Samples for Western analysis were prepared by boiling purified EBs of strains AR39 and CWL029 for 5 min in the protein sample buffer

Samples for Western analysis were prepared by boiling purified EBs of strains AR39 and CWL029 for 5 min in the protein sample buffer. that more readily infects diseased arteries (8) or that its association with vascular disease is confounded by its association with other atherosclerotic risk factors (13). Since seroepidemiological studies indicate that most people are infected by by the age at BTRX-335140 which the clinical manifestations of atherosclerosis usually appear (11), it is likely that if is a causal agent for vascular disease, there is variability in the characteristics of the organism, the host response to infection, or both. Variability in host characteristics could explain why some people might be more prone to develop vascular disease when infected with strains. In this regard, recent genomic studies have shown genetic variation in (16, 24, 29). One of the most prominent genetic differences among the three genomically characterized strains BTRX-335140 of was that one strain (AR39) contained a 4,524-nucleotide single-stranded DNA bacteriophage, Cpn1 (24). Though three phylogenetically related phages were identified in (14, 20, 26), Cpn1 was the first phage to be identified in phage Chp1, encoding the viral structural proteins viral BTRX-335140 protein 1 (Vp1), Vp2, and Vp3, respectively (24). In a recent report of comparative analysis of chlamydia phages, the well-conserved Vp1 protein was predicted to contain a potential receptor binding site (25). Phage-bearing strains of other bacteria are often more pathogenic than phage-free strains (6), and it may be that phage-containing strains of are more strongly correlated with vascular disease. However, the role played by the phage Cpn1 in the disease pathogenesis of is not obvious, since Cpn1 does not carry any known virulence genes, unlike other phages associated with pathogenic bacteria. The development of methodologies to detect this interesting phage is therefore necessary in order to carry out detailed epidemiologic studies of the association of the phage with strains containing the phage Cpn1 from other strains that lack the phage based on the gene encoding Vp1 and to develop an enzyme-linked immunosorbent assay (ELISA) to detect Vp1 antibodies in order to determine whether exposure to strains containing the phage is associated with AAA. MATERIALS AND METHODS Strains of strains used in this study are described in Table ?Table1.1. The human HL cell line was used for growth and propagation of the strains. Elementary bodies (EBs) were purified on discontinuous gradients of Renografin-76 (Squibb Canada, Montreal, Canada) as previously described (28). The purified EBs were resuspended in isotonic sucrose-phosphate-glutamate buffer and stored at ?80C. TABLE 1. strains evaluated for phage CP0543 sequenceDNA polymerase enzyme, and 25 pmol of oligonucleotide primers (for the gene, forward [5-CGCTCCTAGTGGGGGATTTACTGA-3] and reverse [5-CACAGCTTGCTCACCTAAATGGCT-3]; for the 16S RNA gene, forward [5-CGGTAATACGGAGGGTGCTAGC-3] and reverse [5-GAATTAAACCACATGCTCCACTGC-3]; for the CP0543 gene, forward [5-CTGTAAGGTGAAAAGTTTTTA-3] and reverse [5-CAGCTGTAAATGCAGCTTT-3]) in a total volume of 50 l. The PCR cycling conditions were as follows: one cycle of 95C for 3 min and 35 cycles of 94C for 15 s, 55C for 30 s, and 72C for 2 min. This was followed by strand elongation for 10 min at 72C. The sizes of the amplicons were as follows: strains AR39 or CWL029 was isolated using Trizol (Life Technologies) and treated with RNase-free DNase (Roche). These RNA samples were used as BTRX-335140 templates for reverse transcription (RT) in a 20-l reaction mixture containing 2 g of random hexamers (Roche), 1 l of 10 Rabbit polyclonal to PNPLA2 mM deoxyribonucleoside triphosphates, 2 l of 0.1 M dithiothreitol, 1 l of RNasin (Promega), and 200 U of Superscript II (Life Technologies). RT was carried out at 42C for 50 min. PCR was performed with a PTC-200 Peltier Thermal Cycler. The reaction mixture contained.