IL-1 system in apoptosis of murine hippocampal neurons of dentate gyrus induced by trimethyltin administration

Growing evidence suggests that two modes of cell death, known as apoptosis and necrosis, are involved in postanoxic injury. The current opinion on these two types of cell death is that apoptosis and necrosis are not always the uniform and distinct events. The aim of this study was to determine ultrastructural criteria of postanoxic neuronal changes in model of anoxia in vitro . The organotypic cultures of rat hippocampus exposed to 10- and 20-min of anoxic insult revealed the morphological features classic for both necrotic and apoptotic neuronal cell injury. Some neurones exhibited the typical necrotic lysis whereas others clearly reflected an active apoptotic form of cell death consisting of nuclear condensation with early preservation of cell membranes. However, numerous damaged cells shared both apoptotic and necrotic ultrastructural characteristics. These results evidenced the morphological continuum between apoptosis and necrosis under anoxia in vitro .     

Time-resolved single-turnover of ba3 oxidase from Thermus thermophilus

The kinetics of the oxidation of fully-reduced ba₃ cytochrome c oxidase from Thermus thermophilus by oxygen were followed by time-resolved optical spectroscopy and electrometry. Four catalytic intermediates were resolved during this reaction. The chemical nature and the spectral properties of three intermediates (compounds A, P and O) reproduce the general features of aa₃-type oxidases. However the F intermediate in ba₃ oxidase has a spectrum identical to the P state. This indicates that the proton taken up during the P → F transition does not reside in the binuclear site but is rather transferred to the covalently cross-linked tyrosine near that site. The total charge translocation associated with the F → O transition in ba₃ oxidase is close to that observed during the F → O transition in the aa₃ oxidases. However, the P_R → F transition is characterized by significantly lower charge translocation, which probably reflects the overall lower measured pumping efficiency during multiple turnovers.     

Redox properties of novel antioxidant 5,8-Dihydroxycoumarin: implications for its prooxidant cytotoxicity

The aim of this work was to characterize the redox properties of the new antioxidant 5,8-dihydroxycoumarin (5,8-DHC), isolated from sweet grass (Hierochlo? odorata L.), and to determine its impact on its cytotoxic action. Reversible electrochemical oxidation of 5,8-DHC at pH 7.0 was characterized by the midpoint potential (E(p/2)) of 0.23 V vs. the normal hydrogen electrode. 5,8-DHC was slowly autoxidized at pH 7.0, and it was active as a substrate for peroxidase (POD, EC 1.11.1.7) and tyrosinase (TYR, EC 1.14.18.1). Oxidation of 5,8-DHC by POD/H202 yielded the product(s) which reacted with reduced glutathione and supported the oxidation of NADPH by ferredoxin:NADP+ reductase (FNR, EC 1.18.1.2) and NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2). The concentration of 5,8-DHC for 50% survival of bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) during a 24-h incubation was (60 +/- 5.5) microM. Cytotoxicity of 5,8-DHC was decreased by desferrioxamine, catalase, the antioxidant N,N'-diphenyl-p-phenylene diamine, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea and dicumarol, an inhibitor of NQO1. This shows that 5,8-DHC possesses the oxidative stress-type cytotoxicity, evidently due to the action of quinodal oxidation product(s). The protective effect of isoniazide, an inhibitor of cytochrome P-450 2E1, points to hydroxylation of 5,8-DHC as additional toxification route, whereas the potentiating effect of 3,5-dinitrocatechol, an inhibitor of catechol-o-methyltransferase (COMT, EC 2.1.1.6), points to the o-methylation of hydroxylation products as the detoxification route.     

Fungal communities in mycorrhizal roots of conifer seedlings in forest nurseries under different cultivation systems, assessed by morphotyping, direct sequencing and mycelial isolation

Fungi colonising root tips of Pinus sylvestris and Picea abies grown under four different seedling cultivation systems were assessed by morphotyping, direct sequencing and isolation methods. Roots were morphotyped using two approaches: (1) 10% of the whole root system from 30 seedlings of each species and (2) 20 randomly selected tips per plant from 300 seedlings of each species. The first approach yielded 15 morphotypes, the second yielded 27, including 18 new morphotypes. The overall community consisted of 33 morphotypes. The level of mycorrhizal colonisation of roots determined by each approach was about 50%. The cultivation system had a marked effect on the level of mycorrhizal colonisation. In pine, the highest level of colonisation (48%) was observed in bare-root systems, while in spruce, colonisation was highest in polyethylene rolls (71%). Direct internal transcribed spacer ribosomal DNA sequencing and isolation detected a total of 93 fungal taxa, including 27 mycorrhizal. A total of 71 (76.3%) fungi were identified at least to a genus level. The overlap between the two methods was low. Only 13 (13.9%) of taxa were both sequenced and isolated, 47 (50.5%) were detected exclusively by sequencing and 33 (35.5%) exclusively by isolation. All isolated mycorrhizal fungi were also detected by direct sequencing. Characteristic mycorrhizas were Phialophora finlandia, Amphinema byssoides, Rhizopogon rubescens, Suillus luteus and Thelephora terrestris. There was a moderate similarity in mycorrhizal communities between pine and spruce and among different cultivation systems.Electronic supplementary material Supplementary material is available for this article at and accessible for authorised users     

How PspGI, catalytic domain of EcoRII and Ecl18kI acquire specificities for different DNA targets

Restriction endonucleases Ecl18kI and PspGI/catalytic domain of EcoRII recognize CCNGG and CCWGG sequences (W stands for A or T), respectively. The enzymes are structurally similar, interact identically with the palindromic CC:GG parts of their recognition sequences and flip the nucleotides at their centers. Specificity for the central nucleotides could be influenced by the strength/stability of the base pair to be disrupted and/or by direct interactions of the enzymes with the flipped bases. Here, we address the importance of these contributions. We demonstrate that wt Ecl18kI cleaves oligoduplexes containing canonical, mismatched and abasic sites in the central position of its target sequence CCNGG with equal efficiencies. In contrast, substitutions in the binding pocket for the extrahelical base alter the Ecl18kI preference for the target site: the W61Y mutant prefers only certain mismatched substrates, and the W61A variant cuts exclusively at abasic sites, suggesting that pocket interactions play a major role in base discrimination. PspGI and catalytic domain of EcoRII probe the stability of the central base pair and the identity of the flipped bases in the pockets. This ‘double check’ mechanism explains their extraordinary specificity for an A/T pair in the flipping position.     

Specificity of synexin-induced chromaffin granule aggregation

Preparations of synexin (1) exhibit a self-interaction in the absence of chromaffin granules as evidenced by an increase in absorbance at the wave-length used for observing granule aggregation (2). We incorporated this observation into a new formula for calculating the synexin-induced chromaffin granule aggregation. According to this amended analysis, synexin-induced aggregation is specific for chromaffin granules or their membranes. Treatment of intact chromaffin granuleswith trypsin or pronase renders the granules unresponsive to synexin.