Biology of Ixodes (Pholeoixodes) hexagonus under laboratory conditions Part II. Effect of mating on feeding and fecundity of females

The effect of mating on the feeding and fecundity ofIxodes (Pholeoixodes) hexagonus females was studied under controlled laboratory conditions of 22–23°C and 98% relative humidity. The feeding period of mated females was 6–15 days and 11–13 days for unmated females. The mean weight of the engorged mated females was 114.84±45.89 mg, whereas, that of the engorged unmated females was significantly lower (80.61±28.84 mg). During the initial slow feeding period, the weight of mated females increased 6.6 times. At the end of the blood feeding, they had increased their initial weight 35.5 times. Unmated females never entered the rapid engorgement phase and up to 12 days of feeding period their mean weight did not increase more than 9.2 times. The pre-oviposition periods of mated and unmated females were 6–15 days and 4–12 days, respectively. The mean of the egg production efficiency was 40.26±12.47% for mated females and 35.68±12.2% for unmated females. The mean of the mass conversion efficiency was 73.6±13.7% for mated females and 66.48 ±16.55% for unmated females. Sixty per cent of the eggs deposited by mated females hatched whereas only 1% of the eggs deposited by unmated females hatched. These results indicate thatI. hexagonus females possess some predisposition for parthenogenesis and only fertility and not fecundity depends on mating.     

Preparation of soybean seed samples for FT-IR microspectroscopy

Typical preparation of seed samples for infrared (IR) microspectroscopy involves imbibition of the seed for varying time periods followed by cryosectioning. Imbibition, however, may initiate germination even at 4° C with associated changes in the chemistry of the sample. We have found that it is possible to section seeds that are sufficiently hard, such as soybeans, on a standard laboratory microtome without imbibition. The use of dry sectioning of unimbibed seeds is reported here, as well as a comparison of different mounting media and modes of analysis. Glycerol, Tissue-Tek, and ethanol were used as mounting media, and the quality of the resulting spectra was assessed. Ethanol was the preferred mountant, because it dried quickly with no residue and thus did not interfere with the spectrum of interest. Analysis in transmission mode using barium fluoride windows to hold the samples was compared with transmission-reflection analysis with sections mounted on special infrared-reflecting slides. The two modes of analysis performed well in different regions of the spectrum. The mode of analysis (transmission vs. transmission-reflection) should be based on the components of greatest interest in the sample.     

Ability of transovarially and subsequent transstadially infected Ixodes hexagonus ticks to maintain and transmit Borrelia burgdorferi in the laboratory

In a previous study, transstadial and transovarial survival of Borrelia burgdorferi in Ixodes hexagonus and transmission to laboratory mice via the bite of infected females were demonstrated. Here, we report the ability of I. hexagonus progeny infected transovarially to maintain and transmit the spriochaete to the host.Ticks were examined for spirochaetes by direct immunofluorescence antibody test. I. hexagonus larvae derived from the parental transstadially infected females were fed on two white mice: 21/54 (38.9%) of these ticks examined as unfed nymphs were infected. I. hexagonus nymphs were fed on three white mice and examined for spirochaetes after moulting as adults: 7/25 (28%) were found to harbour the spirochaete. The success of B. burgdorferi transmission to the mice by larval and nymphal I. hexagonus was determined by xenodiagnosis using I. ricinus larvae: 20/50 (40%) and 30/99 (30.3%) of the I. ricinus larvae fed on the mice infected by I. hexagonus larvae and nymphs respectively became infected.This study shows that B. burgdorferi can be maintained through transovarial and subsequent transstadial transmissions in I. hexagonus.     

Anti-tumor therapy with macroencapsulated endostatin producer cells

Background  

Theracyte is a polytetrafluoroethylene membrane macroencapsulation system designed to induce neovascularization at the tissue interface, protecting the cells from host's immune rejection, thereby circumventing the problem of limited half-life and variation in circulating levels. Endostatin is a potent inhibitor of angiogenesis and tumor growth. Continuous delivery of endostatin improves the efficacy and potency of the antitumoral therapy. The purpose of this study was to determine whether recombinant fibroblasts expressing endostatin encapsulated in Theracyte immunoisolation devices can be used for delivery of this therapeutic protein for treatment of mice bearing B16F10 melanoma and Ehrlich tumors.     

A mutagenic PCR identifies isolates of Borrelia garinii responsible for Lyme borreliosis

Borrelia garinii is one of the three major Borreliae responsible for Lyme borreliosis in Europe. We have characterized a protein of B. garinii (VS102) and a genomic fragment from the gene encoding this protein was cloned. The DNA sequence of the fragment showed high homology with a known gene of B. burgdorferi sensu stricto. The protein encoded by this gene in B. burgdorferi sensu stricto is a phosphocarrier protein (histidine-containing protein). A mutation T to G polymorphism at codon 57 was found to be specific to B. garinii. A PCR-based approach that allows the rapid detection of this mutation made it possible to specifically discriminate B. garinii from other B. burgdorferi genospecies with high sensitivity and specificity.     

Single-nucleotide polymorphism,linkage disequilibrium and geographic structure in the malaria parasite <Emphasis Type="Italic">Plasmodium vivax</Emphasis>: prospects for genome-wide association studies

Background

The ideal malaria parasite populations for initial mapping of genomic regions contributing to phenotypes such as drug resistance and virulence, through genome-wide association studies, are those with high genetic diversity, allowing for numerous informative markers, and rare meiotic recombination, allowing for strong linkage disequilibrium (LD) between markers and phenotype-determining loci. However, levels of genetic diversity and LD in field populations of the major human malaria parasite P. vivax remain little characterized.

Results

We examined single-nucleotide polymorphisms (SNPs) and LD patterns across a 100-kb chromosome segment of P. vivax in 238 field isolates from areas of low to moderate malaria endemicity in South America and Asia, where LD tends to be more extensive than in holoendemic populations, and in two monkey-adapted strains (Salvador-I, from El Salvador, and Belem, from Brazil). We found varying levels of SNP diversity and LD across populations, with the highest diversity and strongest LD in the area of lowest malaria transmission. We found several clusters of contiguous markers with rare meiotic recombination and characterized a relatively conserved haplotype structure among populations, suggesting the existence of recombination hotspots in the genome region analyzed. Both silent and nonsynonymous SNPs revealed substantial between-population differentiation, which accounted for ~40% of the overall genetic diversity observed. Although parasites clustered according to their continental origin, we found evidence for substructure within the Brazilian population of P. vivax. We also explored between-population differentiation patterns revealed by loci putatively affected by natural selection and found marked geographic variation in frequencies of nucleotide substitutions at the pvmdr-1 locus, putatively associated with drug resistance.

Conclusion

These findings support the feasibility of genome-wide association studies in carefully selected populations of P. vivax, using relatively low densities of markers, but underscore the risk of false positives caused by population structure at both local and regional levels.See commentary: http://www.biomedcentral.com/1741-7007/8/90

     

A xyloglucan-specific endo-beta-1,4-glucanase from Aspergillus aculeatus: expression cloning in yeast, purification and characterization of the recombinant enzyme

A full-length c-DNA encoding a xyloglucan-specific endo -beta-1, 4- glucanase (XEG) has been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast. The colonies expressing functional XEG were identified on agar plates containing azurine-dyed cross-linked xyloglucan. The cDNA encoding XEG was isolated, sequenced, cloned into an Aspergillus expression vector, and transformed into Aspergillus oryzae for heterologous expression. The recombinant enzyme was purified to apparent homogeneity by anion- exchange and gel permeation chromatography. The recombinant XEG has a molecular mass of 23,600, an isoelectric point of 3.4, and is optimally stable at a pH of 3.4 and temperature below 30 degreesC. The enzyme hydrolyzes structurally diverse xyloglucans from various sources, but hydrolyzes no other cell wall component and can therefore be considered a xyloglucan-specific endo -beta-1, 4-glucanohydrolase. XEG hydrolyzes its substrates with retention of the anomeric configuration. The Kmof the recombinant enzyme is 3.6 mg/ml, and its specific activity is 260 micromol/min per mg protein. The enzyme was tested for its ability to solubilize xyloglucan oligosaccharides from plant cell walls. It was shown that treatment of plant cell walls with XEG yields only xyloglucan oligosaccharides, indicating that this enzyme can be a powerful tool in the structural elucidation of xyloglucans.