Vanillic acid metabolism by selected soft-rot,brown-rot,and white-rot fungi

Metabolism of vanillic acid, a product of lignin degradation, has been studied in selected representatives of soft-rot, brown-rot and white-rot fungi. All of the brown-and white-rot species examined decarboxylated vanillate to methoxyhydroquinone oxidatively. Mycelium extracts of all these fungi, except Pleurotus ostreatus contained high levels of an NAD(P)H-dependent vanillate hydroxylase. P. ostreatus also released ¹⁴CO₂ from ¹⁴COOH-vanillate but by a different mechanism possibly involving phenoloxidases. Most of these fungi also contained a dioxygenase which catalysed the intra-diol cleavage of hydroxyquinol (1,2,4-trihydroxybenzene) to form maleylacetate. No 3-O-demethylase activity was detected, and data indicate that in some of the fungi examined cleavage of the aromatic ring occurs without prior removal of the methoxyl group. None of the soft-rot fungi tested contained vanillate hydroxylase or hydroxyquinol 1,2-dioxygenase, but very low levels of protocatechuate 3,4-dioxygenase were detected in mycelium extracts. Vanillate catabolism among members of this group occurs via a different route which may involve ring demethylation although no 3-O-demethylase activity was detected in this study. The enzyme NAD(P)H-quinone oxidoreductase was demonstrated to exist in all the studied groups of fungi.     

Chronic Copper Poisoning in Sheep

Four cases of experimental copper poisoning in sheep were examined. Light microscopical and ultrastructural alterations of erythrocytes were observed a few hours before a significant haemolysis was evident. Heinz body formation in otherwise unchanged red cells was the first morphological alteration observed. The Heinz bodies were predominantly membrane-attached. During the haemolytic crisis severe erythrocytic distortion, structural membrane alteration and Heinz body-containing ghost cells were observed. Erythrophagocytosis was mainly located to the RE cells of the spleen. Intrafollicular necroses were found in all histological sections from the spleen. Pathological changes in liver and kidney were comparable to those of earlier reports, comprising hepatocellular and renal tubular necrosis.     

Optimization of novel monobactams with activity against carbapenem-resistant Enterobacteriaceae – Identification of LYS228

Metallo-β-lactamases (MBLs), such as New Delhi metallo-β-lactamase (NDM-1) have spread world-wide and present a serious threat. Expression of MBLs confers resistance in Gram-negative bacteria to all classes of β-lactam antibiotics, with the exception of monobactams, which are intrinsically stable to MBLs. However, existing first generation monobactam drugs like aztreonam have limited clinical utility against MBL-expressing strains because they are impacted by serine β-lactamases (SBLs), which are often co-expressed in clinical isolates. Here, we optimized novel monobactams for stability against SBLs, which led to the identification of LYS228 (compound 31). LYS228 is potent in the presence of all classes of β-lactamases and shows potent activity against carbapenem-resistant isolates of Enterobacteriaceae (CRE).     

Low efficiency of functional translation of ouabain-resistant α2-Na(+),K(+)-ATPase mRNA in C7-MDCK epithelial cells

Na(+),K(+)-ATPase is a heterodimer consisting of catalytic α1-α4 and regulatory β1-β3 subunits. Recently, we reported that transfection with ouabain-resistant α1R-Na(+),K(+)-ATPase rescues renal epithelial C7-MDCK cells exclusively expressing the ouabain-sensitive α1S-isoform from the cytotoxic action of ouabain. To explore the role of α2 subunit in ion transport and cytotoxic action of ouabain, we compared the effect of ouabain on K(+) ((86)Rb) influx and the survival of ouabain-treated C7-MDCK cells stably transfected with α1R- and α2R-Na(+),K(+)-ATPase. α2R mRNA in transfected cells was ～8-fold more abundant than α1R mRNA, whereas immunoreactive α2R protein content was 5-fold lower than endogenous α1S protein. A concentration of 10?μmol/L ouabain led to complete inhibition of (86)Rb influx both in mock- and α2R-transfected cells, whereas maximal inhibition of (86)Rb influx in α1R-transfectd cells was observed at 1000?μmol/L ouabain. In contrast to the massive death of mock- and α2R-transfected cells exposed to 3?μmol/L ouabain , α1R-cells survived after 24?h incubation with 1000?μmol/L ouabain. Thus, our results show that unlike α1R, the presence of α2R-Na(+),K(+)-ATPase subunit mRNA and immunoreactive protein does not contribute to Na(+)/K(+) pump activity, and does not rescue C7-MDCK cells from the cytotoxic action of ouabain. Our results also suggest that the lack of impact of transfected α2-Na(+),K(+)-ATPase on Na(+)/K(+) pump activity and cell survival can be attributed to the low efficiency of its translation and (or) delivery to the plasma membrane of renal epithelial cells.     

Multi‐factor climate change effects on insect herbivore performance

The impact of climate change on herbivorous insects can have far‐reaching consequences for ecosystem processes. However, experiments investigating the combined effects of multiple climate change drivers on herbivorous insects are scarce. We independently manipulated three climate change drivers (CO₂, warming, drought) in a Danish heathland ecosystem. The experiment was established in 2005 as a full factorial split‐plot with 6 blocks × 2 levels of CO₂ × 2 levels of warming × 2 levels of drought = 48 plots. In 2008, we exposed 432 larvae (n = 9 per plot) of the heather beetle (Lochmaea suturalis Thomson ), an important herbivore on heather, to ambient versus elevated drought, temperature, and CO₂ (plus all combinations) for 5 weeks. Larval weight and survival were highest under ambient conditions and decreased significantly with the number of climate change drivers. Weight was lowest under the drought treatment, and there was a three‐way interaction between time, CO₂, and drought. Survival was lowest when drought, warming, and elevated CO₂ were combined. Effects of climate change drivers depended on other co‐acting factors and were mediated by changes in plant secondary compounds, nitrogen, and water content. Overall, drought was the most important factor for this insect herbivore. Our study shows that weight and survival of insect herbivores may decline under future climate. The complexity of insect herbivore responses increases with the number of combined climate change drivers.     

hnRNP I/PTB can antagonize the splicing repressor activity of SRp30c

The control of alternative pre-mRNA splicing often requires the participation of factors displaying synergistic or antagonistic activities. In the hnRNP A1 pre-mRNA, three elements promote the exclusion of alternative exon 7B, while a fourth intron element (CE9) represses splicing of exon 7B to the downstream exon. We have shown previously that the 5' portion of the 38-nucleotide-long CE9 element is bound by SRp30c, and that this interaction is important for repression in vitro. To determine whether SRp30c alone can impose repression, we tested a high-affinity SRp30c binding site that we identified using the SELEX protocol. We find that multiple high-affinity SRp30c sites are required to replicate the level of repression obtained with CE9, and that both the 5' and the 3' portions of CE9 contribute to SRp30c binding. Performing RNA affinity chromatography with the complete CE9 element recovered hnRNP I/PTB. Surprisingly however, His-tagged PTB reduced the binding of SRp30c to CE9 in a nuclear extract, stimulated splicing to a downstream 3' splice site, and relieved the CE9-mediated splicing repression in vitro. Our in vivo results are consistent with the notion that increasing PTB levels alleviates the repression imposed by CE9 to a downstream 3' splice site. Thus, PTB can function as an anti-repressor molecule to counteract the splicing inhibitory activity of SRp30c.