The effect of purified condensed tannins of forage plants from Botswana on the free-living stages of gastrointestinal nematode parasites of livestock.

The effect of condensed tannins (CT) extracted from forage plants from Botswana on the free-living stages of a number of species of gastrointestinal nematode parasites derived from infected sheep were investigated using in vitro assays. Fresh samples of five different plants (Viscum rotundifolium, Viscum verrucosum, Tapinanthus oleifolius, Grewia flava and Ipomoea sinensis) were collected over two summers (February 2009 and 2010). Fractionation of each crude extract on a Sephadex LH-20 column yielded low molecular weight phenolics and CT-containing fractions. The effect of each purified CT fraction on parasites was evaluated using either egg hatch, larval development or larval migration inhibition assays. Three gastrointestinal nematode species (Haemonchus contortus, Trichostrongylus colubriformis and Teladorsagia circumcincta) derived from infected sheep were evaluated in the study. CT from V. rotundifolium and I. sinensis fractions from samples collected in 2009 and 2010 did not inhibit larval development. However, CT isolated from V. verrucosum, T. oleifolius and G. flava collected in 2009 completely inhibited the development of all parasite species. These CT fractions were more potent in inhibiting larval development of H. contortus than fractions from the same plant species collected in 2010. However, a slight effect on larval migration was observed with some CT extracts. The results suggest that CT extracts of some forage plants from Botswana have anti-parasitic properties in vitro, and that further research is required to determine any in vivo efficacy from feeding the plants to goats in a field situation.

Condensed tannins from Botswanan forage plants are effective priming agents of γδ T cells in ruminants.

The potential impact of extracts from forage plants on γδ T cell activity in ruminants was evaluated using an in vitro immunoassay. This study investigated whether plant extracts could prime γδ T cells via up-regulation of CD25 (interleukin-2 receptor alpha). Purified Sephadex LH-20 fractions, isolated from Viscum rotundifolium, Viscum verrucosum, Tapinanthus oleifolius and Grewia flava, were screened against γδ T cells on kid, lamb and calf peripheral blood lymphocytes. Condensed tannins (CT) from G. flava significantly primed γδ T cells in kids up to 64.75% at 10 µg/mL, which was statistically significant relative to the negative control at 22.66% (p=0.004). CT from T. oleifolius also induced priming of γδ T cells in kids, while fractions from V. rotundifolium and V. verrucosum induced minimal priming of γδ T cells. In contrast, there was no significant priming of γδ T cells from lambs and calves for any of the tested fractions (p>0.05). These findings suggest that CT from a selected range of Botswanan forage plants can stimulate the immune system in vivo in selected ruminant species and may participate in enhancing host innate immune responses.

Use of capillary (zone) electrophoresis for determining felinine and it's application to investigate the stability of felinine.

A rapid capillary electrophoresis method was established to quantify felinine (2-amino-7-hydroxy-5,5-dimethyl-4-thiaheptanoic acid) in cat urine and used to investigate felinine stability. Synthetic felinine was stable in the presence of oxygen while 11% of the natural felinine in urine disappeared after 4 days exposure to air. Both synthetic felinine and the natural felinine (in urine) were stable for up to 3 months when stored at -5 degrees C and 20 degrees C. Thirty percent of the synthetic felinine was lost after 5 hours at 100 degrees C while 95% of the natural felinine disappeared after only 2 hours at the same temperature. The recovery of felinine under certain conditions was greater than 100%. It is possible that acetyl-felinine may be present in the urine and that it is deacetylated during incubation. Overall synthetic felinine was found to be stable but the felinine in cat urine much less so. Other compounds present in the urine may contribute to the decomposition of felinine.

Chemosensitization of fluconazole resistance in Saccharomyces cerevisiae and pathogenic fungi by a D-octapeptide derivative.

Hyperexpression of the Saccharomyces cerevisiae multidrug ATP-binding cassette (ABC) transporter Pdr5p was driven by the pdr1-3 mutation in the Pdr1p transcriptional regulator in a strain (AD/PDR5(+)) with deletions of five other ABC-type multidrug efflux pumps. The strain had high-level fluconazole (FLC) resistance (MIC, 600 microg ml(-1)), and plasma membrane fractions showed oligomycin-sensitive ATPase activity up to fivefold higher than that shown by fractions from an isogenic PDR5-null mutant (FLC MIC, 0.94 microg ml(-1)). In vitro inhibition of the Pdr5p ATPase activity and chemosensitization of cells to FLC allowed the systematic screening of a 1.8-million-member designer D-octapeptide combinatorial library for surface-active Pdr5p antagonists with modest toxicity against yeast cells. Library deconvolution identified the 4-methoxy-2,3,6-trimethylbenzensulfonyl-substituted D-octapeptide KN20 as a potent Pdr5p ATPase inhibitor (concentration of drug causing 50% inhibition of enzyme activity [IC(50)], 4 microM) which chemosensitized AD/PDR5(+) to FLC, itraconazole, and ketoconazole. It also inhibited the ATPase activity of other ABC transporters, such as Candida albicans Cdr1p (IC(50), 30 microM) and Cdr2p (IC(50), 2 microM), and chemosensitized clinical isolates of pathogenic Candida species and S. cerevisiae strains that heterologously hyperexpressed either ABC-type multidrug efflux pumps, the C. albicans major facilitator superfamily-type drug transporter Ben(R)p, or the FLC drug target lanosterol 14 alpha-demethylase (Erg11p). Although KN20 also inhibited the S. cerevisiae plasma membrane proton pump Pma1p (IC(50), 1 microM), the peptide concentrations required for chemosensitization made yeast cells permeable to rhodamine 6G. KN20 therefore appears to indirectly chemosensitize cells to FLC by a nonlethal permeabilization of the fungal plasma membrane.