[PubMed] [Google Scholar] 34. the ICE gene and its protein as a result of increased IRF-1 induction but that this increased ICE was insufficient to cause apoptosis in the RSV-infected cells. ICE might not be able to activate CPP32, which is thought to be the more important protease for apoptosis. Respiratory syncytial computer virus (RSV) contamination in neonates and young infants often causes life-threatening acute bronchiolitis (1, 2). The peculiar tropism of RSV for the bronchiolar epithelium and the fragile anatomy of the infants bronchioles are possible factors in the pathogenesis of acute bronchiolitis (12). However, the precise mechanisms of respiratory epithelial cell death after RSV Mouse monoclonal antibody to Keratin 7. The protein encoded by this gene is a member of the keratin gene family. The type IIcytokeratins consist of basic or neutral proteins which are arranged in pairs of heterotypic keratinchains coexpressed during differentiation of simple and stratified epithelial tissues. This type IIcytokeratin is specifically expressed in the simple epithelia lining the cavities of the internalorgans and in the gland ducts and blood vessels. The genes encoding the type II cytokeratinsare clustered in a region of chromosome 12q12-q13. Alternative splicing may result in severaltranscript variants; however, not all variants have been fully described contamination remain unclear. Recently, two major morphologically and biochemically unique modes of cell death have been explained, apoptosis, or programmed cell death, and necrosis (4, 14, 36). In human viral infections, such as human immunodeficiency computer virus type 1 (9, 29) and LLY-507 influenza (11, 25), apoptosis is usually thought to be the major mode of cell death due to the computer virus contamination. The induction of apoptosis in virus-infected cells is now considered to be the major mechanism for viral clearance by the mammalian immune system (3). However, the participation of apoptosis in other human respiratory viral infections, including RSV, has not yet been fully investigated. Tumor suppressor interferon regulatory factor 1 (IRF-1) plays an essential role in apoptosis (26, 28) and is a transcriptional activator of the interleukin-1-transforming enzyme (ICE) gene (26, 27). ICE is the first mammalian homolog of the cell death gene, polymerase (Promega, Madison, Wis.). Twenty-four cycles of amplification for -actin, 24 for IRF-1, 31 for ICE, and 24 for CPP32 were carried out with a DNA thermal cycler (Perkin-Elmer, Norwalk, Conn.). Each cycle consisted of warming at 95C for 35 s, 55C for 2 min, and 72C for 2 min. Finally, the preparations were incubated at 72C for 15 min. Ten-microliter samples of the RT PCR products were analyzed by electrophoresis on a 2% agarose gel, and the amplified products were visualized by UV fluorescence after staining with ethidium bromide. The UV fluorescence signals of specific PCR products in agarose gels were quantified with a FluorImager SI (Molecular Dynamics, Sunnyvale, Calif.). The specificity of each PCR product was confirmed by determining its predicted size on agarose gels and by Southern blot analysis as explained previously (22) (data not shown). Sequences of the specific oligonucleotide probes are as follows: for -actin, AAAGACCTGTACGCCAACA; for IRF-1, AAGGCCAACTTTCGCTGTGCC; for ICE, ATAGAGAAGGATTTTATCGC; and for CPP32, GCCATCCTTTGAATTTCGCC (15, 31, 32). As a positive control for mRNA for -actin, IRF-1, ICE, and CPP32, total RNA from LLY-507 normal adult peripheral blood mononuclear cells which were incubated with 10 g of concanavalin A per ml for 3 h was used. To quantify relative levels of mRNA, a standard curve was obtained by titration (1/5 dilution) of first strands obtained from 250 ng of RNA from your positive controls explained above (Fig. ?(Fig.1).1). Relative differences in mRNA expression LLY-507 in the test samples were obtained by determination of the standard curves run for the same quantity of cycles as the unknown samples. The intensity of fluorescence of DNA amplified from first strands obtained from 250 ng of RNA was defined arbitrarily as 1,000 mRNA equivalents. Open in a separate windows FIG. 1 Ethidium bromide-stained gels and standard curves of -actin (A), IRF-1 (B), ICE mRNA (C), and CPP32 mRNA (D) generated by RT PCR with control RNA. Two hundred fifty nanograms of total RNA was reverse transcribed, and fivefold dilutions of the first strand were amplified by PCR. The intensity of fluorescence of DNA amplified from your first strand obtained from 250 ng of total RNA was defined arbitrarily as 1,000 mRNA equivalents. The level of -actin in each unknown sample was decided from your actin standard curve, and the levels generally varied less than 30% when the first strand was obtained from the same amount of total RNA (data not shown). The production.