Expression of Lex antigen in Schistosoma japonicum and S.haematobium and immune responses to Lex in infected animals: lack of Lex expression in other trematodes and nematodes

Adults of the human parasitic trematode Schistosoma mansoni, which causes hepatosplenic/intestinal complications in humans, synthesize glycoconjugates containing the Lewis x (Lex) Galbeta1-->4(Fucalpha1-- >3)GlcNAcbeta1-->R, but not sialyl Lewis x (sLex), antigen. We now report on our analyses of Lexand sLexexpression in S.haematobium and S.japonicum, which are two other major species of human schistosomes that cause disease, and the possible autoimmunity to these antigens in infected individuals. Antigen expression was evaluated by both ELISA and Western blot analyses of detergent extracts of parasites using monoclonal antibodies. Several high molecular weight glycoproteins in both S. haematobium and S. japonicum contain the Lexantigen, but no sialyl Lexantigen was detected. In addition, sera from humans and rodents infected with S.haematobium and S.japonicum contain antibodies reactive with Lex. These results led us to investigate whether Lexantigens are expressed in other helminths, including the parasitic trematode Fasciola hepatica , the parasitic nematode Dirofilaria immitis (dog heartworm), the ruminant nematode Haemonchus contortus , and the free-living nematode Caenorhabditis elegans . Neither Lexnor sialyl-Lexis detectable in these other helminths. Furthermore, none of the helminths, including schistosomes, express Lea, Leb, Ley, or the H- type 1 antigen. However, several glycoproteins from all helminths analyzed are bound by Lotus tetragonolobus agglutinin , which binds Fucalpha1-->3GlcNAc, and Wisteria floribunda agglutinin, which binds GalNAcbeta1-->4GlcNAc (lacdiNAc or LDN). Thus, schistosomes may be unique among helminths in expressing the Lexantigen, whereas many different helminths may express alpha1,3-fucosylated glycans and the LDN motif.      

Real-time expression profiling of microRNA precursors in human cancer cell lines

Our previous study described a real-time PCR method to quantify microRNA (miRNA) precursors using SYBR green detection [T. D. Schmittgen, J. Jiang, Q. Liu and L. Yang (2004) Nucleic Acids Res., 32, e43]. The present study adapted the assay to a 384-well format and expanded it to include primers to 222 human miRNA precursors. TaqMan minor groove binder probes were used to discriminate nearly identical members of the let-7 family of miRNA isoforms. The miRNA precursor expression was profiled in 32 human cell lines from lung, breast, colorectal, hematologic, prostate, pancreatic, and head and neck cancers. Some miRNA precursors were expressed at similar levels in many of the cell lines, while others were differentially expressed. Clustering analysis of the miRNA precursor expression data revealed that most of the cell lines clustered into their respective tissues from which each cell line was ostensibly derived. miRNA precursor expression by PCR paralleled the mature miRNA expression by northern blotting for most of the conditions studied. Our study provides PCR primer sequences to all of the known human miRNA precursors as of December 2004 and provides a database of the miRNA precursor expression in many commonly used human cancer cell lines.     

Settlement inhibition of fouling invertebrate larvae by metabolites of the marine bacterium halomonas marina within a polyurethane coating

The marine bacterium, Halomonas marina (ATCC 27129), was shown to inhibit settlement and development of the sessile invertebrates Balanus amphitrite and Bugula neritina. Different bacterial treatments were employed to investigate this interaction. Filmed bacteria and liquid suspensions of whole cells, lysed cells and culture filtrate all reduced settlement of B. amphitrite. Polyurethane coatings containing whole cells were partially inhibitory while lysed cells caused complete inhibition of B. amphitrite larval settlement. In contrast, culture filtrate in a polyurethane matrix stimulated settlement of B. amphitrite larvae. Whole cells, culture filtrate, and lysed cells embedded in a polyurethane coating also controlled B. neritina settlement and maturation.     

Dexamethasone regulates the program of secretory glycoprotein synthesis in hepatoma tissue culture cells

The secretory glycoproteins synthesized by hepatoma tissue culture (HTC) cells were resolved by two-dimensional polyacrylamide gel electrophoresis of media from cells that were grown in the presence of [(3)H]fucose. These cells synthesize and secrete a complex set of fucose-containing glycoproteins. These secretory glycoproteins are distinct from those glycoproteins present in the plasma membrane of HTC cells. Incubation of HTC cells with dexamethasone has a pronounced effect on the quality and quantity (denoted here as the program) of secretory protein synthesis, as assayed by the short-term incorporation of labeled mannose, fucose, or methionine. The synthesis of two mannose- and fucose- containing glycoprotein series, one of 50,000 mol wt and a more heterogeneous series with mol wt of 35,000-50,000, is increased to a high level by the hormone; conversely, the synthesis of other secretory proteins, particularly one with mol wt of 70,000, is decreased or stopped completely. The synthesis of some major secretory proteins is not affected by the hormone. Dexamethasone has less of an effect on the composition of either total cell membrane glycoprotein or plasma membrane glycoprotein. But there is a decrease in the synthesis of a major membrane glycoprotein series with mol wt of 140,000. These effects of dexamethasone are relatively specific to HTC cells. Neither Reuber H-35 cells nor primary cultures of rat hepatocytes show the same response to the steroid. Two variant HTC cell lines, which were selected for their resistance to dexamethasone inhibition of extracellular plasminogen activator activity, respond only partially to the steroid-induced regulation of the secretory and membrane glycoproteins.     

MicroRNAs are mediators of androgen action in prostate and muscle

Androgen receptor (AR) function is critical for the development of male reproductive organs, muscle, bone and other tissues. Functionally impaired AR results in androgen insensitivity syndrome (AIS). The interaction between AR and microRNA (miR) signaling pathways was examined to understand the role of miRs in AR function. Reduction of androgen levels in Sprague-Dawley rats by castration inhibited the expression of a large set of miRs in prostate and muscle, which was reversed by treatment of castrated rats with 3 mg/day dihydrotestosterone (DHT) or selective androgen receptor modulators. Knockout of the miR processing enzyme, DICER, in LNCaP prostate cancer cells or tissue specifically in mice inhibited AR function leading to AIS. Since the only function of miRs is to bind to 3' UTR and inhibit translation of target genes, androgens might induce miRs to inhibit repressors of AR function. In concordance, knock-down of DICER in LNCaP cells and in tissues in mice induced the expression of corepressors, NCoR and SMRT. These studies demonstrate a feedback loop between miRs, corepressors and AR and the imperative role of miRs in AR function in non-cancerous androgen-responsive tissues.     

Real-time PCR quantification of precursor and mature microRNA

microRNAs (miRNAs) are challenging molecules to amplify by PCR because the miRNA precursor consists of a stable hairpin and the mature miRNA is roughly the size of a standard PCR primer. Despite these difficulties, successful real-time RT-PCR technologies have been developed to amplify and quantify both the precursor and mature microRNA. An overview of real-time PCR technologies developed by us to detect precursor and mature microRNAs is presented here. Protocols describe presentation of the data using relative (comparative C(T)) and absolute (standard curve) quantification. Real-time PCR assays were used to measure the time course of precursor and mature miR-155 expression in monocytes stimulated by lipopolysaccharide. Protocols are provided to configure the assays as low density PCR arrays for high throughput gene expression profiling. By profiling over 200 precursor and mature miRNAs in HL60 cells induced to differentiate with 12-O-tetradecanoylphorbol-13-acetate, it was possible to identify miRNAs who's processing is regulated during differentiation. Real-time PCR has become the gold standard of nucleic acid quantification due to the specificity and sensitivity of the PCR. Technological advancements have allowed for quantification of miRNA that is of comparable quality to more traditional RNAs.