The use of Tn5 transposable elements in a gene trapping strategy for the protozoan Leishmania

The use of transposable elements as a gene-trapping strategy is a powerful tool for gene discovery. Herein we describe the development of a transposable system, based on the bacterial Tn5 transposon, which has been used successfully in Leishmania braziliensis. The transposon carries the neomycin phosphotransferase gene, which is expressed only when inserted in-frame with a Leishmania gene present in the target DNA. Four cosmid clones from a L. braziliensis genomic library were used as targets in transposition reactions and four insertional libraries were constructed and transfected in L. braziliensis. Clones resistant to G418 were selected and analysed by immunofluorescence in order to identify the subcellular localisation of the protein coded by the trapped gene. A definitive subcellular localisation for neomycin phosphotransferase/targeted protein fusion was not obtained in any of the four Leishmania clones investigated. However, the constructed transposable element is highly efficient considering the frequency of insertion in large targets and is therefore a useful tool for functional genetic studies in Leishmania. Our data confirm the utility of the Tn5 transposon system for insertion of sequencing priming sites into target DNA. Furthermore, the high frequency of insertion and even distribution are important in studying genomic regions bearing long and polymorphic repetitive sequences.     

Phenylalanine biosynthesis in Arabidopsis thaliana. Identification and characterization of arogenate dehydratases

There is much uncertainty as to whether plants use arogenate, phenylpyruvate, or both as obligatory intermediates in Phe biosynthesis, an essential dietary amino acid for humans. This is because both prephenate and arogenate have been reported to undergo decarboxylative dehydration in plants via the action of either arogenate (ADT) or prephenate (PDT) dehydratases; however, neither enzyme(s) nor encoding gene(s) have been isolated and/or functionally characterized. An in silico data mining approach was thus undertaken to attempt to identify the dehydratase(s) involved in Phe formation in Arabidopsis, based on sequence similarity of PDT-like and ACT-like domains in bacteria. This data mining approach suggested that there are six PDT-like homologues in Arabidopsis, whose phylogenetic analyses separated them into three distinct subgroups. All six genes were cloned and subsequently established to be expressed in all tissues examined. Each was then expressed as a Nus fusion recombinant protein in Escherichia coli, with their substrate specificities measured in vitro. Three of the resulting recombinant proteins, encoded by ADT1 (At1g11790), ADT2 (At3g07630), and ADT6 (At1g08250), more efficiently utilized arogenate than prephenate, whereas the remaining three, ADT3 (At2g27820), ADT4 (At3g44720), and ADT5 (At5g22630) essentially only employed arogenate. ADT1, ADT2, and ADT6 had k(cat)/Km values of 1050, 7650, and 1560 M(-1) S(-1) for arogenate versus 38, 240, and 16 M(-1) S(-1) for prephenate, respectively. By contrast, the remaining three, ADT3, ADT4, and ADT5, had k(cat)/Km values of 1140, 490, and 620 M(-1) S(-1), with prephenate not serving as a substrate unless excess recombinant protein (>150 microg/assay) was used. All six genes, and their corresponding proteins, are thus provisionally classified as arogenate dehydratases and designated ADT1-ADT6.     

Second generation antipsychotics and risk of diabetes type II--comparison between olanzapine and risperidone

Differences in the glucose metabolism were examined and analysed in this study between patients treated with olanzapine and risperidone in comparison with healthy volunteers. The aim of the study was to determine differences of the impaired glucose metabolism in the study groups as well as to point out to the possible mechanisms which bring to these differences. To the group of 15 schizophrenic patients treated with olanzapine, and group of 15 schizophrenic patients treated with risperidone and to 14 healthy volunteers oral glucose tolerancy test is applied in order to determine the level of the impaired glucose tolerance. In the group of the patients treated with olanzapine glucose tolerance was impaired in 33% of the patients, while in the group of the patients treated with risperidone in 20%. Impaired glucose tolerance mostly manifested as hyperinsulinemia. Authors discussed about possible mechanisms responsible for the impaired glucose tolerance in the patients treated with new antipsychotics. Authors conclude that insulin resistance is the main mechanism for development of the diabetes type II in the schizophrenic patients treated with antipsychotics. Insulin resistance is the result of the multiple effects of the antipsychotics, among which most common are: increased body mass and direct involvement of the antipsychotics in the glucose metabolism.     

Evolutionary conflicts: rapid suppression of a male-killer

Conflicts between and within species can drive fast evolutionary change. A recent study has documented remarkably rapid counter-adaptations in the wild to an extreme sex-ratio distortion caused by a bacterial symbiont.     

Chemically inducible expression of the PHB biosynthetic pathway in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Arabidopsis plants were transformed with a multi-gene construct for expression of the polyhydroxybutyrate (PHB) biosynthetic pathway containing a gene switch that can be activated by commercially available non-steroidal ecdysone analogs approved for use on some crops as pesticides. T(1) progeny of transgenic Arabidopsis plants were isolated and screened for PHB production in the presence of ecdysone analogs. T(2) progeny derived from selected T(1) lines were subjected to further analysis by comparing PHB production levels prior to treatment with inducing agent and 21 days after initiation of induction. Significant PHB production was delayed in many of the engineered plants until after induction. PHB levels of up to 14.3% PHB per unit dry weight were observed in young leaves harvested from engineered T(2) plants after applications of the commercial ecdysone analog Mimic. PHB in older leaves reached levels of up to 7% PHB per unit dry weight. This study represents a first step towards engineering a chemically inducible gene switch for PHB production in plants using inducing agents that are approved for field use.     

Mimetics of the tri- and tetrasaccharide epitope of GQ1balpha as myelin-associated glycoprotein (MAG) ligands

The synthesis of phenoxyphenyl, phenoxybenzyl, biphenyl, and phenyltriazole substituted sialic acid derivatives as mimics of the tri- and tetrasaccharide epitopes of GQ1balpha is described. These synthetically easily available sialosides show comparable or even enhanced affinity to MAG compared with the natural tri- and tetrasaccharide epitopes and form a new class of potential MAG antagonists.     

Public information needs after the poisoning of Alexander Litvinenko with polonium-210 in London: cross sectional telephone survey and qualitative analysis

Objectives To identify public perceptions of the risk to health after the poisoning of Alexander Litvinenko with polonium-210 (²¹⁰Po) in London and to assess the impact of public health communications.Design Cross sectional telephone survey and qualitative interviews.Setting London, United Kingdom.Participants 1000 people completed the cross sectional survey and 86 potentially exposed people completed the qualitative interviews.Main outcome measures Perception of risk to personal health after the ²¹⁰Po incident. Qualitative interviews were analysed with an emphasis on information needs.Results 11.7% of the survey sample (n=117) perceived their health to be at risk. Aside from personal variables the main predictors of perceived risk to health were believing that the incident was related to terrorism (odds ratio 2.7, 95% confidence interval 1.5 to 4.6) rather than to espionage, that it was targeted at the wider public rather than one person (5.9, 3.2 to 10.9), and that it could affect people who had not been in the contaminated area (3.2, 2.1 to 5.1). Participants in the qualitative interviews were generally satisfied with the information they had received, although they would have preferred more information about their individual risk of exposure, the results of their urine tests, and the health implications of the incident.Conclusions Perceptions of the public that the ²¹⁰Po incident in London in 2006 was related to espionage helped to reassure them that the risks to personal health were low. In the event of future incidents it is important to ensure that detailed, comprehensible information about the risks of any exposure is available.     

Discovery of 5-HT6 receptor ligands based on virtual HTS

Based on a pharmacophore alignment on a 5-HT(6) ligand applying 4SCan technology, a new lead series was identified and further structurally investigated. K(i)s down to 8 nM were achieved.     

Autoantibody microarrays for biomarker discovery

Identification of autoantigens and the detection of autoantibody reactivity are useful in biomarker discovery and for explaining the role of important biochemical pathways in disease. Despite all of their potential advantages, the main challenge to working with autoantibodies is their sensitivity. Nevertheless, proteomics may hold the key to overcoming this limitation by providing the means to multiplex. Clearly, the ability to detect multiple autoantigens using a platform such as a high-density antigen microarray would improve sensitivity and specificity of detection for autoantibody profiling. The aims of this review are to: briefly describe the current status of antigen-autoantibody microarrays; provide examples of their use in biomarker discoveries; address current limitations; and provide examples and strategies to facilitate their implementation in the clinical setting.