A sequential expression system for high-throughput functional genomic analysis

A method employing sequential rounds of cell-free protein synthesis (CFPS) was developed to identify gene products influencing the complex metabolic systems that result in protein accumulation and folding in vitro. The first round of CFPS creates an array of cell extracts individually enriched with a single gene product expressed in-parallel from linear DNA expression templates (ETs). The cell extract is engineered to enhance template stability and to provide reaction conditions conducive for general protein activation. Following first-round expression, linear templates are selectively degraded and a plasmid template for a reporter enzyme is added to initiate a subsequent round of protein expression. Reporter concentration and activity identify first-round gene products that affect amino acid and nucleic acid stability, energy supply, protein expression, stability, and activation. This sequential CFPS system provides a unique format for the functional genomic identification of broadly diverse metabolic activities.     

Hydrogen sulfide removal by immobilized Thiobacillus novellas on SiO2 in a fluidized bed reactor

The removal of hydrogen sulfide (H2S) from aqueous media was investigated using Thiobacillus novellas cells immobilized on a SiO2 carrier (biosand). The optimal growth conditions for the bacterial strain were 30 degrees C and initial pH of 7.0. The main product of hydrogen sulfide oxidation by T. novellus was identified as the sulfate ion. A removal efficiency of 98% was maintained in the three-phase fluidized-bed reactor, whereas the efficiency was reduced to 90% for the two-phase fluidized-bed reactor and 68% for the two-phase reactor without cells. The maximum gas removal capacity for the system was 254 g H2S/m3/h when the inlet H2S loading was 300 g/m3/h (1,500 ppm). Stable operation of the immobilized reactor was possible for 20 days with the inlet H2S concentration held to 1,100 ppm. The fluidized bed bioreactor appeared to be an effective means for controlling hydrogen sulfide emissions.     

Enzyme Thermistor Analysis of Penicillin in Standard Solutions and in Fermentation Broth

Immobilized penicillinase was applied in an enzyme thermistor for calorimetric analysis of samples containing penicillin G. Standard solutions as well as extracts from fermentation broth were analyzed. The enzyme was applied bound either to porous glass or, when dealing with crude preparations, to the inner surface of nylon tubing. In the fermentation system studied, high concentrations of penicillin were present, thus allowing dilution to reduce the influence of the composition of the medium on the analysis. The useful linear concentration range was from 0.1 to 100 mM. The coefficient of correlation between analytical results obtained with the present method and those from conventional assays was 0.997.     

一种高效的酵母菌培养基的设计

为了设计一种高效酵母菌培养基,以便在需要酵母菌快速生长繁殖时,使用该类培养基,在较短时间内获得大量酵母菌,分别以LB培养基、麦氏培养基作为最基本的培养基加入一定量的大豆提取物,配制成为一定浓度的培养基,并用于酵母菌的培养。根据酵母菌的生长情况,判定大豆提取物对酵母菌的生长繁殖是否有促进作用。实验证明,大豆提取物对酵母菌生长繁殖具有明显的促进作用,尤其是浓度为6％时作用最明显。结果表明,采用LB或麦氏培养基加入6％的大豆提取物对酵母菌的生长促进作用最佳,是一种高效的酵母菌培养基。     

Rebound effects of price differences

Goal, Scope and Background  Traditionally, comparative life cycle assessments (LCA) have not considered rebound effects, for instance in case of significant price differences among the compared products. No justifications have been made for this delimitation in scope. This article shows that price differences and the consequent effects of marginal consumer expenditure may influence the conclusions of comparative LCA significantly. We also show that considerations about rebound effects of price differences can be included in LCAs. Methods  The direct rebound effect of a price difference is marginal consumption. Based on statistical data on private consumption in different income groups (Statistics Denmark 2005a, 2005b), the present article provides an estimate of how an average Danish household will spend an additional 1 DKK for further consumer goods, when the household has gained money from choosing a cheaper product alternative. The approach is to use marginal income changes and the following changes in consumption patterns as an expression for marginal consumption. Secondly, the environmental impact potentials related to this marginal consumption are estimated by the use of environmental impact intensity data from an IO-LCA database (Weidema et al. 2005). Finally, it is discussed whether, and in which ways the conclusions of comparative LCAs can be affected by including the price difference between product alternatives. This is elucidated in a case study of a comparative LCA screening of two different kinds of Danish cheese products (Fricke et al. 2004). Results  Car purchase and driving, use and maintenance of dwelling, clothing purchase and insurance constitutes the largest percentages of the marginal consumption. In a case study of two cheeses, the including the impact potentials related to the price difference results in significant changes in the total impact potentials. Considering the relatively small price difference of the two products, it is likely also to have a significant influence on the results of comparative LCAs more generally. Discussion  The influence of marginal consumption in comparative LCAs is relevant to consider in situations with large differences in the price of the product alternatives being compared, and in situations with minor differences in the impact potentials related to the alternatives. However, different uncertainties are linked to determining the pattern for marginal consumption and the environmental impact potential related to this. These are first of all related to the method used, but also include inaccurate data of consumption in households, aggregation and weighting of income groups, aggregation of product groups, estimation and size of the price difference, and the general applicability of the results. Conclusion  Incorporating marginal consumption in consequential LCAs is possible in practice. In the case study used, including the rebound effects of the price difference has a significant influence on the result of the comparative LCA, as the result for the impact categories acidification and nutrient enrichment changes in favour of the expensive product. Recommendations and Perspectives  It is recommended that the rebound effects of price differences should be included more frequently in LCAs. In order to ensure this, further research in marginal consumption and investment patterns and IO data for different countries or regions is required. Furthermore, this study does not consider the economic distributional consequences of buying an expensive product instead of a cheaper product (e.g. related to how the profit is spent by those who provided the product). It should also be noted, that more expensive products not necessarily result in less consumption, as those who provided the product also will spend the money they have earned from the sale. Ideally, these consequences should also be further investigated. Likewise, the development of databases to include marginal consumption in PC-tools is needed. In general, considerations of marginal consumption would favour expensive product alternatives, depending, however, on the type of consumer. ESS-Submission Editor: Dr. David Hunkeler (david.hunkeler@aquaplustech.ch)     

Characterization of xylanase from Lentinus edodes M290 cultured on waste mushroom logs

Extracellular enzymes from Lentinus edodes M290 on normal woods (Quercus mongolica) and waste logs from oak mushroom production were comparatively investigated. Endoglucanase, cellobiohydrolase, beta-glucosidase, and xylanase activities were higher on waste mushroom logs than on normal woods after L. edodes M290 inoculation. Xylanase activity was especially different, with a three times higher activity on waste mushroom logs. When the waste mushroom logs were used as a carbon source, a new 35 kDa protein appeared. After the purification, the optimal pH and temperature for xylanase activity were determined to be 4.0 and 50 degrees C, respectively. More than 50% of the optimal xylanase activity was retained when the temperature was increased from 20 to 60 degrees C, after a 240 min reaction. At 40 degrees C, the xylanase maintained 93% of the optimal activity, after a 240 min reaction. The purified xylanase showed a very high homology to the xylanase family 10 from Aspergillus terreus by LC/MS-MS analysis. The highest Xcorr (1.737) was obtained from the peptide KWI SQGIPIDGIG SQTHLGSGGS WTVK originated from Aspergillus terreus, indicating that the 35 kDa protein was xylanase. This protein showed low homology to a previously reported L. edodes xylanase sequence.     

Enhancement of 7-methoxyresorufin O-demethylation activity of human cytochrome P450 1A2 by molecular breeding

Alkylresorufins are model substrates for cytochrome P450 (P450) 1A2. The ability of human P450 1A2 to catalyze 7-methoxyresorufin O-demethylation was improved by screening of random mutant libraries (expressed in Escherichia coli) on the basis of 7-methoxyresorufin O-demethylation. After three rounds of mutagenesis and screening, the triple mutant E163K/V193M/K170Q yielded a kcat > five times faster than wild type P450 1A2 in steady-state kinetic analysis using either isolated membrane fractions or purified, reconstituted enzymes. The enhanced catalytic activity was not attributed to changes in substrate affinity. The kinetic hydrogen isotope effect of the triple mutant did not change from wild type enzyme and suggests that C-H bond cleavage is rate-limiting in both enzymes. Homology modeling, based on an X-ray structure of rabbit P450 2C5, suggests that the locations of mutated residues are not close to the substrate binding site and therefore that structural elements outside of this site play roles in changing the catalytic activity. This approach has potential value in understanding P450 1A2 and generating engineered enzymes with enhanced catalytic activity.     

An Imported Case of Cystic Echinococcosis in the Liver

A 25-year-old Uzbek male presented with right upper abdominal pain for 20 days. On radiologic studies, a huge cystic mass was noticed in the right liver which was suspected as parasitic. The patient received right hepatic segmentectomy (segment 7), and the surgically resected mass was confirmed as cystic echinococcosis (CE), measuring 10.5 cm in its diameter. The inner surface of the cyst was bile-stained. The patient was discharged on the 8th hospital day, and was rechecked 6 months after the surgical intervention without any evidence of recurrence. The present report describes findings of an imported case of CE which represented ultrasound images of the ''ball of wool''.     

Detection of an intermediate during the unfolding process of the dimeric ketosteroid isomerase

Failure to detect the intermediate in spite of its existence often leads to the conclusion that two-state transition in the unfolding process of the protein can be justified. In contrast to the previous equilibrium unfolding experiment fitted to a two-state model by circular dichroism and fluorescence spectroscopies, an equilibrium unfolding intermediate of a dimeric ketosteroid isomerase (KSI) could be detected by small angle X-ray scattering (SAXS) and analytical ultracentrifugation. The sizes of KSI were determined to be 18.7A in 0M urea, 17.3A in 5.2M urea, and 25.1A in 7M urea by SAXS. The size of KSI in 5.2M urea was significantly decreased compared with those in 0M and 7M urea, suggesting the existence of a compact intermediate. Sedimentation velocity as obtained by ultracentrifugation confirmed that KSI in 5.2M urea is distinctly different from native and fully-unfolded forms. The sizes measured by pulse field gradient nuclear magnetic resonance (NMR) spectroscopy were consistent with those obtained by SAXS. Discrepancy of equilibrium unfolding studies between size measurement methods and optical spectroscopies might be due to the failure in detecting the intermediate by optical spectroscopic methods. Further characterization of the intermediate using (1)H NMR spectroscopy and Kratky plot supported the existence of a partially-folded form of KSI which is distinct from those of native and fully-unfolded KSIs. Taken together, our results suggest that the formation of a compact intermediate should precede the association of monomers prior to the dimerization process during the folding of KSI.     

Inhibition of IFN-gamma receptor signaling by hepatitis C virus non-structural protein NS4B]

The function of NS4B is incompletely understood. The aim of the study is to understand the influence of NS4B on anti-viral response. After cell line stably expressing NS4B established, the influence of IFN-alpha of different concentration on VSV was studied using plaque assay; cell expression profiling caused by NS4B was studied using DNA microarray, and the IFNGR1 fluorescence intensity was analyzed. Our data showed that HCV-NS4B could suppress immuno-associated gene expression, in particular, IFN-gamma receptor signal transduction-related genes. Taken together, NS4B could play some roles in HCV resistance to IFN therapy.