Ioannis Papapostolou

Sclerotial metamorphosis in filamentous fungi is induced by oxidative stress

Sclerotium-forming filamentous fungi are of great agricultural and biological interest because they can be viewed as models of simple metamorphosis. They differentiate by asexually producing sclerotia but the processes involved in sclerotial metamorphosis were poorly understood. In 1997, it was shown for the first time that the sclerotial differentiation state in Sclerotium rolfsii concurred with increasing levels of lipid peroxides. This finding prompted the development of a theory supporting that sclerotial metamorphosis is induced by oxidative stress. Growth factors that reduce or increase oxidative stress are expected to inhibit or promote sclerotium metamorphosis, respectively. This theory has been verified by a series of published data on the effect of certain hydroxyl radical scavengers on sclerotial metamorphosis, on the identification and quantification of certain endogenous antioxidants (such as ascorbic acid, β-carotene) in relation to the fungal undifferentiated and differentiated states, and on their inhibiting effect on sclerotial metamorphosis as growth nutrients. In 2004-2005, we developed assays for the measurement of certain redox markers of oxidative stress, such as the thiol redox state, the small-sized fragmented DNA, and the superoxide radical. These new advances allowed us to initiate studies on the exact role of glutathione, hydrogen peroxide, and superoxide radical on sclerotial metamorphosis. The emerging data, combined with similar data from other better-studied fungi, allowed us to make some preliminary postulations on the ROS-dependent biochemical signal transduction pathways in sclerotiogenic filamentous fungi.

Mechanism of Coomassie brilliant blue G-250 binding to proteins: a hydrophobic assay for nanogram quantities of proteins

We investigated the mechanism of Coomassie brilliant blue G-250 (CBB) binding to proteins in order to develop a protein assay with the maximum possible sensitivity. We found that the neutral ionic species of CBB binds to proteins by a combination of hydrophobic interactions and heteropolar bonding with basic amino acids. On the basis of these findings, we developed a very sensitive hydrophobic assay for proteins (at the nanogram level) using the hydrophobic reagents ammonium sulfate and trichloroacetic acid under pH conditions that increase neutral species concentration in the assay reagent in order to enhance the binding of more CBB dye molecules per protein molecule than in previous CBB-based assays.

Superoxide radical detection in cells, tissues, organisms (animals, plants, insects, microorganisms) and soils

A simple protocol is presented for the assessment of superoxide radical in organisms (animal/plant tissues, microorganisms, cell cultures, biological/culture fluids) and soils, through the quantification of 2-hydroxyethidium (2-OH-E+), its specific reaction product with hydroethidine (HE). It is an alternative to the quantification of 2-OH-E+ by HPLC (restricted to cell cultures), offering the advantage of the in vivo assessment of superoxide radical in a wide range of experimental systems. The protocol includes alkaline-acetone extraction of the sample, purification by microcolumn cation exchange and hydrophobic chromatographies, and fluorescence detection of the isolated 2-OH-E+/HE-oxidation products mixture before and after consumption of 2-OH-E+ by a horseradish peroxidase/hydrogen peroxide system. The protocol is sensitive at <1 pmol 2-OH-E+ per mg protein (extended to the femto level when using large samples) in biological systems, and in soils at 9 pmol superoxide radical per gram of soil. The protocol includes a cytochrome c-based subprotocol for superoxide radical detection in soils at 770 pmol g−1 soil. For processing ten samples and depending on the experimental material used (soil or biological), the approximate procedure time would be 2–7 h.

Beneficial effect of the oxygen free radical scavenger amifostine (WR-2721) on spinal cord ischemia/reperfusion injury in rabbits

BackgroundParaplegia is the most devastating complication of thoracic or thoraco-abdominal aortic surgery. During these operations, an ischemia-reperfusion process is inevitable and the produced radical oxygen species cause severe oxidative stress for the spinal cord. In this study we examined the influence of Amifostine, a triphosphate free oxygen scavenger, on oxidative stress of spinal cord ischemia-reperfusion in rabbits.MethodsEighteen male, New Zealand white rabbits were anesthetized and spinal cord ischemia was induced by temporary occlusion of the descending thoracic aorta by a coronary artery balloon catheter, advanced through the femoral artery. The animals were randomly divided in 3 groups. Group I functioned as control. In group II the descending aorta was occluded for 30 minutes and then reperfused for 75 min. In group III, 500 mg Amifostine was infused into the distal aorta during the second half-time of ischemia period. At the end of reperfusion all animals were sacrificed and spinal cord specimens were examined for superoxide radicals by an ultra sensitive fluorescent assay.ResultsSuperoxide radical levels ranged, in group I between 1.52 and 1.76 (1.64 ± 0.10), in group II between 1.96 and 2.50 (2.10 ± 0.23), and in group III (amifostine) between 1.21 and 1.60 (1.40 ± 0.19) (p = 0.00), showing a decrease of 43% in the Group of Amifostine. A lipid peroxidation marker measurement ranged, in group I between 0.278 and 0.305 (0.296 ± 0.013), in group II between 0.427 and 0.497 (0.463 ± 0.025), and in group III (amifostine) between 0.343 and 0.357 (0.350 ± 0.007) (p < 0.00), showing a decrease of 38% after Amifostine administration.ConclusionBy direct and indirect methods of measuring the oxidative stress of spinal cord after ischemia/reperfusion, it is suggested that intra-aortic Amifostine infusion during spinal cord ischemia phase, significantly attenuated the spinal cord oxidative injury in rabbits.

Hydrogen peroxide is involved in the sclerotial differentiation of filamentous phytopathogenic fungi.

Aims:  The purpose of this study was to investigate the role of H2O2 and the related oxidative stress markers catalase (CAT) and lipid peroxidation in the sclerotial differentiation of the phytopathogenic filamentous fungi Sclerotium rolfsii, Sclerotinia minor, Sclerotinia sclerotiorum and Rhizoctonia solani.

Protocol for the quantitative assessment of DNA concentration and damage (fragmentation and nicks)

An ultrasensitive protocol is presented for the quantitative assessment of fragmented and nicked dsDNA using PicoGreen and consists of four methods. The first quantifies the concentration of DNA, whereas the second (quantitative complement of the Comet assay) quantifies the degree of DNA fragmentation seen in a typical DNA agarose electrophoresis gel. Both methods have sensitivity of 5 pg of DNA. The third method (quantitative counterpart of the electrophoresis-based qualitative apoptotic and necrotic DNA assays) quantifies the polyethylene glycol-fractionated small-size (0–1 kb) fragmented DNA. This method also detects up to 5 pg of damaged DNA and requires a minimum sample of quantity 0.2 ml of 2.5 μg ml−1. The fourth method measures the percentage of DNA nicks by alkaline DNA unwinding and requires up to 15 pg of DNA sample. The time required for processing 10 DNA samples is 1/2, 1, 13 and 1 h for the first, second, third and fourth method, respectively.

Evidence for photochemical production of reactive oxygen species in desert soils

The combination of intense solar radiation and soil desiccation creates a short circuit in the biogeochemical carbon cycle, where soils release significant amounts of CO2 and reactive nitrogen oxides by abiotic oxidation. Here we show that desert soils accumulate metal superoxides and peroxides at higher levels than non-desert soils. We also show the photogeneration of equimolar superoxide and hydroxyl radical in desiccated and aqueous soils, respectively, by a photo-induced electron transfer mechanism supported by their mineralogical composition. Reactivity of desert soils is further supported by the generation of hydroxyl radical via aqueous extracts in the dark. Our findings extend to desert soils the photogeneration of reactive oxygen species by certain mineral oxides and also explain previous studies on desert soil organic oxidant chemistry and microbiology. Similar processes driven by ultraviolet radiation may be operating in the surface soils on Mars.

Superoxide radical induces sclerotial differentiation in filamentous phytopathogenic fungi: a superoxide dismutase mimetics study

This study shows that the superoxide radical (O(2) *( -)), a direct indicator of oxidative stress, is involved in the differentiation of the phytopathogenic filamentous fungi Rhizoctonia solani, Sclerotinia sclerotiorum, Sclerotium rolfsii and Sclerotinia minor, shown by using superoxide dismutase (SOD) mimetics to decrease their sclerotial differentiation. The production rate of O(2) *(-) and SOD levels in these fungi, as expected, were significantly lowered by the SOD mimetics, with concomitant decrease of the indirect indicator of oxidative stress, lipid peroxidation.

Superoxide radical is involved in the sclerotial differentiation of filamentous phytopathogenic fungi: identification of a fungal xanthine oxidase

This study shows that the direct indicator of oxidative stress superoxide radical (O·₂⁻) is involved in the sclerotial differentiation of the phytopathogenic filamentous fungi Rhizoctonia solani, Sclerotinia sclerotiorum, Sclerotium rolfsii, and Sclerotinia minor. The production rate of O·₂⁻ and the antioxidant enzyme superoxide dismutase (SOD) levels in the sclerotiogenic fungi were significantly higher and lower, respectively, than those of their non-differentiating counterpart strains, which strongly suggests that the oxidative stress of the sclerotium differentiating fungi is higher than that of the non-differentiating ones. Xanthine oxidase (XO), which was detected for the first time in fungi in general, was localized in the cytoplasmic membrane. The contribution of XO in the overall O·₂⁻production was very significant, reaching 30-70% among the strains, especially in the transition developmental stage between the undifferentiated and the differentiated state, suggesting a sclerotium triggering and a phytopathogenic role of XO during plant infection. The additional finding that these fungi secrete extracellular SOD can be related to their protection from the response of plants to produce O·₂⁻ at infection sites.

Time-related alterations of superoxide radical levels in diverse organs of bile duct-ligated rats

The time-related alterations of superoxide radical measured in vivo by employing an ultrasensitive fluorescent assay in the liver, intestine, kidney and brain of rats with experimentally induced obstructive jaundice was investigated. Eighteen rats were randomly divided into Group A, rats subjected to sham operation, and Group B, rats subjected to bile duct ligation (BDL). Three rats from each group were subsequently killed at different time points post-operatively (1, 5 and 10 days). As compared to sham-operated, BDL rats showed a gradual increase with time of superoxide radical in the intestine, liver, kidney and brain: for animals sacrificed on the 1st, 5th and 10th day the increase was 45%, 50% and 96% in the liver, 76%, 81% and 118% in the intestine, 64%, 71% and 110% in the kidney and 76%, 95% and 142% in the brain, respectively. This study provides direct evidence of an early appearance of oxidative stress in diverse organs, implying a uniform systemic response to biliary obstruction and emphasizing the need of early bile flow restoration.