Pycnogenol® and vitamin E inhibit ethanol‐induced apoptosis in rat cerebellar granule cells

Pycnogenol® (PYC), a patented combination of bioflavonoids extracted from the bark of French maritime pine (Pinus maritima), scavenges free radicals and promotes cellular health. The protective capacity of PYC against ethanol toxicity of neurons has not previously been explored. The present study demonstrates that in postnatal day 9 (P9) rat cerebellar granule cells the antioxidants vitamin E (VE) and PYC (1) dose dependently block cell death following 400, 800, and 1600 mg/dL ethanol exposure (2) inhibit the ethanol‐induced activation of caspase‐3 in the same model system; and (3) reduce neuronal membrane disruption as assayed by phosphatidylserine translocation to the cell surface. These results suggest that both PYC and VE have the potential to act as therapeutic agents, antagonizing the induction of neuronal cell death by ethanol exposure. © 2004 Wiley Periodicals, Inc. J Neurobiol 59: 261–271, 2004     

Melanocortin‐1 receptor structure and functional regulation

The melanogenic actions of the melanocortins are mediated by the melanocortin‐1 receptor (MC1R). MC1R is a member of the G‐protein‐coupled receptors (GPCR) superfamily expressed in cutaneous and hair follicle melanocytes. Activation of MC1R by adrenocorticotrophin or α‐melanocyte stimulating hormone is positively coupled to the cAMP signaling pathway and leads to a stimulation of melanogenesis and a switch from the synthesis of pheomelanins to the production of eumelanic pigments. The functional behavior of the MC1R agrees with emerging concepts in GPCR signaling including dimerization, coupling to more than one signaling pathway and a high agonist‐independent constitutive activity accounting for inverse agonism phenomena. In addition, MC1R displays unique properties such as an unusually high number of natural variants often associated with clearly visible phenotypes and the occurrence of endogenous peptide antagonists. Therefore MC1R is an ideal model to study GPCR function. Here we review our current knowledge of MC1R structure and function, with emphasis on information gathered from the analysis of natural variants. We also discuss recent data on the regulation of MC1R function by paracrine and endocrine factors and by external stimuli such as ultraviolet light.     

Affinity chromatography of Ankistrodesmus braunii nitrate reductase using blue dextran-sepharose (author's transl)

Blue Dextran has been coupled covalently to Sepharose-4B to purify the enzymatic complex NAD(P)H-nitrate reductase (EC 1.6.6.2) from the green alga Ankistrodesmus braunii by affinity chromatography. The optimum conditions for the accomplishment of the chromatographic process have been determined. The adsorption of nitrate reductase on Blue Dextran Sepharose is optimum when a phosphate buffer of low ionic strength and pH 6.5-7.0 is used. Once the enzyme has been bound to Blue Dextran Sepharose, it can be specifically eluted by addition of NADH and FAD to the washing buffer. However, none of the nucleotides added separately is able to promote the elution of the enzyme from the column. The elution can be also achieved, but not specifically, by increasing the ionic strength of the buffer with KCl. These results have made possible a procedure for the purification of A. braunii nitrate reductase which led to electrophoretic homogeneity, with an overall yield of 70% and a specific activity of 49 units/mg of protein.     

Isolation and characterization of microsatellites in Pacific white shrimp Penaeus (Litopenaeus) vannamei

Five polymorphic microsatellite loci were characterized for Penaeus (Litopenaeus) vannamei. Loci were isolated using a partial Sau3A1 genomic library by the sequencing of randomly selected clones and by a biotinylated (CT)₁₀ and (GT)₁₀ probes screening procedure. The last strategy resulted in the most useful data. About 40% of the clones showed a previously reported satellite/microsatellite (PVS1), reducing the chance of finding new microsatellite regions. Whereas two of the microsatellite loci with more than 10 alleles will be useful for mating analysis in a breeding program, the others might prove useful for population genetic studies.     

Active site mutations in DNA topoisomerase I distinguish the cytotoxic activities of camptothecin and the indolocarbazole, rebeccamycin.

DNA topoisomerase I (Top1p) catalyzes topological changes in DNA and is the cellular target of the antitumor agent camptothecin (CPT). Non-CPT drugs that target Top1p, such as indolocarbazoles, are under clinical development. However, whether the cytotoxicity of indolocarbazoles derives from Top1p poisoning remains unclear. To further investigate indolocarbazole mechanism, rebeccamycin R-3 activity was examined in vitro and in yeast. Using a series of Top1p mutants, where substitution of residues around the active site tyrosine has well-defined effects on enzyme catalysis, we show that catalytically active, CPT-resistant enzymes remain sensitive to R-3. This indolocarbazole did not inhibit yeast Top1p activity, yet was effective in stabilizing Top1p-DNA complexes. Similar results were obtained with human Top1p, when Ser or His were substituted for Asn-722. The mutations altered enzyme function and sensitivity to CPT, yet R-3 poisoning of Top1p was unaffected. Moreover, top1delta, rad52delta yeast cells expressing human Top1p, but not catalytically inactive Top1Y723Fp, were sensitive to R-3. These data support hTop1p as the cellular target of R-3 and indicate that distinct drug-enzyme interactions at the active site are required for efficient poisoning by R-3 or CPT. Furthermore, resistance to one poison may potentiate cell sensitivity to structurally distinct compounds that also target Top1p.     

Short hairpin RNAs against eotaxin or interleukin‐5 decrease airway eosinophilia and hyper‐responsiveness in a murine model of asthma

Background

Eosinophilia plays the major role in the pathogenesis of asthma and correlates with the up‐regulation of eotaxin, which, together with interleukin (IL)‐5, is important for differentiation, chemo‐attraction, degranulation, and survival of eosinophils in local tissue. In a previous study, we found that administration of lentivirus‐delivered short hairpin RNA (shRNA) to suppress the expression of IL‐5 inhibited airway inflammation. The present study aimed to investigate the role of eotaxin shRNA and the synergistic effect of eotaxin and IL‐5 shRNAs on airway inflammation in an ovalbumin (OVA)‐induced murine model of asthma.

Methods

Lentivirus‐delivered shRNAs were used to suppress the expression of eotaxin and/or IL‐5 in local tissue in an OVA‐induced murine asthma model.

Results

Intra‐tracheal administration of lentivirus containing eotaxin shRNA expressing cassette (eoSEC3.3) efficiently moderated the characteristics of asthma, including airway hyper‐responsiveness, cellular infiltration of lung tissues, and eotaxin and IL‐5 levels in bronchio‐alveolar lavage fluid. Administration of lentiviruses expressing IL‐5 or eotaxin shRNAs (IL5SEC4 + eoSEC3.3) also moderated the symptoms of asthma in a mouse model.

Conclusions

Local delivery of lentiviruses expressing IL‐5 and eotaxin shRNAs provides a potential tool in moderating airway inflammation and also has the potential for developing clinical therapy based on the application of shRNAs of chemokines and cytokines involved in T helper 2 cell inflammation and eosinophilia. Copyright © 2008 John Wiley & Sons, Ltd.     

How Can the Eco‐efficiency of a Region be Measured and Monitored?

The concept of eco-efficiency is commonly referred to as a business link to sustainable development. In this article, ecoefficiency is examined at a regional level as an approach to promoting the competitiveness of economic activities in the Finnish Kymenlaakso region and mitigating their harmful impacts on the environment. The aim is to develop appropriate indicators for monitoring changes in the eco-efficiency of the region. A starting point is to produce indicators for the environmental and economic dimensions of regional development and use them for measuring regional eco-efficiency. The environmental impact indicators are based on a life-cycle assessment method, producing different types of environmental impact indicators: pressure indicators (e.g., emissions of CO₂), impact category indicators (e.g., CO₂ equivalents in the case of climate change), and a total impact indicator (aggregating different impact category indicator results into a single value). Environmental impact indicators based on direct material input, total material input, and total material requirement of the Kymenlaakso region are also assessed. The economic indicators used are the gross domestic product, the value added, and the output of the main economic sectors of Kymenlaakso. In the eco-efficiency assessment, the economic and environmental impact indicators are monitored in the same graph. In a few cases eco-efficiency ratios can also be calculated (the economic indicators are divided by the environmental indicators). Output (= value added + intermediate consumption) is used as an economic indicator related to the environmental impact indicators, which also cover the upstream processes of the region's activities. In the article, we also discuss the strengths and weaknesses of using the different environmental impact indicators.