2002a)

2002a). named ixodegrins that display homology to variabilin, a GPIIb/IIIa antagonist from the tick and in eastern and western North America, respectively (Barbour, 1998). Humans usually acquire Lyme disease when an infected nymphal-stage sp. tick attaches and transmits the spirochete (Burgdorfer et al., 1985). and transmit other zoonotic agents Orexin 2 Receptor Agonist besides the Lyme disease spirochete, such as (both species) or (only) (Barbour, 1998). Transmission is facilitated by tick saliva that operates not only as a carrier for salivary glands. We report the full-length clone of 87 novel sequences and discuss their potential role in modulating host inflammatory and immune responses. Materials and methods Reagents All water used was of 18 M quality and was produced using a MilliQ apparatus (Millipore, Bedford, MA, USA). Organic compounds were obtained from Sigma (St. Louis, MO, USA) or as stated Orexin 2 Receptor Agonist otherwise. Ixodes pacificus Salivary gland cDNA library construction and sequencing Ticks were collected in northern California by dragging low vegetation with a tick-drag. Salivary glands were excised and kept at ?80C until use. The mRNA from two pairs of salivary glands was obtained using a Micro-Fast Track mRNA isolation kit (Invitrogen, San Diego, CA, USA) according to the manufacturers instructions. The PCR-based cDNA library was made following the instructions for the SMART cDNA library construction kit (Clontech, Palo Alto, CA, USA) as described in detail in the supplemental data in Francischetti et al. (2004b). Cycle sequencing reactions using the DTCS labeling kit (Beckman Coulter, Fullerton, CA, USA) were performed as reported (Francischetti Rabbit Polyclonal to GTF3A et al., 2004b) and can be Orexin 2 Receptor Agonist found as supplemental data at http://www.ncbi.nlm.nih.gov/projects/omes/ in the section Poisonous Animals. cDNA sequence clustering and bioinformatics Other procedures were as reported in detail in the supplemental data described in Francischetti et al (2004b) and can be found as supplemental data at http://www.ncbi.nlm.nih.gov/projects/omes/ in the section Poisonous Animals. Structural bioinformatics and molecular modeling Molecular model of the histamine-binding protein-like lipocalin gi 51011604 superimposed with the crystal structure of histamine-binding protein. The 3D-PSSM web server V2.6.0, found at http://www.sbg.bio.ic.ac.uk/ server was used to generate a model of gi 51011604 based on sequence alignment using PSI Blast, secondary structure prediction and search of a fold database of known structures (Kelley et al., 2000). Electronic version of the manuscript The electronic version of the manuscript containing figures and table with hyperlinks can be found at http://www.ncbi.nlm.nih.gov/projects/omes/, in the section Salivary transcriptomes (sialome) of vector arthropods (and are the respective vectors for in the eastern and western U.S. (Fig. 1). After attachment to the host, infected ticks transmit after 1C2 days of blood-feeding (Barbour, 1998) Orexin 2 Receptor Agonist via saliva, a secretion that contains a cocktail of bioactive molecules (Ribeiro and Francischetti, 2003). Actually, the identification of the transcripts and proteins present in the salivary gland of ticks such as (Valenzuela et al., 2002), (Santos et al., 2004), and (Nene et al., 2004) have been identified recently. Here we identified secretory genes from the salivary gland of by constructing a unidirectional PCR-based cDNA library (see Materials and Orexin 2 Receptor Agonist methods). Next, 735 cDNA were randomly sequenced followed by bioinformatics analysis that included: clustering at high stringency levels, BLAST search against the non-redundant and protein motifs databases, and submission of the translated sequences to the Signal P server (see Materials and methods). This initial approach allowed us to obtain a fingerprint of the protein families or clusters present in this particular salivary gland. Several sequences were then selected based on novelty or the protein family it assigns for and extension of their corresponding cDNA were performed until the poly A was reached. Among.