Reprogramming of somatic cells to induced pluripotent stem cells rewrites the code of cell fate at the chromatin level. Yet, little is known about this process physically. Here, we describe a fluorescence recovery after photobleaching method to assess the dynamics of heterochromatin/euchromatin and show significant heterochromatin loosening at the initial stage of reprogramming. We identify growth arrest and DNA damage-inducible protein a (Gadd45a) as a chromatin relaxer in mouse embryonic fibroblasts, which also enhances somatic cell reprogramming efficiency. We show that residue glycine 39 (G39) in Gadd45a is essential for interacting with core histones, opening chromatin and enhancing reprogramming. We further demonstrate that Gadd45a destabilizes histone-DNA interactions and facilitates the binding of Yamanaka factors to their targets for activation. Our study provides a method to screen factors that impact on chromatin structure in live cells, and identifies Gadd45a as a chromatin relaxer.

The Tsdaf-21 cDNA was cloned into pET-32a and subsequently expressed in Escherichia coli. The recombinant protein TsDAF-21 was purified and evaluated as an antigen in Western blot. The serum from mouse 14, 21, and 28 days after being infected with Trichinella spiralis recognized rTsDAF-21, but the serum from mouse 7 days post infection of T. spiralis could not react with rTsDAF-21. It implied that the antibody of TsDAF-21 did appear in the host 14 days after infection of T. spiralis. Western blot analysis revealed that the expression of TsDAF-21 in the muscle larvae incubated at room temperature for 8 h and at 37 °C for 4 and 8 h was increased compared to the muscle larvae incubated at 4 °C for 4 and 8 h. The results imply that the expression of TsDAF-21 could be induced at high temperature, not by the cold stress. The expression of TsDAF-21 could be attenuated under the different concentrations of geldanamycin (GA) treatment. Western blot showed that the expression of TsDAF-21 was reduced in the muscle larvae of T. spiralis treated with GA compared to Ampicillin and Gentamycin sulfate treated as control, and this inhibition effect was GA dependent. Other antibiotic treatments did not show any inhibition effects. Immunolocalization showed that TsDAF-21 was expressed in the different stages of T. spiralis including newborn larvae, muscle larvae, and adult worms. TsDAF-21 was mostly localized in nuclei of the cells in the worm. These results suggest that the expression of TsDAF-21 could be induced by the heat stress and attenuated by GA treatment, and TsDAF-21 might be a diagnostic marker for Trichinellosis.

A full-length of complementary deoxyribonucleic acid (Tscdc42) encoding a putative Rho-family small GTPase gene cdc42 was isolated from Trichinella spiralis, an economically important parasitic nematode of zoonosis. The uninterrupted open reading frame of TsCDC42 encodes a predicted protein of 147 amino acids and containing a highly conserved domain of CDC42. Comparison with selected sequences from the free-living nematode Caenorhabditis elegans, Drosophila melanogaster, Xenopus laevis, Danio rerio, Mus musculus, and human showed that Tscdc42 is highly conserved. The highest identity of TsCDC42 with CDC42 from Drosophila is 67%, the similarity is up to 73%, the identity of TsCDC42 with the CDC42 homologue of C. elegans is 62%, and the similarity is up to 71%. Phylogenetic analysis of the amino acid sequence data, using the neighbor-joining and maximum parsimony methods, revealed that TsCDC42 is closely related to the molecule inferred from the cdc-42 gene of C. elegans. The transcript of TsCDC42 was analyzed during different stages of the worm.

No abstract

Hookworm infection is still a public health problem in developing countries. Aspartic protease plays an important role in parasite invasion and migration in the host. Aspartic protease gene from Ancylostoma caninum has been reported (Biochem Biophys Res Commun 227:294-302, 1996), but the activity of Acasp in eggs and larvae stage has not been studied. This paper reported the cloning of Acasp, expression in Escherichia coli, and characterization in eggs and larval stage. The mRNA encoding Acasp was detected using reverse transcriptase polymerase chain reaction in L4 and adult worm. A polyclonal antiserum against E. coli expressed recombinant Acasp was produced and used to detect and localize the Acasp in various developmental stages of A. caninum. Results from Western blot and indirect fluorescent immunoassay showed that the Acasp was present in the embryo, larvae, as well as in the adult worms. The recombinant Acasp exhibited the protease activity by the gelatin gel digestion assay.