Eight-week-old normal male or osteoprotegerin knockout mice were used for the LPS experiments

nt plasmid with gene specific primers containing the T7 promoter recognition sites . The PCR product was purified using a Gene Jet PCR Purification Kit and used to synthesize dsRNA with a T7 Ribo Max Express RNA Kit according to the manufacturer’s protocol. The dsRNA synthesis 6 / 20 ABC-Mediated Heme and Pesticide Detoxification was evaluated by 1.5% agarose gel electrophoresis, and the concentration was determined order Oleandrin spectrophotometrically at 260 nm in a NanoDrop 1000 instrument. The dsRNA of a tick-unrelated gene, MSP1 from Plasmodium falciparum, was used as a negative control. This gene was obtained from a plasmid kindly provided by Dr. Gerhard Wunderlich. The 1110 bp sequence was recovered after digestion of the plasmids with Fast Digest SpEI, and the dsRNA of MSP1 was synthesized as described above. Partially engorged tick females of the POA strain were manually removed from experimentally infested bovines. Groups of 15 tick females were immobilized on a glass plate covered with double-sided adhesive tape, and dsRNA solutions were injected into the hemocoel. These ticks were artificially fed using microhematocrit capillary tubes filled with blood from non-infested bovines collected in the presence of sodium citrate. Initially, females were fed with 50 L of blood supplemented with either the fluorescent heme analog ZnPP diluted in DMSO or DMSO alone, as a control for autofluorescence. After this initial meal, the females were fed until repletion with blood alone, without metalloporphyrin or DMSO. The females were allowed to feed for approximately 28 h, and then kept in separate vials at 2728C and 8090% relative humidity. After 24 h or 48 h after feeding, the females were dissected, and the midguts were collected for RNA PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19757813 extraction and microscopy. Statistical analysis Multiple comparisons were performed by a one-way ANOVA analysis of variance and an a posteriori Tukey’s test for pair-wise comparisons. Single comparisons were performed PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19754877 by T test analysis with Mann-Whitney test, using GraphPad Prism version 4.0 for Windows. Results Digest cells of the tick midgut degrade hemoglobin and direct heme into an intracellular pathway that involves digestive vacuoles and ultimately leads to heme accumulation in hemosomes. To test whether this heme intracellular transport is dependent on an ABC transporter, digest cells isolated from the midgut of R. microplus were incubated with Rhodamine 123, a PgP protein transporter substrate that has been validated for membrane transport studies. The fluorescent dye was found to be associated with both digestive vacuoles and hemosomes after incubation. Furthermore, uptake was markedly inhibited by preincubation with 10M CsA, a commonly used ABC inhibitor, which suggests the participation of a transporter of this type. This conclusion was also supported using immunocytochemistry with a commercially available polyclonal antibody against vertebrate PgP-1 ABC-transporter. The anti-PgP-1 antibody specifically labeled digest cell membranes. Further demonstration of the presence of an ABC ATPase activity in the digestive vesicle membrane was obtained by a cerium-based histochemical method. In this method, phosphate released by ATPase activity complex with cerium, a heavy metal atom whose phosphate salt is highly insoluble. Digestive vesicles exhibited high ATPase activity associated with their membranes which is inhibited by CsA. Some residual activity, however, was still found even in the presence of CsA. Hemosome