mice developing acute campylobacteriosis similar to humans. Starting 4 days prior peroral C. jejuni-infection, mice received synthetic ascorbate via the drinking water until the end of the experiment. At day 6 post-infection, ascorbate-treated mice harbored slightly lower colonic pathogen loads and suffered from less severe C. jejuni-induced enterocolitis as compared to placebo control animals. Ascorbate treatment did not only alleviate macroscopic sequelae of infection, but also dampened apoptotic and inflammatory immune cell responses in the intestines that were accompanied by less pronounced pro-inflammatory cytokine secretion. Remarkably, the anti-inflammatory effects of ascorbate pretreatment in C. jejuni-infected mice were not restricted to the intestinal tract but could also be observed in extra-intestinal compartments including liver, kidneys and lungs. In conclusion, due to the potent anti-inflammatory effects observed in the clinical murine C. jejuni-infection model, ascorbate constitutes a promising novel option for prophylaxis and treatment of acute campylobacteriosis

Retrospective Evaluation of Clinical Experience With Intravenous Ascorbic Acid in Patients With Cancer.

BACKGROUND: Intravenous ascorbic acid (IV AA) has been used extensively in cancer patients throughout the United States. Currently, there are limited data on the safety and clinical effects of IV AA. The purpose of this study was to expand the current literature using a retrospective analysis of adverse events and symptomatic changes of IV AA in a large sample of cancer patients. METHODS: We conducted a retrospective chart review of all patients receiving IV AA for cancer at the Thomas Jefferson University Hospital over a 7-year period. We assessed all reports of adverse events, laboratory findings, and hospital or emergency department admissions. We also reviewed quality-of-life data, including fatigue, nausea, pain, appetite, and mood. RESULTS: There were 86 patients who received a total of 3034 doses of IV AA ranging from 50 to 150g. In all, 32 patients received only ascorbic acid as part of their cancer management (1197 doses), whereas 54 patients received ascorbic acid in conjunction with chemotherapy (1837 doses). The most common adverse events related to ascorbic acid were temporary nausea and discomfort at the injection site. All events reported in the ascorbic acid alone group were associated with less than 3% of the total number of infusions. Patients, overall, reported improvements in fatigue, pain, and mood while receiving ascorbic acid. CONCLUSIONS: The results of this retrospective analysis support the growing evidence that IV AA is generally safe and well tolerated in patients with cancer, and may be useful in symptom management and improving quality of life

Inhibition of DNA methyltransferase leads to increased genomic 5-hydroxymethylcytosine levels in hematopoietic cells.

5-Hydroxymethylcytosine (5hmC) is produced from 5-methylcytosine (5mC) by Ten-eleven translocation (TET) dioxygenases. The epigenetic modification 5hmC has crucial roles in both cellular development and differentiation. The 5hmC level is particularly high in the brain. While 5mC is generally associated with gene silencing/reduced expression, 5hmC is a more permissive epigenetic mark. To understand its physiological function, an easy and accurate quantification method is required. Here, we have developed a novel LC-MS/MS-based approach to quantify both genomic 5mC and 5hmC contents. The method is based on the liberation of nucleobases by formic acid. Applying this method, we characterized the levels of DNA methylation and hydroxymethylation in mouse brain and liver, primary hepatocytes, and various cell lines. Using this approach, we confirm that the treatment of different cell lines with the DNA methyltransferase inhibitor 5-aza-2\u27-deoxycytidine leads to a decrease in 5mC content. This decrease was accompanied by an increase in 5hmC levels in cell lines of hematopoietic origin. Finally, we showed that ascorbate elevates the levels of 5hmC and augments the effect of 5-aza-2\u27-deoxycytidine without significantly influencing 5mC levels

Reduction of a-tocopherylquinone Model Compound With Various Reductant

In order to study the possibility of tranformation of a-tocopherylquinone (TQ) into a more oxidiseable compound and also to find out the recycling effect in the cells, an experiment was conducted by reducing the model compound 2-(3- hydroxy-3-methylbutyl)-3,5,6-trimethyl-1,4-benzoquinone (PQ) with various reductants. In the experiment it was shown that glutathione did not reduce PQ,nor NADH by itself, so the effective reductant in the NADH/FAD combination must have been FADH2. Thus there is a probability that in a biological system, the most probable reductant for TQ would be a flavin enzyme rather that ascorbic acid or glutathione. The non-physiological dithiothreitol was as effective as NADH/ FAD which is interesting because of its similarity to the physiologically important reduced lipoic acid. The reactivity of the various reductants used in this experiment decrease in the order of dithiothreitol ~ NADH/FAD (8/10) > sodium dithionite > NADH/FAD (2:10) > sodium ascorbate > ascorbic acid (Fig.8)

Oxidative Stress Associated with Chilling Injury in Immature Fruit: Postharvest Technological and Biotechnological Solutions

Immature, vegetable-like fruits are produced by crops of great economic importance, including cucumbers, zucchini, eggplants and bell peppers, among others. Because of their high respiration rates, associated with high rates of dehydration and metabolism, and their susceptibility to chilling injury (CI), vegetable fruits are highly perishable commodities, requiring particular storage conditions to avoid postharvest losses. This review focuses on the oxidative stress that affects the postharvest quality of vegetable fruits under chilling storage. We define the physiological and biochemical factors that are associated with the oxidative stress and the development of CI symptoms in these commodities, and discuss the different physical, chemical and biotechnological approaches that have been proposed to reduce oxidative stress while enhancing the chilling tolerance of vegetable fruits

Rate-limiting Step Preceding Cytochrome c Release in Cells Primed for Fas-mediated Apoptosis Revealed by Analysis of Cellular Mosaicism of Respiratory Changes

In the present work, Jurkat cells undergoing anti-Fas antibody (anti-Fas)-triggered apoptosis exhibited in increasing proportion a massive release of cytochrome c from mitochondria, as revealed by double-labeling confocal immunofluorescence microscopy. The cytochrome c release was followed by a progressive reduction in the respiratory activity of the last respiratory enzyme, cytochrome c oxidase (COX), and with a little delay, by a decrease in overall endogenous respiration rate, as measured in vivo in the whole cell population. Furthermore, in vivo titration experiments showed that an ~30% excess of COX capacity over that required to support endogenous respiration, found in naive cells, was maintained in anti-Fas-treated cells having lost ~40% of their COX respiratory activity. This observation strongly suggested that only a subpopulation of anti-Fas-treated cells, which maintained the excess of COX capacity, respired. Fractionation of cells on annexin V-coated paramagnetic beads did indeed separate a subpopulation of annexin V-binding apoptotic cells with fully released cytochrome c and completely lacking respiration, and a nonbound cell subpopulation exhibiting nearly intact respiration and in their great majority preserving the mitochondrial cytochrome c localization. The above findings showed a cellular mosaicism in cytochrome c release and respiration loss, and revealed the occurrence of a rate-limiting step preceding cytochrome c release in the apoptotic cascade. Furthermore, the striking observation that controlled digitonin treatment caused a massive and very rapid release of cytochrome c and complete loss of respiration in the still respiring anti-Fas-treated cells, but not in naive cells, indicated that the cells responding to digitonin had already been primed for apoptosis, and that this treatment bypassed or accelerated the rate-limiting step most probably at the level of the mitochondrial outer membrane