Tissues were stained with vector red dye and photographed under a dissecting scope. A. the immature stages of development, apparently being transported into the intrauterine larva from your mother via the milk gland ducts. Transferrin is also detected in the vitellogenic ovary and the adult male testes, further supporting its classification as a vitellogenic protein. Similar to reports in other insects, transferrin mRNA levels increase upon bacterial challenge in tsetse suggesting that transferrin may play an additional role in immunity. Although transferrin expression is induced following TMC353121 bacterial challenge, it is significantly reduced in tsetse transporting midgut trypanosome infections. Analysis of tsetse that have cured the parasite challenge shows normal levels of NRAMP2 (DMT1). The molecular characterization of the 2108 bp full-length cDNA predicted a secretory protein with a molecular mass of about 72 kDa (Strickler-Dinglasan et al. 2006). The putative GmmTsf has apparently retained the signature amino acids found conserved in invertebrate transferrins and similarly lacks iron-binding residues in its C-terminal lobe when compared to vertebrate transferrins (Dunkov and Georgieva 2006; Harizanova et al. 2005; Jamroz et al. 1993; Yoshiga et al. 1999). Despite the rigid blood feeding requirement seen TMC353121 in both sexes in tsetse adults, the expression of was found to be female specific and was restricted to excess fat body/milk gland tissue portion and absent from your midgut. Here we statement on a detailed analysis of the temporal expression of mRNA and protein, as well as the tissue and sex-specific nature of its synthesis during development. Using a transferrin specific antibody generated against recombinant GmmTsf, we compare its protein levels during development in different tissues in male and female flies and further localize its synthesis via immunohistochemical analysis. We also statement around the immune-related expression profile of from pathogen challenged flies and from flies with midgut trypanosome infections. We discuss the implications TMC353121 of our findings with respect to transferrins postulated role as an iron-binding, vitellogenic and immune-responsive protein. 2. Materials and methods 2.1. Biological material The (cultured 105 Ytat1.1 procyclic form parasites/ml. Newly emerged teneral flies also were given a blood meal made up of 105 K12 cells. 2.2. Northern blot analysis Newly eclosed females were mated at day 5 and collected in groups of three per day for the 30 day time course and snap frozen in liquid nitrogen. Total RNA was isolated from individual flies using Trizol Reagent (Invitrogen, Carlsbad, CA) according to manufacturers instructions. Ten micrograms of RNA from each sample was analyzed on a 1.5% agarose/formaldehyde gel and transferred to a nylon membrane (Hybond-N+, Amersham Biosciences, Piscataway, NJ) by capillary blotting. Probes were generated by PCR Dig Probe Synthesis Kit (Roche Applied Science, Indianapolis, IN) utilizing gene specific primers probe as an internal loading control and hybridization signals for were normalized to the transmission using Kodak 1D 3. 6. 1. Imaging Software. To analyze expression in mothers and their intrauterine larvae during the course of pregnancy, offspring were dissected from your uterus of the pregnant females during different stages of pregnancy; i.e., 1st instar larvae, 2nd instar larvae and 3rd instar larvae, and RNA was isolated using the TRIzol? reagent (Invitrogen, Carlsbad, CA). Tissue specific expression analysis was accomplished using midgut, fatbody/milk gland, reproductive tract and carcass dissected from mated flies during all stages of the reproductive cycle. Microscopically dissected tissues were collected in phosphate buffered saline (pH 7.4) and RNA Rabbit Polyclonal to CSGLCAT was isolated using the TRIzol protocol. To detect impact of trypanosome contamination on transcript large quantity, trypanosome infections were established in adult flies by challenging newly emerged (teneral) flies with a blood meal made up of 1106 Ytat 1.1 cells/ml supplemented with 0.05 M N-acetyl glucosamine. At 22-24 days post contamination and 48 hr post routine blood feeding, midguts were dissected and microscopically examined for parasite infections and RNA was.
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