Radical scavenging and electron-transfer reactions in Polyporus Versicolor laccase: A pulse radiolysis study

The interaction of the radicals OH^?, t-BuO^?, e_aq^?, CO₂^XXX and O₂^XXX with the copper oxidase. laccase. from Polyporus, has been studied by the pulse-radiolysis technique. Each of these radicals formed transient adducts with a broad absorption maximum around 310 nm. Analysis of the optical properties and of the very fast rates of formation of these compounds shows that each radical interacts with a limited number of sites on the polypeplide part of the protein amongst R-S-S-R. histidine and aromatic residues. Interaction with the carbonyl group of some of the peptide bonds is also possible. The few target sites are probably hit simultaneously and electron transfer between these sites may also occur. In all cases, in a subsequent step, intramolecular electron transfer from the polypeptide radical adducts leads to a partial reduction of the blue type-1 Cu²⁺ with rates varying between 10³ and 10⁴ s^?1. Further reduction of the type-1 Cu²⁺ occurs through a slow intermolecular reaction between two laccase radical transient adducts. In the case of CO^XXX₂ and O^XXX₂, this slow reduction could alternatively be due to an intermolecular reaction between laccase and CO^XXX₂ or O^XXX₂. The oxidant radicals OH^?. Br^XXX₂ and (SCN)^XXX₂, which formed radical adducts with fully ascorbate-reduced laccase, did not induce any type-1 copper reoxidation.     

Complexity in water and carbon dioxide fluxes following rain pulses in an African savanna

The idea that many processes in arid and semi-arid ecosystems are dormant until activated by a pulse of rainfall, and then decay from a maximum rate as the soil dries, is widely used as a conceptual and mathematical model, but has rarely been evaluated with data. This paper examines soil water, evapotranspiration (ET), and net ecosystem CO₂ exchange measured for 5 years at an eddy covariance tower sited in an Acacia–Combretum savanna near Skukuza in the Kruger National Park, South Africa. The analysis characterizes ecosystem flux responses to discrete rain events and evaluates the skill of increasingly complex “pulse models”. Rainfall pulses exert strong control over ecosystem-scale water and CO₂ fluxes at this site, but the simplest pulse models do a poor job of characterizing the dynamics of the response. Successful models need to include the time lag between the wetting event and the process peak, which differ for evaporation, photosynthesis and respiration. Adding further complexity, the time lag depends on the prior duration and degree of water stress. ET response is well characterized by a linear function of potential ET and a logistic function of profile-total soil water content, with remaining seasonal variation correlating with vegetation phenological dynamics (leaf area). A 1- to 3-day lag to maximal ET following wetting is a source of hysteresis in the ET response to soil water. Respiration responds to wetting within days, while photosynthesis takes a week or longer to reach its peak if the rainfall was preceded by a long dry spell. Both processes exhibit nonlinear functional responses that vary seasonally. We conclude that a more mechanistic approach than simple pulse modeling is needed to represent daily ecosystem C processes in semiarid savannas. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fractionation of the rat ventral prostate with respect to isolation and exocytosis of the prostatic secretion protein

The ventral prostrate was fractionated into one mitochondrial and three microsomal fractions. The different fractions were characterized morphologically and chemically. An interesting finding was that upon homogenization the endoplasmic reticulum membranes often turned ‘inside-out’ giving rise to microsomes with ribosomes attached to the inside of the vesicles. The secretion of the protatic secretion was studied by means of isotopic pulse labeling using radioactive leucine. Peak radioactivity in the secretory fluid was obtained at 2 h after injection with a relativity rapid fall. The radioactivity in the secretory fluid displayed a continuous increase up to 8 h followed by a plateau. When prostatic secretion was purified from secretory fluid and microsomes using a Con A-Sepharose column it showed a typical precursor-product relationship with an early peak at 60 min in microsomal prosatatic secretion protein followed by a peak in secretory fluid at 4 h. Vinblastine blocked the release of labeled secretion protein into the secretory fluid, a phenomenon characteristic for secretory proteins which are exocytosed by means of fusion between secretory granules and the plasma membrane. Following intravenous injection of [³H]estramustine, accumulation was seen in the secretory fluid. Some estramustine probably binds to newly synthesized protatic secretion protein and follows the same route of intracellular transport and extracellular discharge as does prostatic secretion protein.     

Physiology and cryosensitivity of coral endosymbiotic algae (Symbiodinium)

Coral throughout the world are under threat. To save coral via cryopreservation methods, the Symbiodinium algae that live within many coral cells must also be considered. Coral juvenile must often take up these important cells from their surrounding water and when adult coral bleach, they lose their endosymbiotic algae and will die if they are not regained. The focus of this paper was to understand some of the cryo-physiology of the endosymbiotic algae, Symbiodinium, living within three species of Hawaiian coral, Fungia scutaria, Porites compressa and Pocillopora damicornis in Kaneohe Bay, Hawaii. Although cryopreservation of algae is common, the successful cryopreservation of these important coral endosymbionts is not common, and these species are often maintained in live serial cultures within stock centers worldwide. Freshly-extracted Symbiodinium were exposed to cryobiologically appropriate physiological stresses and their viability assessed with a Pulse Amplitude Fluorometer. Stresses included sensitivity to chilling temperatures, osmotic stress, and toxic effects of various concentrations and types of cryoprotectants (i.e., dimethyl sulfoxide, propylene glycol, glycerol and methanol). To determine the water and cryoprotectant permeabilities of Symbiodinium, uptake of radio-labeled glycerol and heavy water (D₂O) were measured. The three different Symbiodinium subtypes studied demonstrated remarkable similarities in their morphology, sensitivity to cryoprotectants and permeability characteristics; however, they differed greatly in their sensitivity to hypo- and hyposmotic challenges and sensitivity to chilling, suggesting that standard slow freezing cryopreservation may not work well for all Symbiodinium. An appendix describes our H₂O:D₂O water exchange experiments and compares the diffusionally determined permeability with the two parameter model osmotic permeability.     

Dioxathiadiaza-heteropentalenes. New Photosystem-I electron acceptors

The redox properties of some dioxathiadiaza-heteropentalenes have been studied by pulse radiolysis and cyclic voltammetry. The midpoint potential at pH 7 for reduction of this class of compounds to the corresponding anion radical is comparable with the first reduction potential of the bipyridinium herbicides. The heteropentalenes act as Photosystem-I electron acceptors at concentrations of the order of 1 · 10^?6 M. The herbicidal properties of the heteropentalenes are similar to those of the bipyridinium herbicides.     

Suberoylanilide hydroxamic acid radiosensitizes tumor hypoxic cells in vitro through the oxidation of nitroxyl to nitric oxide

The pharmacological effects of hydroxamic acids are partially attributed to their ability to serve as HNO and/or NO donors under oxidative stress. Previously, it was concluded that oxidation of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) by the metmyoglobin/H₂O₂ reaction system releases NO, which was based on spin trapping of NO and accumulation of nitrite. Reinvestigation of this system demonstrates the accumulation of N₂O, which is a marker of HNO formation, at similar rates under normoxia and anoxia. In addition, the yields of nitrite that accumulated in the absence and the presence of O₂ did not differ, implying that the source of nitrite is other than autoxidation of NO. In this system metmyoglobin is instantaneously and continuously converted into compound II, leading to one-electron oxidation of SAHA to its respective transient nitroxide radical. Studies using pulse radiolysis show that one-electron oxidation of SAHA (pK_a=9.56±0.04) yields the respective nitroxide radical (pK_a=9.1±0.2), which under all experimental conditions decomposes bimolecularly to yield HNO. The proposed mechanism suggests that compound I oxidizes SAHA to the respective nitroxide radical, which decomposes bimolecularly in competition with its oxidation by compound II to form HNO. Compound II also oxidizes HNO to NO and NO to nitrite. Given that NO, but not HNO, is an efficient hypoxic cell radiosensitizer, we hypothesized that under an oxidizing environment SAHA might act as a NO donor and radiosensitize hypoxic cells. Preincubation of A549 and HT29 cells with 2.5 μM SAHA for 24 h resulted in a sensitizer enhancement ratio at 0.01 survival levels (SER_0.01) of 1.33 and 1.59, respectively. Preincubation of A549 cells with oxidized SAHA had hardly any effect and, with 2 mM valproic acid, which lacks the hydroxamate group, resulted in SER_0.01=1.17. Preincubation of HT29 cells with SAHA and Tempol, which readily oxidizes HNO to NO, enhanced the radiosensitizing effect of SAHA. Pretreatment with SAHA blocked A549 cells at the G1 stage of the cell cycle and upregulated γ-H2AX after irradiation. Overall, we conclude that SAHA enhances tumor radioresponse by multiple mechanisms that might also involve its ability to serve as a NO donor under oxidizing environments.     

Competition for phosphorus between two dinoflagellates: A toxic Alexandrium minutum and a non-toxic Heterocapsa triquetra

The understanding of the dominance of one species with respect to others is a pertinent challenge in HAB growth dynamics studies and the nutrient supply mode is one of the factors potentially involved. The competition for phosphorus (P) between a toxic species, Alexandrium minutum, and a non-toxic species, Heterocapsa triquetra, was studied (1) along a gradient of P depletion, (2) testing different P depletion degrees before a single PO₄ supply and (3) experimenting different PO₄ supply frequencies. In conditions of PO₄ depletion, H. triquetra stopped growing after two days both in monospecific and mixed batch cultures whereas A. minutum grew progressively from day 2 until the end of the experiment. This time-lag growth of A. minutum is associated to its ability to store P intracellularly and then mobilize it for cell division when P depletion becomes severe. Heterocapsa triquetra outcompeted A. minutum when it was submitted to less than three days of P depletion before the pulse. In contrast, A. minutum outcompeted H. triquetra after more than three days of depletion. This transition was related to the capacity for A. minutum to increase its cell PO₄ uptake rate in a higher proportion to face potential PO₄ supply. As a result of this physiological acclimatation to P starvation, A. minutum consumed the whole PO₄ pulse supplied after 3 to 10 days of P depletion. This resulted in a reduction of H. triquetra growth. These two acclimatations were confirmed in a P limited semi-continuous culture experiment testing several PO₄ supply frequencies (1, 2, 4, 6 day intervals). These experiments revealed that A. minutum is a “storage specialist” species for P, which uptakes PO₄ pulses for luxury consumption, survives depletion periods and, then, utilizes P for cell growth. In contrast, H. triquetra is more a “velocity adapted” species, which utilizes PO₄ just after supply to increase their cell division rate.