Inhibition of Cryptosporidium parvum infection of a mammalian cell culture by recombinant scFv antibodies

Two phage display antibody libraries (Tomlinson I and J) were screened against the whole oocysts of Cryptosporidium parvum to select for scFv (single chain variable fragment) antibodies. Three scFv antibodies were selected that bound to C. parvum oocysts as determined by monoclonal phage ELISA. DNA sequencing revealed that clone A11 lacked the majority of its V (H) chain. Clone B10 had a stop codon in the first framework region of the V (H) chain. We changed this stop codon to Gly by site-directed mutagenesis, and designated the variant mutB10. Clone B9 had a complete scFv gene with no internal stop codons. These antibody genes were individually subcloned into the pET-20b expression vector for soluble scFv antibody production. C. parvum infectivity was determined by infection of HCT-8 tissue culture monolayers and quantified by the foci detection method. By incubating C. parvum oocysts with individual scFv antibodies for 1 h at 37 degrees C prior to infecting the HCT-8 cells with the oocyst-scFv mixture, the infectivity of C. parvum was reduced in a dose-dependant manner. At the highest soluble scFv concentration tested (4 nmol), the mean number of infectious foci was reduced by 82%, 73% and 94% for the A11, B9 and mutB10 scFv, respectively. This inhibition of oocyst infectivity was abolished when the scFvs were exposed to boiling water. The results showed that the 3 selected scFvs bound to C. parvum oocysts, and their ability to neutralize infectivity may have potential therapeutic potential against cryptosporidiosis.     

The High-Molecular-Weight Cytochrome c Cyc2 of Acidithiobacillus ferrooxidans Is an Outer Membrane Protein

A high-molecular-weight c-type cytochrome, Cyc2, and a putative 22-kDa c-type cytochrome were detected in the membrane fraction released during spheroplast formation from Acidithiobacillus ferrooxidans. This fraction was enriched in outer membrane components and devoid of cytoplasmic membrane markers. The genetics, as well as the subcellular localization of Cyc2 at the outer membrane level, therefore make it a prime candidate for the initial electron acceptor in the respiratory pathway between ferrous iron and oxygen.     

Ethnobotanical comparison Of “Pau Brasil” (Brosimum Rubescens Taub.) forests in a Xavante Indian and a non-Xavante community in eastern Mato Grosso State, Brazil

A monodominant forest of Brosimum rubescens Taub. located in an Indian Reservation was compared with a similar forest located on a farm owned by non-Xavante settlers, in terms of its phytosociology and the patterns of plant use. In both areas, 60 (10 X 10 m) nested-plots were established in a representative portion of the forest. All woody plants were identified, and their common and scientific names and uses were recorded. The ethnobotanical study was conducted by open-interviews initially and ranking at a later stage for a total of two years of study. The Xavante people use more species, 56% of the 57 species fit in five categories of direct use while the settlers have direct use for 50% of the 44 species found in the forest. The Xavante culture has strong links with the native biodiversity, valuing the multiple use of the species while the settlers use them mostly for timber. The species with higher IVI in the phytosociological study were also the most valued in both communities. Brosimum wood is used for the making of traditional clubs by the Xavante, the fruits are edible and attract wildlife for hunting. The non-Xavante people have been heavily logging these trees for fence posts used in the large farms of the region.     

How to quantify information loss due to phase ambiguity in haplotype case-control studies

Assigning haplotypes in a case-control study is a challenging problem. We proposed a method to quantify the information loss due to missing phase information. We determined which individuals were responsible for the information loss, and calculated how much information could be gained when the ambiguous individuals could be resolved by adding additional parental information.     

Heteroplasmy and evidence for recombination in the mitochondrial control region of the flatfish Platichthys flesus

The general assumption that mitochondrial DNA (mtDNA) does not undergo recombination has been challenged recently in invertebrates. Here we present the first direct evidence for recombination in the mtDNA of a vertebrate, the flounder Platichthys flesus. The control region in the mtDNA of this flatfish is characterized by the presence of a variable number of tandem repeats and a high level of heteroplasmy. Two types of repeats were recognized, differing by two C-T point mutations. Most individuals carry a pure "C" or a pure "T" array, but one individual showed a compound "CT" array. Such a compound array is evidence for recombination in the mtDNA control region from the flounder.     

Development of a high throughput Pseudomonas aeruginosa epithelial cell adhesion assay

Pseudomonas aeruginosa colonizes the lungs of cystic fibrosis and mechanically ventilated patients by binding to specific carbohydrate residues on the surface of lung epithelial cells. Studies have shown that blocking this interaction may have therapeutic effects in vivo. To test compounds that may have an effect on the binding of P. aeruginosa to epithelial cells, we have developed a pseudomonal adhesion assay that is compatible with high throughput technology. This assay utilizes a 96-well culture plate assay and P. aeruginosa strains that have been modified to bioluminesce. This method has proven to be a rapid, sensitive and reproducible system for screening agents that inhibit bacterial adhesion.     

An optimized enumeration method for sorbitol-fermenting Bifidobacteria in water samples

With increased focus on watershed protection under the Surface Water Treatment Rule, indicators that discriminate among sources of microbial inputs (microbial source tracking) are needed to supplement the quantitative information provided by total and fecal coliform measurements for drinking water monitoring. Bifidobacteria are found in the digestive tract and feces of humans and other animals, and also in sewage. Sorbitol is a food additive used exclusively in food intended for human consumption. Therefore, the presence of sorbitol-fermenting Bifidobacteria in environmental waters can be indicative of sources of human fecal contamination. A series of media were evaluated using ATCC cultures of B. breve and B. adolescentis, feces from different animals, and domestic wastewater samples. The media evaluated were Human Bifid Sorbitol agar (HBSA), modified Human Bifid Sorbitol agar, Beerens Medium, modified Beerens Medium, Reinforced Clostridial agar, BIM-25 Medium, and modified BIM-25 Medium. Variables such as sample preservation, incubation time, different pH indicators, plating technique, and discontinuous exposure to sorbitol were also evaluated. A series of biochemical tests were used to confirm positive colonies enumerated on the various media. Membrane filtration and enumeration of sodium sulfite preserved samples on HBSA containing bromocresol purple using loose lidded plates for 48 h provided the best recoveries for presumptive positive colonies. A number of sorbitol-fermenters that were not Bifidobacteria were able to grow on all media tested, resulting in false-positives. Therefore, plating on HBSA should be followed by a confirmation step when monitoring for sorbitol-fermenting Bifidobacteria in environmental waters. A year-long sampling survey of a managed reservoir in Massachusetts provided field validation of the proposed methodology for sorbitol-fermenting Bifidobacteria as a human-related source tracking indicator tool.     

Regulation of WRN helicase activity in human base excision repair

Werner syndrome patients are deficient in the Werner protein (WRN), which is a multifunctional nuclear protein possessing 3'-5' exonuclease and ATP-dependent helicase activities. Studies of Werner syndrome cells and biochemical studies of WRN suggest that WRN plays a role in several DNA metabolic pathways. WRN interacts with DNA polymerase beta (pol beta) and stimulates pol beta strand displacement synthesis on a base excision repair (BER) intermediate in a helicase-dependent manner. In this report, we examined the effect of the major human apurinic/apyrimidinic endonuclease (APE1) and of pol beta on WRN helicase activity. The results show that WRN alone is able to unwind several single strand break BER intermediates. However, APE1 inhibits WRN helicase activity on these intermediates. This inhibition is likely due to the binding of APE1 to nicked apurinic/apyrimidinic sites, suggesting that APE1 prevents the promiscuous unwinding of BER intermediates. This inhibitory effect was relieved by the presence of pol beta. A model involving the pol beta-mediated hand-off of WRN protein is proposed based on these results.