Extensive multiple test centre evaluation of the VecTest malaria antigen panel assay

To determine which species and populations of Anopheles transmit malaria in any given situation, immunological assays for malaria sporozoite antigen can replace traditional microscopical examination of freshly dissected Anopheles. We developed a wicking assay for use with mosquitoes that identifies the presence or absence of specific peptide epitopes of circumsporozoite (CS) protein of Plasmodium falciparum and two strains of Plasmodium vivax (variants 210 and 247). The resulting assay (VecTest Malaria) is a rapid, one-step procedure using a 'dipstick' test strip capable of detecting and distinguishing between P. falciparum and P. vivax infections in mosquitoes. The objective of the present study was to test the efficacy, sensitivity, stability and field-user acceptability of this wicking dipstick assay. In collaboration with 16 test centres world-wide, we evaluated more than 40 000 units of this assay, comparing it to the standard CS ELISA. The 'VecTest Malaria' was found to show 92% sensitivity and 98.1% specificity, with 97.8% accuracy overall. In accelerated storage tests, the dipsticks remained stable for > 15 weeks in dry conditions up to 45 degrees C and in humid conditions up to 37 degrees C. Evidently, this quick and easy dipstick test performs at an acceptable level of reliability and offers practical advantages for field workers needing to make rapid surveys of malaria vectors.     

Glutaredoxins catalyze the reduction of glutathione by dihydrolipoamide with high efficiency

Glutaredoxins (Grx) are small (approximately 12kDa) proteins which catalyze thiol disulfide oxidoreductions involving glutathione (GSH) and disulfides in proteins or small molecules. Here, we present data which demonstrate the ability of glutaredoxins to catalyze the reduction of oxidized glutathione (GSSG) by dihydrolipoamide (DHL), an important biological redox catalyst and synthetic antioxidant. We have designed a new assay method to quantify the rate of reduction of GSSG and other disulfides by reduced lipoamide and have tested a set of eight recombinant Grx from human, rat, yeast, and E. coli. Lipoamide dependent activity is highest with the large atypical E. coli Grx2 (k(cat)=3.235 min(-1)) and lowest for human mitochondrial Grx2a (k(cat)=96 min(-1)) covering a wider range than k(cat) for the standard reduction of hydroxyethyldisulfide (HED) by GSH (290-2.851 min(-1)). The lipoamide/HED activity ratio was highest for yeast Grx2 (1.25) and E. coli Grx2 and lowest for E. coli Grx1 (0.13). These results suggest a new role for Grxs as ancillary proteins that could shunt reducing equivalents from main catabolic pathways to recycling of GSSG via a lipoyl group, thus serving biochemical functions which involve GSH but without NAD(P)H consumption.     

Morphologic differentiation of HL-60 cells is associated with appearance of RPTPbeta and induction of Helicobacter pylori VacA sensitivity

Phorbol 12-myristate 13-acetate (PMA) induces differentiation of human leukemic HL-60 cells into cells with macrophage-like characteristics and enhances the susceptibility of HL-60 cells to the Helicobacter pylori VacA toxin (de Bernard, M., Moschioni., M., Papini, E., Telford, J. L., Rappuoli, R., and Montecucco, C. (1998) FEBS Lett. 436, 218-222). We examined the mechanism by which HL-60 cells acquire sensitivity to VacA, in particular, looking for expression of RPTPbeta, a VacA-binding protein postulated to be the VacA receptor (Yahiro, K., Niidome, T., Kimura, M., Hatakeyama, T., Aoyagi, H., Kurazono, H., Imagawa, K., Wada, A., Moss, J., and Hirayama, T. (1999) J. Biol. Chem. 274, 36693-36699). PMA induced expression of RPTPbeta mRNA and protein as determined by RNase protection assay and indirect immunofluorescence studies, respectively. Vitamin D(3) and interferon-gamma, which stimulate differentiation of HL-60 cells into monocyte-like cells, also induced VacA sensitivity and expression of RPTPbeta mRNA, whereas 1. 2% Me(2)SO and retinoic acid, which stimulated the maturation of HL-60 into granulocyte-like cells, did not. RPTPbeta antisense oligonucleotide inhibited induction of VacA sensitivity and expression of RPTPbeta. Double immunostaining studies also indicated that newly expressed RPTPbeta colocalized with VacA in PMA-treated HL-60 cells. In agreement with these data, BHK-21 cells, which are insensitive to VacA, when transfected with the RPTPbeta cDNA, acquired VacA sensitivity. All data are consistent with the conclusion that acquisition of VacA sensitivity by PMA-treated HL-60 cells results from induction of RPTPbeta, a protein that functions as the VacA receptor.     

Isolation and cloning of Streptomyces terminal fragments

Streptomyces species have a linear chromosome of approximately 8 Mb in size. Many strains also carry linear plasmids. Most of these linear elements contain terminal proteins covalently bound to the 5 ends of the DNA. Using a method for the visualisation of terminal DNA fragments in agarose gels, it was possible to see three fragments in S. rimosus and five fragments in S. avermitilis. The method was also used to clone the 298 bp BamHI fragment carrying the left end of plasmid SLP2. Analysis of the sequence showed that the end resembled other Streptomyces chromosome and plasmid ends, but there were eight palindromes (instead of seven) and a tandem duplication of a 14 bp sequence.     

Solution structure of the recombinant human oncoprotein p13 MTCP1

The human oncoprotein p13 ^MTCP1 is coded by the MTCP1 gene, a gene involved in chromosomal translocations associated with T-cell prolymphocytic leukemia, a rare form of human leukemia with a mature T-cell phenotype. The primary sequence of p13 ^MTCP1 is highly and only homologous to that of p14 ^TCL1 , a product coded by the gene TCL1 which is also involved in T-cell prolymphocytic leukemia. These two proteins probably represent the first members of a new family of oncogenic proteins. We present the three-dimensional solution structure of the recombinant p13 ^MTCP1 determined by homonuclear proton two-dimensional NMR methods at 600 MHz. After proton resonance assignments, a total of 1253 distance restraints and 64 dihedral restraints were collected. The solution structure of p13 ^MTCP1 is presented as a set of 20 DYANA structures. The rmsd values with respect to the mean structure for the backbone and all heavy atoms for the conformer family are 1.07 ± 0.19 and 1.71 ± 0.17 Å, when the structured core of the protein (residues 11–103) is considered. The solution structure of p13 ^MTCP1 consists of an orthogonal -barrel, composed of eight antiparallel -strands which present an original arrangement. The two -pleated loops which emerge from this barrel might constitute the interaction surface with a potential molecular partner.     

Isolation and characterization of a novel human bladder cancer cell line: BK10

Summary Molecular studies of bladder carcinomas have aided in determining causative genetic events and the prognosis of cancers endowed with certain abnormalities. In vitro bladder cancer characterization of key cytogenetic alterations is useful for study of molecular changes that may promote oncogenic events. In our laboratory, a novel human bladder cancer cell line, BK10, has been established in vitro and passaged for more than 20 mo. This new bladder cancer cell line (BK10) was derived from bladder tissue containing grade III-IV/IV transitional cell carcinoma. Bladder cancer tissue was obtained at the time of radical cystoprostatectomy extirpation. Cell cultures derived from this surgical sample exhibited an epithelial morphology and expressed epithelial cytokeratins. Immunostains of BK10 were negative for prostate specific antigen (PSA), fibronectin, smooth muscle actin alpha, and desmin. Karyotypic analysis revealed an aneuploid chromosomal content 〈4n〉 with many numerical and structural abnormalities previously linked to bladder oncogenesis. Translocations occurred in chromosomes 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 13, 14, 15, 16, 17, 19, 20, 21, 22, X and Y. G-banding analysis revealed rearrangements involving chromosomes 9q and 17p, and the location of the ab11 oncogene and the p53 gene, respectively. The availability of this bladder cancer cell line will provide a useful too for the further study of bladder carcinoma oncogenesis and gene therapy.     

Population history,gene flow,and bottlenecks in island populations of a secondary seed disperser,the southern grey shrike (Lanius meridionalis koenigi)

Studying the population history and demography of organisms with important ecological roles can aid understanding of evolutionary processes at the community level and inform conservation. We screened genetic variation (mtDNA and microsatellite) across the populations of the southern grey shrike (Lanius meridionalis koenigi) in the Canary Islands, where it is an endemic subspecies and an important secondary seed disperser. We show that the Canarian subspecies is polyphyletic with L. meridionalis elegans from North Africa and that shrikes have colonized the Canary Islands from North Africa multiple times. Substantial differences in genetic diversity exist across islands, which are most likely the product of a combination of historical colonization events and recent bottlenecks. The Eastern Canary Islands had the highest overall levels of genetic diversity and have probably been most recently and/or frequently colonized from Africa. Recent or ongoing bottlenecks were detected in three of the islands and are consistent with anecdotal evidence of population declines due to human disturbance. These findings are troubling given the shrike's key ecological role in the Canary Islands, and further research is needed to understand the community‐level consequences of declines in shrike populations. Finally, we found moderate genetic differentiation among populations, which largely reflected the shrike's bottleneck history; however, a significant pattern of isolation‐by‐distance indicated that some gene flow occurs between islands. This study is a useful first step toward understanding how secondary seed dispersal operates over broad spatial scales.     

Physical Activity Differentially Affects the Cecal Microbiota of Ovariectomized Female Rats Selectively Bred for High and Low Aerobic Capacity

The gut microbiota is considered a relevant factor in obesity and associated metabolic diseases, for which postmenopausal women are particularly at risk. Increasing physical activity has been recognized as an efficacious approach to prevent or treat obesity, yet the impact of physical activity on the microbiota remains under-investigated. We examined the impacts of voluntary exercise on host metabolism and gut microbiota in ovariectomized (OVX) high capacity (HCR) and low capacity running (LCR) rats. HCR and LCR rats (age = 27wk) were OVX and fed a high-fat diet (45% kcal fat) ad libitum and housed in cages equipped with (exercise, EX) or without (sedentary, SED) running wheels for 11wk (n = 7-8/group). We hypothesized that increased physical activity would hinder weight gain, increase metabolic health and shift the microbiota of LCR rats, resulting in populations more similar to that of HCR rats. Animals were compared for characteristic metabolic parameters including body composition, lipid profile and energy expenditure; whereas cecal digesta were collected for DNA extraction. 16S rRNA gene-based amplicon Illumina MiSeq sequencing was performed, followed by analysis using QIIME 1.8.0 to assess cecal microbiota. Voluntary exercise decreased body and fat mass, and normalized fasting NEFA concentrations of LCR rats, despite only running one-third the distance of HCR rats. Exercise, however, increased food intake, weight gain and fat mass of HCR rats. Exercise clustered the gut microbial community of LCR rats, which separated them from the other groups. Assessments of specific taxa revealed significant (p<0.05) line by exercise interactions including shifts in the abundances of Firmicutes, Proteobacteria, and Cyanobacteria. Relative abundance of Christensenellaceae family was higher (p = 0.026) in HCR than LCR rats, and positively correlated (p<0.05) with food intake, body weight and running distance. These findings demonstrate that exercise differentially impacts host metabolism and gut microbial communities of female HCR and LCR rats without ovarian function.     

S100 to receptor for advanced glycation end-products binding assay: Looking for inhibitors

Secreted by tumor and stromal cells, S100 proteins exert their biological functions via the interaction with surface receptors. The most described receptor is the receptor for advanced glycation end-products (RAGE), thereby participating in the S100-dependent cell migration, invasion, tumor growth, angiogenesis and metastasis. Several approaches have been described for determining this interaction. Here we describe an easy, specific and highly reproducible ELISA-based method, by optimizing several parameters such as the binding and blocking buffer, interaction time and concentrations, directed to screen chemical and biological inhibitors of this interaction for S100A4, S100A7 and S100P proteins. The efficiency of the protocol was validated by using well described neutralizing agents of the RAGE receptor and of the S100A4 activity. The methodology described here will allow future works with other members of the S100 protein family and their receptors.