Construction and Characterisation of a Genetically Engineered Escherichia coli Strain for the Epoxide Hydrolase-catalysed Kinetic Resolution of Epoxides

The Rhodotorula glutinis epoxide hydrolase, Eph1, was produced in the heterologous host Escherichia coli BL21(DE3) in order to develop a highly effective epoxide hydrolysis system. A 138-fold increase in Eph1 activity was found in cell extracts of the recombinant E. coli when compared to cell extracts of Rhodotorula glutinis, despite the formation of Eph1 inclusion bodies. Optimization of cultivation conditions and co-expression of molecular chaperones resulted in a further increase in activity and a reduction of the inclusion bodies formation, respectively. Compared to Rhodotorula glutinis cells and cell extracts, a total increase in Eph1 activity of over 200 times was found for both Escherichia coli cells and crude enzyme preparations of these cells. The improved conditions for recombinant Eph1 production were used to demonstrate the Eph1-catalysed kinetic resolution of a new Eph1 substrate, 1-oxaspiro[2.5]octane-2-carbonitrile.     

Evaluation of suitable DNA regions for molecular identification of high value medicinal plants in genus Kaempferia

DNA barcoding coupled high resolution melting (Bar-HRM) is an emerging method for species discrimination based on DNA dissociation kinetics. The aim of this work was to evaluate the suitability of different primer sets, derived from selected DNA regions, for Bar-HRM analysis of species in Kaempferia (Zingiberaceae). Four primer pairs were evaluated (rbcL, rpoC, trnL and ITS1). It was observed that the ITS1 barcode was the most useful DNA barcoding region overall for species discrimination out of all of the regions and primers assessed. Thus, the primer pair derived from the ITS1 region was the single most effective region for the identification of the tested species, whereas the rbcL primer pair gave the lowest resolution. Our Bar-HRM developed here would not only be useful for identification of Kaempferia plant specimens lacking essential parts for morphological identification but will be useful for authenticating products in powdered form of a high value medicinal species Kaempferia parviflora, in particular.     

Community Member Viewpoints on the Ría Celestún Biosphere Reserve,Yucatan, Mexico: Suggestions for Improving the Community/Natural Protected Area Relationship

The results of an analysis of the viewpoints of Celestún community members regarding the Ría Celestún Biosphere Reserve, Mexico, are used as the basis for recommendation to improve the community/natural protected area (NPA) relationship. Participant observation was used to record the opinions of 80 people (8–83 years of age) between April and August 2002. Twenty semistructured interviews were held with local young people randomly selected from the participant observation group and their fathers; one interview was held with a municipal officer. Results indicate that the informants perceived a lack of communication between community members and the Reserve concerning its objectives and action areas, minimal community participation in Reserve regulation development, an absence of alternative productive activities and unjustified restriction of natural resources. These perceived conditions can potentially generate conflict between the community and the Reserve administration, preventing fulfillment of the Reserve’s environmental protection mission and negatively affecting community welfare. It is suggested that communication between the Reserve and the community be mutual and free-flowing, that Reserve objectives be identified in conjunction with the community, that community members be included in Reserve activities and administration, that interdisciplinary teams be formed to encourage communication and participation and that alternative productive activities be developed. Particular emphasis is placed on the need to listen to and address the concerns of young people because they will define the community/Reserve relationship in the near future.

Enantiomeric resolution of amino acids by thin-layer chromatography

A simple and rapid method of separating optical isomers of amino acids on a reversed-phase TLC plate, without using impregnated plates or a chiral mobile phase, is described. Amino acids derivatized with 1-fluoro-2,4-dinitrophenyl-5- -alanine amide were spotted on a reversed phase pre-coated TLC plate. Enantiomers of glutamate and aspartate were separated most effectively with solvent consisting of 25% acetonitrile in triethylamine-phosphate buffer (50 mM, pH 5.5) (v/v). Separation of - and -serine was achieved with 30% of acetonitrile solvent. The enantiomers of threonine, proline and alanine were separated with 35% of acetonitrile solvent, and those of methionine, valine, phenylalanine and leucine with 40% of acetonitrile solvent. The possibility of using TLC for quantitative determination of amino acid enantiomers was shown by the quantitative recovery of - and -alanine from the TLC plate in the range of 0.56–4.48 nmol.     

A search for endogenous mechanisms of anti-inflammation uncovers novel chemical mediators: missing links to resolution

Multicellular responses to infection, injury, or inflammatory stimuli lead to the formation and release of a wide range of local chemical mediators by the host. The integrated response of the host is essential in health and disease, thus it is important to achieve a more complete understanding of the local cellular and molecular events that govern the formation and actions of local mediators that can serve as endogenous counter-regulatory functions in effector cells of the immune system or endogenous local mediators of resolution. Since these compounds in theory and in experimental models of inflammation appear to control the duration and magnitude of inflammation, knowledge of their elucidation could provide new avenues for appreciating the molecular phenotypes of many inflammatory diseases. The first of these endogenous local counter-regulators recognized were the lipoxins, which are trihydroxytetraene-containing lipid mediators that can be formed during cell–cell interactions via transcellular biosynthesis. Since this circuit of lipoxin formation and action appears to be of physiological relevance for the resolution of inflammation, therapeutic modalities targeted at this system are likely to have fewer unwanted side effects acting as agonists than the inhibitor approach currently used in anti-inflammatory therapies. This chapter provides an overview of the recent knowledge about the biosynthesis and bioactions of the novel anti-inflammatory lipid mediators, resolvins, docosatrienes, and neuroprotectins, and their aspirin-triggered counterparts. These novel families of lipid-derived mediators, which carry anti-inflammatory, pro-resolving, and protective properties, were originally isolated during spontaneous resolution. These new pathways open new opportunities for appreciating the role of neutrophils in the generation of potent protective lipid mediators and protective host signaling.     

Options for a rational dialogue on the acceptance of biotechnology

This paper explores how framing discussions about biotechnology in different ways influences how scientists, policy makers, and members of the public communicate with each other, to either inhibit or promote acceptance. It argues for the use of new techniques to create a rational dialogue with the public, based on scientific knowledge, that allows them to fully participate as informed stakeholders in debates about new technologies. Several different outlooks on the role of science in society are presented. Effectively communicating these different positions to the public requires innovative approaches. Drawing on advances in scientific communication practices, we briefly describe four principles that consider not only the scientific content, but also its delivery. New views need to be delivered with confidence, to be sensible within existing frameworks of understanding and not overwhelming, to reveal both potentially positive and negative aspects, and to promote interactive discussion with stakeholders. The authors conclude using the regional initiative in Southern Africa--the African Policy Dialogues on Biotechnology--as an illustration of this approach, where discussions started by first seeking agreement about establishing a process for dialogue, rather than initially trying to achieve consensus on any substantive points.     

Geographic evaluation of conservation status of African forest squirrels (Sciuridae) considering land use change and climate change: the importance of point data

Known occurrences based on natural history museum voucher specimens for three genera of African forest squirrels were used to develop a detailed, fine-scale distributional under- standing of each species. Considerations of species’ autecology, effects of land use change, and effects of global climate change were all included in our analyses, and negative effects of land use and climate change on species’ distributional areas were roughly equivalent across the species surveyed. We describe geographic patterns of distribution and endemism, and identify areas of potential occurrence of unknown species. Comparing with coarse grid-based approaches currently in vogue in African biodiversity conservation efforts, we suggest that the point-based method offers significant advantages in fine resolution and avoiding loss of information, and yet are feasibly implemented for many vertebrate groups.     

Resolution of 2-octanol by SBA-15 immobilized Pseudomonas sp. lipase

Lipase from Pseudomonas sp. (PSL) was immobilized on SBA-15 (a highly ordered hexagonal array mesoporous silica molecular sieve) through physical adsorption and the immobilized PSL was used in resolution of (R,S)-2-octanol with vinyl acetate as acyl donor. Enhanced activity and enantioselectivity were observed for the immobilized PSL compared with those of the free one. The effects of reaction conditions, such as solvents, temperature, water activity and substrate ratio were investigated. Under the optimum conditions, the residual (S)-2-octanol was recovered with 99% enantiomeric excess at 52% conversion. The results also indicated that the immobilized PSL maintained 90% of its initial activity even after reusing it five times.     

产γ_内酰胺水解酶菌株的筛选及发酵条件研究

(-)γ-内酰胺是合成两种抗艾滋病药物(-)carbovir和(-)abacavir的重要原料,具有重要的应用价值,微生物酶法拆分( /-)γ-内酰胺生产(-)γ-内酰胺的方法具有良好的应用前景。以N-乙酰苯丙氨酸为唯一碳源,从土壤中筛选得到了69株具有酰胺水解酶活性的菌株,利用液相色谱分析方法确定了其中20株有较高的酰胺水解酶活性,利用手性色谱分析的方法进一步得到了一株具有较高立体选择性,能拆分( /-)γ-内酰胺而获得(-)γ-内酰胺的菌株L29-9,对该菌株的产酶培养基的碳、氮源及初始pH值等进行了研究。结果表明:当选择碳源柠檬酸2g/L、氮源酵母提取物5g/Lp、H 7.0、培养时间40h时,通过完整细胞转化,在30℃下经过12h的反应,产物(-)γ-内酰胺产率达40%,ee值99.5%。