The yeast metacaspase is implicated in oxidative stress response in frataxin-deficient cells

Friedreich ataxia is the most common recessive neurodegenerative disease and is caused by reduced expression of mitochondrial frataxin. Frataxin depletion causes impairment in iron-sulfur cluster and heme biosynthesis, disruption of iron homeostasis and hypersensitivity to oxidants. Currently no pharmacological treatment blocks disease progression, although antioxidant therapies proved to benefit patients. We show that sensitivity of yeast frataxin-deficient cells to hydrogen peroxide is partially mediated by the metacaspase. Metacaspase deletion in frataxin-deficient cells results in recovery of antioxidant capacity and heme synthesis. In addition, our results suggest that metacaspase is associated with mitochondrial respiration, intracellular redox control and genomic stability.     

Nod factors stimulate seed germination and promote growth and nodulation of pea and vetch under competitive conditions

Nod factors are lipochitooligosaccharide (LCO) produced by soil bacteria commonly known as rhizobia acting as signals for the legume plants to initiate symbiosis. Nod factors trigger early symbiotic responses in plant roots and initiate the development of specialized plant organs called nodules, where biological nitrogen fixation takes place. Here, the effect of specific LCO originating from flavonoid induced Rhizobium leguminosarum bv. viciae GR09 culture was studied on germination, plant growth and nodulation of pea and vetch. A crude preparation of GR09 LCO significantly enhanced symbiotic performance of pea and vetch grown under laboratory conditions and in the soil. Moreover, the effect of GR09 LCOs seed treatments on the genetic diversity of rhizobia recovered from vetch and pea nodules was presented.     

The plastid genome-encoded Ycf4 protein functions as a nonessential assembly factor for photosystem I in higher plants

Photosystem biogenesis in the thylakoid membrane is a highly complicated process that requires the coordinated assembly of nucleus-encoded and chloroplast-encoded protein subunits as well as the insertion of hundreds of cofactors, such as chromophores (chlorophylls, carotenoids) and iron-sulfur clusters. The molecular details of the assembly process and the identity and functions of the auxiliary factors involved in it are only poorly understood. In this work, we have characterized the chloroplast genome-encoded ycf4 (for hypothetical chloroplast reading frame no. 4) gene, previously shown to encode a protein involved in photosystem I (PSI) biogenesis in the unicellular green alga Chlamydomonas reinhardtii. Using stable transformation of the chloroplast genome, we have generated ycf4 knockout plants in the higher plant tobacco (Nicotiana tabacum). Although these mutants are severely affected in their photosynthetic performance, they are capable of photoautotrophic growth, demonstrating that, different from Chlamydomonas, the ycf4 gene product is not essential for photosynthesis. We further show that ycf4 knockout plants are specifically deficient in PSI accumulation. Unaltered expression of plastid-encoded PSI genes and biochemical analyses suggest a posttranslational action of the Ycf4 protein in the PSI assembly process. With increasing leaf age, the contents of Ycf4 and Y3IP1, another auxiliary factor involved in PSI assembly, decrease strongly, whereas PSI contents remain constant, suggesting that PSI is highly stable and that its biogenesis is restricted to young leaves.     

Tissue-specific differences in the accumulation of sequence rearrangements with age

Mitotic homologous recombination (HR) is a critical pathway for the accurate repair of DNA double strand breaks (DSBs) and broken replication forks. While generally error-free, HR can occur between misaligned sequences, resulting in deleterious sequence rearrangements that can contribute to cancer and aging. To learn more about the extent to which HR occurs in different tissues during the aging process, we used Fluorescent Yellow Direct Repeat (FYDR) mice in which an HR event in a transgene yields a fluorescent phenotype. Here, we show tissue-specific differences in the accumulation of recombinant cells with age. Unlike pancreas, which shows a dramatic 23-fold increase in recombinant cell frequency with age, skin shows no increase in vivo. In vitro studies indicate that juvenile and aged primary fibroblasts are similarly able to undergo HR in response to endogenous and exogenous DNA damage. Therefore, the lack of recombinant cell accumulation in the skin is most likely not due to an inability to undergo de novo HR events. We propose that tissue-specific differences in the accumulation of recombinant cells with age result from differences in the ability of recombinant cells to persist and clonally expand within tissues.     

Lipoic acid ameliorates oxidative stress and renal injury in alloxan diabetic rabbits

The therapeutic potential of lipoic acid (LA) in diabetes and diabetic nephropathy treatment was elucidated. Alloxan diabetic rabbits were treated daily for three weeks with either 10 or 50 mg of LA per kg body weight (i.p.). The following parameters were measured: 1) serum glucose, urea, creatinine and hydroxyl free radical (HFR) levels; 2) blood glutathione redox state; 3) urine albumin concentration; 4) hepatic and renal HFR levels, GSH/GSSG ratios, cysteine contents and the activities of the enzymes of glutathione metabolism; and 5) the activity of renal NADPH oxidase. Histological studies of kidneys were also performed. The treatment of diabetic rabbits with 50 mg of LA resulted in lethal hypoglycaemia in 50% of animals studied. Although the low dose of LA did not change serum glucose concentration, it decreased serum urea and creatinine concentrations, attenuated diabetes-induced decline in GSH/GSSG ratio and abolished hydroxyl free radicals accumulation in serum, liver and kidney cortex. LA did not change the activities of the enzymes of glutathione metabolism, but it elevated hepatic content of cysteine, which limits the rate of glutathione biosynthesis. Moreover, LA lowered urine albumin concentration and attenuated glomerulopathy characteristic of diabetes. However, it did not affect diabetes-stimulated activity of renal NADPH oxidase. In view of these data, it is concluded that low doses of LA might be useful for the therapy of diabetes and diabetic nephropathy. Beneficial action of LA seems to result mainly from direct scavenging of HFR and restoring glutathione redox state due to elevation of intracellular cysteine levels.     

Sites of generation of reactive oxygen species in homogenates of brain tissue determined with the use of respiratory substrates and inhibitors

Reactive oxygen species (ROS) have been widely implicated in the pathogenesis of various neurological diseases and aging. But the exact sites of ROS generation in brain tissue remained so far elusive. Here, we provide direct experimental evidence that at least 50% of total ROS generation in succinate-oxidizing homogenates of brain tissue can be attributed to complex I of mitochondrial respiratory chain. Applying quantitative methods for ROS detection we observed in different preparations from human, rat and mouse brain (digitonin-permeabilized tissue homogenates and isolated mitochondria) a linear relationship between rate of oxygen consumption and ROS generation with succinate as mitochondrial substrate. This quantitative relationship indicates, that under the particular conditions of oxygen saturation about 1% of the corresponding respiratory chain electron flow is redirected to form superoxide. Since we observed in mouse and rat brain mitochondria a unique dependency of both forward and reverse electron flow-dependent mitochondrial H(2)O(2) production on NAD redox state, we substantiated previous evidence that the FMN moiety of complex I is the major donor of electrons for the single electron reduction of molecular oxygen.     

Selenium and glutathione peroxidases in blood of patients with different stages of chronic renal failure

In patients with chronic renal failure (CRF) Se concentration in blood components is usually lower as compared with healthy controls. One of the five known forms of Se-dependent glutathione peroxidases (GSH-Px), the plasma GSH-Px, is synthesized primarily in the kidney. In CRF patients, plasma GSH-Px activity is reduced and the reduction increases with the progress of the disease.

The Se concentration in blood components was measured spectrofluorometrically with 2,3-diaminonaphthalene as complexing reagent. Activities of GSH-Px in red cells and in plasma were assayed by the coupled method with t-butyl hydroperoxide as substrate. The study group consisted of 150 patients in different stages of CRF. The results were compared with the values for 30 healthy subjects.

Se concentrations in whole blood and plasma of the entire group of patients were significantly lower (p < 0.01) as compared with the healthy subjects. In the incipient stage, however, the Se levels in all blood components were non-significantly lower. In whole blood and plasma the Se levels gradually decreased, reaching in the end stage values that were lower by 29 to 32% (p < 0.0001) as compared with the control group. Total protein and albumin levels in plasma of patients were significantly lower (p < 0.0001) as compared with healthy subjects and they decreased linearly with the progress of the disease. Positive and highly significant correlations were noted between total plasma protein and plasma Se concentrations (p < 0.0001) as well as between plasma albumin and plasma Se concentrations (p < 0.0001).

Red cell GSH-Px activity in the entire group of patients was lower (p < 0.05) than in the control group and did not change significantly with the progress of the disease. In plasma, however, GSH-Px activity of the entire group was lower by 33% (p < 0.0001) as compared with healthy subjects and decreased gradually with increasing renal failure. Highly significant, inverse correlations were seen between creatinine levels and plasma GSH-Px activities (p < 0.0001) as well as between urea nitrogen levels and plasma GSH-Px activities (p < 0.0001) when all stages of the disease were included.

In conclusion, patients with CRF exhibit lower Se levels in blood components as compared with healthy subjects. In whole blood and plasma these levels decrease with the progress of the disease. Plasma GSH-Px activity in patients was extremely reduced and it dramatically decreased with the progress of the illness.     

Pioglitazone, a PPAR-gamma ligand, exerts cytostatic/cytotoxic effects against cancer cells, that do not result from inhibition of proteasome

Thiazolidinediones are oral antidiabetic agents that activate peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and exert potent antioxidant and anti-inflammatory properties. It has also been shown that PPAR-gamma agonists induce G0/G1 arrest and apoptosis of malignant cells. Some of these effects have been suggested to result from inhibition of proteasome activity in target cells. The aim of our studies was to critically evaluate the cytostatic/cytotoxic effects of one of thiazolidinediones (pioglitazone) and its influence on proteasome activity. Pioglitazone exerted dose-dependent cytostatic/cytotoxic effects in MIA PaCa-2 cells. Incubation of tumor cells with pioglitazone resulted in increased levels of p53 and p27 and decreased levels of cyclin D1. Accumulation of polyubiquitinated proteins within cells incubated with pioglitazone suggested dysfunction of proteasome activity. However, we did not observe any influence of pioglitazone on the activity of isolated proteasome and on the proteolytic activity in lysates of pioglitazone-treated MIA PaCa-2 cells. Further, treatment with pioglitazone did not cause an accumulation of fluorescent proteasome substrates in transfected HeLa cells expressing unstable GFP variants. Our results indicate that pioglitazone does not act as a direct or indirect proteasome inhibitor.