Expression of the histone chaperone SET/TAF-Iβ during the strobilation process of Mesocestoides corti (Platyhelminthes, Cestoda)

SUMMARYThe histone chaperone SET/TAF-Iβ is implicated in processes of chromatin remodelling and gene expression regulation. It has been associated with the control of developmental processes, but little is known about its function in helminth parasites. In Mesocestoides corti, a partial cDNA sequence related to SET/TAF-Iβ was isolated in a screening for genes differentially expressed in larvae (tetrathyridia) and adult worms. Here, the full-length coding sequence of the M. corti SET/TAF-Iβ gene was analysed and the encoded protein (McSET/TAF) was compared with orthologous sequences, showing that McSET/TAF can be regarded as a SET/TAF-Iβ family member, with a typical nucleosome-assembly protein (NAP) domain and an acidic tail. The expression patterns of the McSET/TAF gene and protein were investigated during the strobilation process by RT-qPCR, using a set of five reference genes, and by immunoblot and immunofluorescence, using monospecific polyclonal antibodies. A gradual increase in McSET/TAF transcripts and McSET/TAF protein was observed upon development induction by trypsin, demonstrating McSET/TAF differential expression during strobilation. These results provided the first evidence for the involvement of a protein from the NAP family of epigenetic effectors in the regulation of cestode development.

First evidence of the P-glycoprotein gene expression and multixenobiotic resistance modulation in earthworm

Summary Multixenobiotic resistance (MXR) is an important mechanism of cellular efflux mediated by ATP binding cassette (ABC) transporters that bind and actively remove toxic substrates from the cell. This study was the first to identify ABC transporter P-glycoprotein (P-gp/ABCB1) as a representative of the MXR phenotype in earthworm (Eisenia fetida). The identified partial cDNA sequence of ABCB1 overlapped with ABCB1 homologues of other organisms from 58.5 % to 72.5 %. We also studied the effect of five modulators (verapamil, cyclosporine A, MK571, probenecid, and orthovanadate) on the earthworm’s MXR activity by measuring the accumulation of model substrates rhodamine B and rhodamine 123 in whole body tissue of the adult earthworm. MK571, orthovanadate, and verapamil significantly inhibited MXR activity, and rhodamine 123 turned out to better reflect MXR activity in that species than rhodamine B. Our results show that E. fetida can serve well as a test organism for environmental pollutants that inhibit MXR activity.

Expression, purification and identification of gibberellin-induced cysteine-rich protein ofGymanadenia conopsea

Primers bearing restriction enzyme sites forEcoR I andHind III were designed according to the known partial cDNA sequence for gibberellin-induced cysteine-rich protein and were then used to amplify the full-length open reading frame (ORF) and signal peptide-truncated fragment of thegcgasagene. Two fragments with lengths 319 and 238 bp were obtained and were further cloned into plasmid pET-32(a). Following transformation intoEscherichia coliBL21(DE3), the fusion proteins were observed to appear at ~26.0 and 25.2 kDa after induction from 1 mmol/l isopropyl-beta-D-thiogalactopyronoside (IPTG). The results of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transmission electron microscopy (TEM) of an ultra-thin section revealed that the presence of signal peptide gave rise to the formation of an inclusion body located in the periplasmic space; however, the absence of signal peptide greatly enhanced the solubility of the target protein. The expressed soluble protein was further purified by Ni2+-NTA affinity chromatography and gel filtration methods.

Cloning and characterization of an orphan seven transmembrane receptor from Schistosoma mansoni

SUMMARYA partial cDNA sequence was obtained from the human blood fluke, Schistosoma mansoni using a signal sequence trap approach. The full-length cDNA was cloned and termed Sm-7TM. The corresponding open reading frame had 7 membrane spanning domains and shared identity with a small, novel group of seven transmembrane (7TM) receptors from vertebrates and invertebrates, including the human ee3 receptor – a heptahelical protein implicated in neuronal signalling. Phylogenetic analysis of this novel family showed that the Sm-7TM ORF formed a clade with exclusively invertebrate sequences. Based on topology modelling with ee3, Sm-7TM was predicted to possess an intracellular C-terminal tail, which was expressed as a soluble thioredoxin fusion protein (Sm-7TMC) in Escherichia coli and purified using metal ion-affinity chromatography. A polyclonal antiserum against this domain was used to detect Sm-7TM in detergent-soluble parasite extracts and to immunolocalize the receptor to the tegument of adult S. mansoni.