The Phytophthora diseases of stone fruit trees in Greece

Phytophthora collar and crown rots are serious soilborne diseases which for a long time have caused considerable losses in stone fruit orchards in Greece. A number of Phytophthora species are notorious for being the cause of crown and root rots in Greek stone trees, including P. cactorum, P. citricola, P. cryptogea, P. drechsleri, P. nicotianae, P. citrophthora, P. syringae and P. megasperma. The most important Phytophthora species is P. cactorum, while P. syringae and P. citrophthora may be locally significant. The economic consequences from death of peach trees and yield losses caused by this disease in Imathia County are serious.     

Eng2 Is a Component of a Dynamic Protein Complex Required for Endocytic Uptake in Fission Yeast

Eng2 is a glucanase required for spore release, although it is also expressed during vegetative growth, suggesting that it might play other cellular functions. Its homology to the Saccharomyces cerevisiae Acf2 protein, previously shown to promote actin polymerization at endocytic sites in vitro, prompted us to investigate its role in endocytosis. Interestingly, depletion of Eng2 caused profound defects in endocytic uptake, which were not due to the absence of its glucanase activity. Analysis of the dynamics of endocytic proteins by fluorescence microscopy in the eng2Δ strain unveiled a previously undescribed phenotype, in which assembly of the Arp2/3 complex appeared uncoupled from the internalization of the endocytic coat and resulted in a fission defect. Strikingly also, we found that Eng2‐GFP dynamics did not match the pattern of other endocytic proteins. Eng2‐GFP localized to bright cytosolic spots that moved around the cellular poles and occasionally contacted assembling endocytic patches just before recruitment of Wsp1, the Schizosaccharomyces pombe WASP. Interestingly, Csh3‐YFP, a WASP‐interacting protein, interacted with Eng2 by co‐immunoprecipitation and was recruited to Eng2 in bright cytosolic spots. Altogether, our work defines a novel endocytic functional module, which probably couples the endocytic coat to the actin module.      

An Immunogenic Peptide in the A-box of HMGB1 Protein Reverses Apoptosis-induced Tolerance through RAGE Receptor

Apoptotic cells trigger immune tolerance in engulfing phagocytes. This poorly understood process is believed to contribute to the severe immunosuppression and increased susceptibility to nosocomial infections observed in critically ill sepsis patients. Extracellular high mobility group box 1 (HMGB1) is an important mediator of both sepsis lethality and the induction of immune tolerance by apoptotic cells. We have found that HMGB1 is sensitive to processing by caspase-1, resulting in the production of a fragment within its N-terminal DNA-binding domain (the A-box) that signals through the receptor for advanced glycation end products (RAGE) to reverse apoptosis-induced tolerance. In a two-hit mouse model of sepsis, we show that tolerance to a secondary infection and its associated mortality were effectively reversed by active immunization with dendritic cells treated with HMGB1 or the A-box fragment, but not a noncleavable form of HMGB1. These findings represent a novel link between caspase-1 and HMGB1, with potential therapeutic implications in infectious and inflammatory diseases.     

Effect of substrate nitrogen on lignin degradation by Pleurotus ostreatus

In order to determine the effect of substrate nitrogen (N) on ligninolytic activity, Pleurotus ostreatus was grown in solid media containing either growth-limiting (1mM) or excess (10mM) NH₄Cl. After 25 days, ¹⁴C–CO₂ production from ¹⁴C-cornstover lignin in low-N medium was 3 times that in nitrogen (N)-rich medium. Supplementation of low-N medium with glucose (0.3%) further enhanced ligninolytic activity. Decolorization of an aromatic, polymeric dye, Poly R-481, in solid media was also greatest under N-limiting conditions.     

Extracellular tyrosinase from the fungus Trichoderma reesei shows product inhibition and different inhibition mechanism from the intracellular tyrosinase from Agaricus bisporus

Tyrosinase (EC 1.14.18.1) is a widely distributed type 3 copper enzyme participating in essential biological functions. Tyrosinases are potential biotools as biosensors or protein crosslinkers. Understanding the reaction mechanism of tyrosinases is fundamental for developing tyrosinase-based applications. The reaction mechanisms of tyrosinases from Trichoderma reesei (TrT) and Agaricus bisporus (AbT) were analyzed using three diphenolic substrates: caffeic acid, L-DOPA (3,4-dihydroxy-l-phenylalanine), and catechol. With caffeic acid the oxidation rates of TrT and AbT were comparable; whereas with L-DOPA or catechol a fast decrease in the oxidation rates was observed in the TrT-catalyzed reactions only, suggesting end product inhibition of TrT. Dopachrome was the only reaction end product formed by TrT- or AbT-catalyzed oxidation of L-DOPA. We produced dopachrome by AbT-catalyzed oxidation of L-DOPA and analyzed the TrT end product (i.e. dopachrome) inhibition by oxygen consumption measurement. In the presence of 1.5 mM dopachrome the oxygen consumption rate of TrT on 8 mM L-DOPA was halved. The type of inhibition of potential inhibitors for TrT was studied using p-coumaric acid (monophenol) and caffeic acid (diphenol) as substrates. The strongest inhibitors were potassium cyanide for the TrT-monophenolase activity, and kojic acid for the TrT-diphenolase activity. The lag period related to the TrT-catalyzed oxidation of monophenol was prolonged by kojic acid, sodium azide and arbutin; contrary it was reduced by potassium cyanide. Furthermore, sodium azide slowed down the initial oxidation rate of TrT- and AbT-catalyzed oxidation of L-DOPA or catechol, but it also formed adducts with the reaction end products, i.e., dopachrome and o-benzoquinone.     

A Comparative Evaluation of the Response to Peroxynitrite by a Brain Endothelial Cell Line and Control of the Effects by Drug Targeting

The potent oxidant peroxynitrite (ONOO⁻) is formed after the combination of nitric oxide with superoxide and has been closely associated with the pathology of inflammatory disease. In particular, the generation of ONOO⁻ has been linked to central nervous system disorders including Alzheimer’s and Parkinson’s disease, multiple sclerosis and bacterial and viral meningitis. Specifically, ONOO⁻ has been implicated in the loss of blood–brain barrier (BBB) integrity during neuroinflammation, but the precise mechanisms through which the molecule acts to mediate neurovascular breakdown have not been established. The disruptive effects of ONOO⁻ could be mediated by either direct or indirect actions on the endothelial cells that comprise the major component of the BBB. The current study has comparatively assessed the direct toxic effects of ONOO⁻ on the brain endothelial cell line, b.End3 and C6 astrocytoma and NA neuroblastoma preparations. b.End3 cells were relatively resistant to ONOO⁻-induced cell death compared with C6 and NA cultures. The indirect involvement of ONOO⁻ in neuroendothelial disruption was pharmacologically determined via adhesion molecule expression and immunocompetent cell attachment to b.End3 cells. ONOO⁻-targeted drugs, including the selective free radical scavenger, uric acid, the decomposition catalyst 5,10,15,20-tetrakis (4-sulphonatophenyl) porphyrinatoiron (III) (FeTPPS) and the poly(ADP-ribose) polymerase inhibitor N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-(N,N-dimethylamino) acetamide hydrochloride (PJ34) revealed that ONOO⁻ was only partly involved in E-selectin, ICAM-1 and VCAM-1 expression on b.End3 cells and also cytokine-induced T-lymphocyte attachment to the cell line. The results indicate that ONOO⁻ contributes to b.End3 cell disruption but is not exclusively responsible for the breakdown of neuroendothelial function.     

Modifying Apolipoprotein A-I by Malondialdehyde,but Not by an Array of Other Reactive Carbonyls,Blocks Cholesterol Efflux by the ABCA1 Pathway

Dysfunctional high density lipoprotein (HDL) is implicated in the pathogenesis of cardiovascular disease, but the underlying pathways remain poorly understood. One potential mechanism involves covalent modification by reactive carbonyls of apolipoprotein A-I (apoA-I), the major HDL protein. We therefore determined whether carbonyls resulting from lipid peroxidation (malondialdehyde (MDA) and hydroxynonenal) or carbohydrate oxidation (glycolaldehyde, glyoxal, and methylglyoxal) covalently modify lipid-free apoA-I and inhibit its ability to promote cellular cholesterol efflux by the ABCA1 pathway. MDA markedly impaired the ABCA1 activity of apoA-I. In striking contrast, none of the other four carbonyls were effective. Liquid chromatography-electrospray ionization-tandem mass spectrometry of MDA-modified apoA-I revealed that Lys residues at specific sites had been modified. The chief adducts were MDA-Lys and a Lys-MDA-Lys cross-link. Lys residues in the C terminus of apoA-I were targeted for cross-linking in high yield, and this process may hinder the interaction of apoA-I with lipids and ABCA1, two key steps in reverse cholesterol transport. Moreover, levels of MDA-protein adducts were elevated in HDL isolated from human atherosclerotic lesions, suggesting that lipid peroxidation might render HDL dysfunctional in vivo. Taken together, our observations indicate that MDA damages apoA-I by a pathway that generates lysine adducts at specific sites on the protein. Such damage may facilitate the formation of macrophage foam cells by impairing cholesterol efflux by the ABCA1 pathway.     

Search for novel antifungal agents by monitoring fungal metabolites in presence of synthetically designed fluconazole derivatives using NMR spectroscopy

Resistance to currently available antifungal drugs necessitates development of new drugs using rapid, robust and automated methods to test a large number of newly synthesized drugs in less time. We have compared the effect of ketoconazole, fluconazole and its synthesized analogues on Candida albicans ATCC 10231. A metabolic profile of C.albicans ATCC 10231 in presence of drugs has been compared using ¹H NMR. Signals from metabolites have been monitored with time. MIC determined using conventional methods has been compared with Metabolic End Point (MEP) obtained from NMR spectroscopy. Results indicate that the activity of the fluconazole derivatives is in the order fluconazole p-methoxybenzoate > fluconazole = fluconazole benzoate > fluconazole toluate > fluconazole p-nitrobenzoate.     

Association of mitochondrial nitric oxide synthase activity with respiratory chain complex I

The present study shows that rat liver and brain mitochondrial nitric oxide synthase (mtNOS) are functionally associated with mitochondrial respiratory chain complex I. When complex I is activated, mtNOS exerts high activity and generates nitric oxide, whereas inactivation of complex I leads mtNOS to abandon its NOS activity. Functional association of mtNOS with complex I is potentially important in regulating mtNOS activity and mitochondrial functions.