High-performance thin-layer chromatography-bioautography for multiple antibiotic residues in cow's milk

An analytical method to identify and quantify multiple antibiotic residues (chloramphenicol, ampicillin, benzylpenicillin, dicloxacillin and erythromycin) in cow's milk by high-performance thin-layer chromatography (HPTLC) combined with bioautography was developed. The test microorganism used for bioautography was Bacillus subtilis ATCC 6633. Antibiotic residues were extracted with acetonitrile, fat eliminated with petroleum ether and residues isolated with dichloromethane The sensitivity of the method guarantees the detection of the above-mentioned antibiotics at levels below maximum residue limits (MRL) allowed for milk. Percentage recoveries ranged between 90 and 100%, with coefficients of variation between 7.2 and 21.3%. Some advantages of this methodology over thin-layer chromatography (TLC)/bioautography are also discussed.     

The 37 kDa/67 kDa laminin receptor is required for PrP(Sc) propagation in scrapie-infected neuronal cells

The accumulation of PrP^Sc in scrapie-infected neuronal cells has been prevented by three approaches: (i) transfection of ScMNB cells with an antisense laminin receptor precursor (LRP) RNA-expression plasmid, (ii) transfection of ScN2a cells and ScGT1 cells with small interfering RNAs (siRNAs) specific for the LRP mRNA, and (iii) incubation of ScN2a cells with an anti-LRP/LR antibody. LRP antisense RNA and LRP siRNAs reduced LRP/LR expression and inhibited the accumulation of PrP^Sc in these cells. The treatments also reduced PrP^c levels. The anti-LRP/LR antibody, W3, abolished PrP^Sc accumulation and reduced PrP^c levels after seven days of incubation. Cells remained free of PrP^Sc after being cultured for 14 additional days without the antibody, whereas the PrP^c level was restored. Our results demonstrate the necessity of the laminin receptor (LRP/LR) for PrP^Sc propagation in cultured cells and suggest that LRP/LR-specific antibodies could be used as powerful therapeutic tools in the treatment of transmissible spongiform encephalopathies.     

Mosquito larval consumption of toxic arborescent leaf-litter,and its biocontrol potential

Previously we described the mosquito larvicidal properties of decomposed leaf-litter from deciduous trees, especially the alder Alnus glutinosa (L) Gaertn., due to toxic polyphenols and other secondary compounds. To further examine the biocontrol potential of toxic leaf-litter for mosquito control, feeding rates of third-instar mosquito larvae were assessed for examples of three genera: Anopheles stephensi Liston, Aedes aegypti (L) and Culex pipiens L. (Diptera: Culicidae). When immersed in a suspension of non-toxic leaf-litter particles (approximately 0.4 mm), pre-starved larvae of all three species ingested sufficient material in 30 min to fill the anterior gut lumen (thorax plus two to three abdominal segments). Gut filling peaked after 1-2 h ingestion time, filling the intestine up to six to seven abdominal segments for Ae. aegypti, but maxima of five abdominal segments for Cx. pipiens and An. stephensi. Using three methods to quantify consumption of three materials by third-instar larvae of Ae. aegypti, the average amount of leaf-litter (non-toxic 0.4 mm particles) ingested during 3 h was determined as approximately 20 microg/larva (by dry weight and by lignin spectrophotometric assay). Consumption of humine (approximately 100 microm particles extracted from leaf-litter) during 3 h was approximately 80 microg/larva for Ae. aegypti, but only approximately 30 microg/larva for Cx. pipiens and 15 microg/larva for An. stephensi, with good concordance of determinations by dry weight and by radiometric assay. Cellulose consumption by Ae. aegypti was intermediate: approximately 40 microg/larva determined by radiometric assay. Apparent differences between the amounts of these materials ingested by Ae. aegypti larvae (humine four-fold, cellulose two-fold more than leaf-litter) may be attributed to contrasts in palatability (perhaps related to particle size or form), rather than technical discrepancies, because there was good concordance between results of both methods used to determine the amounts of humine and leaf-litter ingested. Bioassays of toxic leaf-litter (decomposed 10 months) with 4-h exposure period (ingestion time) ranked the order of sensitivity: Ae. aegypti (LC50 < 0.03 g/L) > An. stephensi (LC50 = 0.35 g/L) > Cx. pipiens (LC20 > 0.4 g/L). When immersed in the high concentration of 0.5 g/L toxic leaf-litter (0.4 mm particles), as little as 15-30 min ingestion time (exposure period) was sufficient to kill the majority of larvae of all three species, as soon as the gut lumen was filled for only the first few abdominal segments. Possibilities for mosquito larval control with toxic leaf-litter products and the need for standardized ingestion bioassays of larvicidal particles are discussed.     

Atomic structure of the major capsid protein of rotavirus: implications for the architecture of the virion

The structural protein VP6 of rotavirus, an important pathogen responsible for severe gastroenteritis in children, forms the middle layer in the triple-layered viral capsid. Here we present the crystal structure of VP6 determined to 2 A resolution and describe its interactions with other capsid proteins by fitting the atomic model into electron cryomicroscopic reconstructions of viral particles. VP6, which forms a tight trimer, has two distinct domains: a distal beta-barrel domain and a proximal alpha-helical domain, which interact with the outer and inner layer of the virion, respectively. The overall fold is similar to that of protein VP7 from bluetongue virus, with the subunits wrapping about a central 3-fold axis. A distinguishing feature of the VP6 trimer is a central Zn(2+) ion located on the 3-fold molecular axis. The crude atomic model of the middle layer derived from the fit shows that quasi-equivalence is only partially obeyed by VP6 in the T = 13 middle layer and suggests a model for the assembly of the 260 VP6 trimers onto the T = 1 viral inner layer.     

Structural polymorphism of the major capsid protein of rotavirus

Rotaviruses are important human pathogens with a triple-layered icosahedral capsid. The major capsid protein VP6 is shown here to self-assemble into spherical or helical particles mainly depending upon pH. Assembly is inhibited either by low pH (<3.0) or by a high concentration (>100 mM) of divalent cations (Ca(2+) and Zn(2+)). The structures of two types of helical tubes were determined by electron cryomicroscopy and image analysis to a resolution of 2.0 and 2.5 nm. In both reconstructions, the molecular envelope of VP6 fits the atomic model determined by X-ray crystallography remarkably well. The 3-fold symmetry of the VP6 trimer, being incompatible with the helical symmetry, is broken at the level of the trimer contacts. One type of contact is maintained within all VP6 particles (tubes and virus), strongly suggesting that VP6 assemblies arise from different packings of a unique dimer of trimers. Our data show that the protonation state and thus the charge distribution are important switches governing the assembly of macromolecular assemblies.     

Establishment and characterization of a new continuous cell line from Lutzomyia longipalpis (Diptera: psychodidae) and its susceptibility to infections with arboviruses and Leishmania chagasi

Embryonic tissue explants of the sand fly Lutzomyia longipalpis (Lutz & Neiva 1912) the main vector of Leishmania chagasi (Cunha and Chagas), were used to obtain a continuous cell line (Lulo). The tissues were seeded in MM/VP12 medium and these were incubated at 28 masculineC. The first subculture was obtained 45 days after explanting and 96 passages have been made to date. Lulo is composed of epithelioid cells, showed a 0.04 generations/hour exponential growth rate and population doubling time at 24.7 h. The cell line isoenzymatic profiles were determined by using PGI, PGM, MPI and 6-PGDH systems, coinciding with patterns obtained from the same species and colony's pupae and adults. The species karyotype characteristics were recognized (2n = 8), in which pair 1 is subtelocentric and pairs 2, 3 and 4 are metacentric. Lulo was free from bacterial, fungal, mycoplasmic and viral infection. Susceptibility to five arbovirus was determined, the same as Lulo interaction with Leishmania promastigotes.     

Novel Bread Wheat Lines Enriched in Carotenoids Carrying Hordeum chilense Chromosome Arms in the ph1b Background

The use of crop wild relative species to improve major crops performance is well established. Hordeum chilense has a high potential as a genetic donor to increase the carotenoid content of wheat. Crosses between the 7H^ch H. chilense substitution lines in wheat and the wheat pairing homoeologous1b (ph1b) mutant allowed the development of wheat-H. chilense translocation lines for both 7H^chα and 7H^chβ chromosome arms in the wheat background. These translocation lines were characterized by in situ hybridization and using molecular markers. In addition, reverse phase chromatography (HPLC) analysis was carried out to evaluate the carotenoid content and both 7H^chα∙7AL and 7AS∙7H^chβ disomic translocation lines. The carotenoid content in 7H^chα∙7AL and 7AS∙7H^chβ disomic translocation lines was higher than the wheat-7H^ch addition line and double amount of carotenoids than the wheat itself. A proteomic analysis confirmed that the presence of chromosome 7H^ch introgressions in wheat scarcely altered the proteomic profile of the wheat flour. The Psy1 (Phytoene Synthase1) gene, which is the first committed step in the carotenoid biosynthetic pathway, was also cytogenetically mapped on the 7H^chα chromosome arm. These new wheat-H. chilense translocation lines can be used as a powerful tool in wheat breeding programs to enrich the diet in bioactive compounds.     

Chromosomal Evolution and Patterns of Introgression in Helianthus

Knowledge of the nature and extent of karyotypic differences between species provides insight into the evolutionary history of the genomes in question and, in the case of closely related species, the potential for genetic exchange between taxa. We constructed high-density genetic maps of the silverleaf sunflower (Helianthus argophyllus) and Algodones Dune sunflower (H. niveus ssp. tephrodes) genomes and compared them to a consensus map of cultivated sunflower (H. annuus) to identify chromosomal rearrangements between species. The genetic maps of H. argophyllus and H. niveus ssp. tephrodes included 17 linkage groups each and spanned 1337 and 1478 cM, respectively. Comparative analyses revealed greater divergence between H. annuus and H. niveus ssp. tephrodes (13 inverted segments, 18 translocated segments) than between H. annuus and H. argophyllus (10 inverted segments, 8 translocated segments), consistent with their known phylogenetic relationships. Marker order was conserved across much of the genome, with 83 and 64% of the H. argophyllus and H. niveus ssp. tephrodes genomes, respectively, being syntenic with H. annuus. Population genomic analyses between H. annuus and H. argophyllus, which are sympatric across a portion of the natural range of H. annuus, revealed significantly elevated genetic structure in rearranged portions of the genome, indicating that such rearrangements are associated with restricted gene flow between these two species.