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.

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.

The combination of intense solar radiation and soil desiccation creates a short circuit in the biogeochemical carbon cycle, where soils release significant amounts of CO 2 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.

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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.