KIN, a mammalian nuclear protein immunologically related to E. coli RecA protein

A polypeptide of about 120 kDa, called KIN, has been identified in rat FR 3T3 cells by immunoblotting using affinity-purified antibodies against the RecA protein of Escherichia coli (38 kDa). The KIN protein as shown by fluorescent light microscopy and electron microscopy is essentially concentrated in the nucleus. Its level is higher in proliferating than in quiescent cells. Cell treatment with mitomycin C increases the level of the KIN protein. We sought similar proteins in other mammalian cells. Proteins with the same electrophoretic mobility were detected in mouse, monkey and human cell lines as well as in rat and mouse embryos.     

The Smart Aerial Release Machine,a Universal System for Applying the Sterile Insect Technique

Background

Beyond insecticides, alternative methods to control insect pests for agriculture and vectors of diseases are needed. Management strategies involving the mass-release of living control agents have been developed, including genetic control with sterile insects and biological control with parasitoids, for which aerial release of insects is often required. Aerial release in genetic control programmes often involves the use of chilled sterile insects, which can improve dispersal, survival and competitiveness of sterile males. Currently available means of aerially releasing chilled fruit flies are however insufficiently precise to ensure homogeneous distribution at low release rates and no device is available for tsetse.

Methodology/Principal Findings

Here we present the smart aerial release machine, a new design by the Mubarqui Company, based on the use of vibrating conveyors. The machine is controlled through Bluetooth by a tablet with Android Operating System including a completely automatic guidance and navigation system (MaxNav software). The tablet is also connected to an online relational database facilitating the preparation of flight schedules and automatic storage of flight reports. The new machine was compared with a conveyor release machine in Mexico using two fruit flies species (Anastrepha ludens and Ceratitis capitata) and we obtained better dispersal homogeneity (% of positive traps, p<0.001) for both species and better recapture rates for Anastrepha ludens (p<0.001), especially at low release densities (<1500 per ha). We also demonstrated that the machine can replace paper boxes for aerial release of tsetse in Senegal.

Conclusions/Significance

This technology limits damages to insects and allows a large range of release rates from 10 flies/km² for tsetse flies up to 600 000 flies/km² for fruit flies. The potential of this machine to release other species like mosquitoes is discussed. Plans and operating of the machine are provided to allow its use worldwide.     

Automatic analysis of DNA microarray images using mathematical morphology

MOTIVATION: DNA microarrays are an experimental technology which consists in arrays of thousands of discrete DNA sequences that are printed on glass microscope slides. Image analysis is an important aspect of microarray experiments. The aim of this step is to reduce an image of spots into a table with a measure of the intensity for each spot. Efficient, accurate and automatic analysis of DNA spot images is essential in order to use this technology in laboratory routines. RESULTS: We present an automatic non-supervised set of algorithms for a fast and accurate spot data extraction from DNA microarrays using morphological operators which are robust to both intensity variation and artefacts. The approach can be summarised as follows. Initially, a gridding algorithm yields the automatic segmentation of the microarray image into spot quadrants which are later individually analysed. Then the analysis of the spot quadrant images is achieved in five steps. First, a pre-quantification, the spot size distribution law is calculated. Second, the background noise extraction is performed using a morphological filtering by area. Third, an orthogonal grid provides the first approach to the spot locus. Fourth, the spot segmentation or spot boundaries definition is carried out using the watershed transformation. And fifth, the outline of detected spots allows the signal quantification or spot intensities extraction; in this respect, a noise model has been investigated. The performance of the algorithm has been compared with two packages: ScanAlyze and Genepix, showing its robustness and precision.     

Phosphoprotein profiling by PA-GeLC-MS/MS

A significant consequence of protein phosphorylation is to alter protein-protein interactions, leading to dynamic regulation of the components of protein complexes that direct many core biological processes. Recent proteomic studies have populated databases with extensive compilations of cellular phosphoproteins and phosphorylation sites and a similarly deep coverage of the subunit compositions and interactions in multiprotein complexes. However, considerably less data are available on the dynamics of phosphorylation, composition of multiprotein complexes or that define their interdependence. We describe a method to identify candidate phosphoprotein complexes by combining phosphoprotein affinity chromatography, separation by size, denaturing gel electrophoresis, protein identification by tandem mass spectrometry, and informatics analysis. Toward developing phosphoproteome profiling, we have isolated native phosphoproteins using a phosphoprotein affinity matrix, Pro-Q Diamond resin (Molecular Probes-Invitrogen). This resin quantitatively retains phosphoproteins and associated proteins from cell extracts. Pro-Q Diamond purification of a yeast whole cell extract followed by 1-D PAGE separation, proteolysis and ESI LC-MS/MS, a method we term PA-GeLC-MS/MS, yielded 108 proteins, a majority of which were known phosphoproteins. To identify proteins that were purified as parts of phosphoprotein complexes, the Pro-Q eluate was separated into two fractions by size, <100 kDa and >100 kDa, before analysis by PAGE and ESI LC-MS/MS and the component proteins queried against databases to identify protein-protein interactions. The <100 kDa fraction was enriched in phosphoproteins indicating the presence of monomeric phosphoproteins. The >100 kDa fraction contained 171 proteins of 20-80 kDa, nearly all of which participate in known protein-protein interactions. Of these 171, few are known phosphoproteins, consistent with their purification by participation in protein complexes. By comparing the results of our phosphoprotein profiling with the informational databases on phosphoproteomics, protein-protein interactions and protein complexes, we have developed an approach to examining the correlation between protein interactions and protein phosphorylation.     

Fragment-based screening of the donor substrate specificity of human blood group B galactosyltransferase using saturation transfer difference NMR

Saturation transfer difference NMR experiments on human blood group B alpha-(1,3)-galactosyltransferase (GTB) for the first time provide a comprehensive set of binding epitopes of donor substrate analogs in relation to the natural donor UDP-Gal. This study revealed that the enzyme binds several UDP-activated sugars, including UDP-Glc, UDP-GlcNAc, and UDP-GalNAc. In all cases, UDP is the dominant binding epitope. To identify the minimum requirements for specific binding, a detailed analysis utilizing a fragment-based approach was employed. The binding of donor substrate to GTB is essentially controlled by the base as a "molecular anchor." Uracil represents the smallest fragment that is recognized, whereas CDP, AMP, and GDP do not exhibit any significant binding affinity for the enzyme. The ribose and beta-phosphate moieties increase the affinity of the ligands, whereas the pyranose sugar apparently weakens the binding, although this part of the molecule controls the specificity of the enzyme. Accordingly, UDP represents the best binder. The binding affinities of UDP-Gal, UDP-Glc, and UMP are about the same, but lower than that of UDP. Furthermore, we observed that beta-D-galactose and alpha-D-galactose bind weakly to GTB. Whereas beta-D-galactose binds to the acceptor and donor sites, it is suggested that alpha-D-galactose occupies a third hitherto unknown binding pocket. Finally, our experiments revealed that modulation of enzymatic activity by metal ions critically depends on the total enzyme concentration, raising the question as to which of the bivalent metal cations Mg(2+) and Mn(2+) is more relevant under physiological conditions.     

Snake population venomics and antivenomics of Bothrops atrox: Paedomorphism along its transamazonian dispersal and implications of geographic venom variability on snakebite management

We describe two geographically differentiated venom phenotypes across the wide distribution range of Bothrops atrox, from the Colombian Magdalena Medio Valley through Puerto Ayacucho and El Paují, in the Venezuelan States of Amazonas and Orinoquia, respectively, and S?o Bento in the Brazilian State of Maranh?o. Colombian and Venezuelan venoms show an ontogenetic toxin profile phenotype whereas Brazilian venoms exhibit paedomorphic phenotypes. Venoms from each of the 16 localities sampled contain both population-specific toxins and proteins shared by neighboring B. atrox populations. Mapping the molecular similarity between conspecific populations onto a physical map of B. atrox range provides clues for tracing dispersal routes that account for the current biogeographic distribution of the species. The proteomic pattern is consistent with a model of southeast and southwest dispersal and allopatric fragmentation northern of the Amazon Basin, and trans-Amazonian expansion through the Andean Corridor and across the Amazon river between Monte Alegre and Santarém. An antivenomic approach applied to assess the efficacy towards B. atrox venoms of two antivenoms raised in Costa Rica and Brazil using Bothrops venoms different than B. atrox in the immunization mixtures showed that both antivenoms immunodepleted very efficiently the major toxins (PIII-SVMPs, serine proteinases, CRISP, LAO) of paedomorphic venoms from Puerto Ayacucho (Venezuelan Amazonia) through S?o Bento, but had impaired reactivity towards PLA(2) and P-I SVMP molecules abundantly present in ontogenetic venoms. The degree of immunodepletion achieved suggests that each of these antivenoms may be effective against envenomations by paedomorphic, and some ontogenetic, B. atrox venoms.     

Genus- and species-specific identification of mycoplasmas by 16S rRNA amplification.

Systematic computer alignment of mycoplasmal 16S rRNA sequences allowed the identification of variable regions with both genus- and species-specific sequences. Species-specific sequences of Mycoplasma collis were elucidated by asymmetric amplification and dideoxynucleotide sequencing of variable regions, using primers complementary to conserved regions of 16S rRNA. Primers selected for Mycoplasma pneumoniae, M. hominis, M. fermentans, Ureaplasma urealyticum, M. pulmonis, M. arthritidis, M. neurolyticum, M. muris, and M. collis proved to be species specific in the polymerase chain reaction. The genus-specific primers reacted with all mycoplasmal species investigated as well as with members of the genera Ureaplasma, Spiroplasma, and Acholeplasma. No cross-reaction was observed with members of the closely related genera Streptococcus, Lactobacillus, Bacillus, and Clostridium or with any other microorganism tested. On the basis of the high copy number of rRNA, a highly sensitive polymerase chain reaction assay was developed in which the nucleic acid content equivalent to a single organism could be detected.