Purification of Pregnancy Zone Protein and Its Receptor Binding Domain from Human Plasma

A new significantly improved method for purification of pregnancy zone protein (PZP), α₂-macroglobulin (α₂M), and the C-terminal PZP receptor binding domain is presented. Several steps in an earlier procedure have been deleted, and modifications in the gradients in the DEAE step leave most of the contaminants bound to a DEAE-Sephacel gel. This procedure makes possible the rapid, simultaneous purification of both of these closely related unstable proteins in native form from human plasma, with no thiolester cleavage or formation of tetrameric PZP. The final preparations of both α₂M and PZP are pure as determined by nonreducing and reducing polyacrylamide gel electrophoresis following silver staining and no cross-contamination can be observed. The yield has been significantly improved and typically more than 500 mg PZP can be obtained from 1 liter pregnancy plasma. Furthermore, the stability of PZP at different temperatures on storage was studied. In liquid nitrogen PZP can be maintained in native dimeric form with intact thiolester for many years. The storage of native PZP with intact functional properties during and after purification is an obligatory prerequisite to elucidate the biological role of PZP. The receptor binding domain of PZP can be cleaved from the PZP–methylamine complex by papain and isolated from the other peptides by S-200 gel filtration. The cleavage site was determined and the C-terminal fragment was identified with several site-specific monoclonal antibodies against PZP.     

Block by phencyclidine of acetylcholine and barium induced adrenal catecholamine secretion

Activation of the sympathetic system by phencyclidine (PCP) should result in catecholamine release from the adrenals. However, adrenalectomy does not reduce PCP-induced hypertension. In an attempt to rectify this inconsistency, the direct effects of PCP on the bovine adrenal medulla were examined. At (3×10^?6M), PCP reduced the acetylcholine-(ACh)-induced catecholamine release by 50%. Surprisingly, barium-induced secretion of catecholamines was also reduced by PCP. ACh-induced catecholamine release was not altered by 10^?3M 4-aminopyridine (4 AP), the potassium channel blocker. Thus, calcium antagonist actions of PCP and consequent block of catecholamine secretion from adrenal medulla may explain the lack of effect of adrenalectomy on PCP-induced hypertension. Possible contributions of calcium and/or potassium channel blockade to other manifestations of PCP overdosage are discussed.     

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.     

Esters of trehalose from Corynebacterium diphtheriae: A modified purification procedure and studies on the structure of their constituent hydroxylated fatty acids

The purification procedure of 6,6′-diesters of trehalose from Corynebacterium diphtheriae was modified and the isolated substance was analysed by mass spectrometry as its permethylated derivative. The fatty acid moiety released from the glycolipid after alkaline hydrolysis was studied by mass spectral analysis of the O-methylated and O-acetylated methyl ester derivatives. By argentation thin-layer chromatography, three species of O-acetylated methyl esters were recognized, corresponding to saturated, mono-unsaturated and di-unsaturated α-branched-β-hydroxylated fatty acids. The double bond was located by ozonolysis of the O-acetylated methyl ester derivatives, by gas chromatography of the reaction product and mass spectrometry of the effluent from the gas chromatograph. The main components of each species of α-branched-β-hydroxylated fatty acids found in the gly colipid fraction of C. diphtheriae were 2-tetradecyl-3-hydroxyoctadecanoic acid (C₃₂H₆₄O₃, corynomycolic acid), 2-tetradecyl-3-hydroxy-11-octadecenoic acid (C₃₂H₆₂O₃, corynomycolenic acid), 2-tetradec-7′-enyl-3-hydroxy octadecanoic acid (C₃₂H₆₂O₃) and 2-tetradec-7′-enyl-3-hydroxy-11-octadecenoic acid (C₃₂H₆₀O₃, corynomycoldienic acid). The glycolipid fraction from C. diphtheriae is obviously a complex mixture of 6,6′-diesters of trehalose.     

Characterization of Viticultural Terroirs using a Simple Field Model Based on Soil Depth – II. Validation of the Grape Yield and Berry Quality in the Anjou Vineyard (France)

Soil and climate are major constituents of the French notion of Terroir. This concept implies that there is a strong relationship between the composition of the grape, the characteristics of the wine and the territory of production. To study this link, a new method of characterization of the Terroir, including geological and pedological factors, was investigated. It uses a field model based on depth and clay content of soil, together with the degree of weathering of the parent rock. Consequently, for every type of parent rock belonging to a given geologic stage, there are a series of soils that show different stages of pedological evolution. According to the model, three kinds of soils are distinguished with regards to the weathering intensity of the parent rock, that are named weakly weathered rock (WWR), moderately weathered rock (MWR) and strongly weathered rock (SWR). By hypothesis, each soil type is considered as a homogeneous unit for vine production from the viewpoint of ecophysiological factors. Each terroir unit defined by this method is called a Basic Terroir Unit (BTU). To validate this hypothesis, experimental plots planted with Chenin and Cabernet Franc vines were studied over three consecutive seasons (2000–2002), in the Anjou vineyard (Loire Valley – France). The major BTUs developed on the two most important geological systems of Anjou (Brioverian and Ordovician–Devonian), were studied. Results showed that the berries of vines cultivated in WWR were significantly smaller, richer in sugars and anthocyanins and had a Total Phenolic Index higher than those of the vines cultivated in SWR. They also had a lower titratable acidity. Cabernet Franc vines cultivated in MWR had berries with sugar and anthocyanin contents but also total phenolics very close to those of WWR. With Chenin vines there was a good relationship between the global pool of free aromas of berries and the BTU type. The study showed significant relationships between the quality of grapes and the measured values of several ecophysiological variables such as the water supply regime or the timing of budburst.