Isolation of a novel rmn1 gene genetically linked to spnab2 with respect to mRNA export in fission yeast

In fission yeast, Schizosaccharomyces pombe, the spnab2 gene encodes an ortholog of the budding yeast nuclear abundant poly(A)⁺ RNA-binding protein 2 (Nab2) that is an essential protein required for both mRNA biogenesis and nuclear export of mRNA to the cytoplasm. We have previously isolated three mutants (SLnab1–3) that showed synthetic lethality under the repressed condition of spnab2 expression. In this study, we isolated a novel rmn1 gene as a multicopy suppressor that complemented the defects in growth and mRNA export of SLnab1 mutant cells. The rmn1 gene contained three introns and encoded a 589 amino-acid protein with the RNA recognition motif (RRM) in the central region. The Δrmn1 null mutant was viable but showed a s light mRNA export defect. However, its over-expression caused a deleterious effect on growth accompanied by intense accumulation of poly(A)⁺ RNA in the nucleus. The combination of Δrmn1 with Δspnab2 or Δspmex67 also inhibited growth. In addition, Rmn1p was associated with Rae1p in vivo. These results suggest that rmn1 is a novel gene that is functionally linked to spnab2.     

Schistosoma mansoni Annexin 2: Molecular characterization and immunolocalization

We here describe the cloning and characterization of the Schistosoma mansoni Annexin 2, previously identified in the tegument by proteomic studies, and as an up-regulated gene in schistosomulum stage by microarray data. In silico analysis predicts a conserved core containing four repeat domains of Annexin (ANX) and a variable N-terminal region similar to that described for mammalian isoforms. Real-time RT-PCR and Western blot analysis determined that S. mansoni Annexin 2 is significantly up-regulated in the transition from free-living cercaria to schistosomulum and adult worm parasitic stages. Immunolocalization experiments and tegument membrane preparations confirmed Annexin 2 as a protein mainly localized in the tegument of schistosomula and adult worms. Furthermore, it binds to the tegument surface membranes in a calcium-dependent manner. These results suggest that S. mansoni Annexin 2 is closely associated to the tegument arrangement, being a potential target for immune intervention.     

Sequence-specific interaction between ABD-B homeodomain and castor gene in Drosophila

We have examined the effect of bithorax complex genes on the expression of castor gene. During the embryonic stages 12-15, both Ultrabithorax and abdominal-A regulated the castor gene expression negatively, whereas Abdominal-B showed a positive correlation with the castor gene expression according to real-time PCR. To investigate whether ABD-B protein directly interacts with the castor gene, electrophoretic mobility shift assays were performed using the recombinant ABD-B homeodomain and oligonucleotides, which are located within the region 10 kb upstream of the castor gene. The results show that ABD-B protein directly binds to the castor gene specifically. ABD-B binds more strongly to oligonucleotides containing two 5''-TTAT-3'' canonical core motifs than the probe containing the 5''-TTAC-3'' motif. In addition, the sequences flanking the core motif are also involved in the protein-DNA interaction. The results demonstrate the importance of HD for direct binding to target sequences to regulate the expression level of the target genes. [BMB Reports 2014; 47(2): 92-97]     

High level transactivation by the ecdysone receptor complex at the core recognition motif.

Ecdysteroid signaling in insects is mediated by the ecdysone receptor complex that is composed of a heterodimer of the ecdysone receptor and Ultraspiracle. The DNA binding specificity plays a critical role of defining the repertoire of target genes that respond to the hormone. We report here the determination of the preferred core recognition motif by a binding site selection procedure. The consensus sequence consists of a perfect palindrome of the heptameric half-site sequence GAGGTCA that is separated by a single A/T base pair. No binding polarity of the ecdysone receptor/Ultraspiracle heterodimer to the core recognition motif was observed. This core motif mediated the highest level of ligand-induced transactivation when compared to a series of synthetic ecdysone response elements and to the natural element of the Drosophila hsp27 gene. This is the first report of a palindromic sequence identified as the highest affinity DNA binding site for a heterodimeric nuclear hormone receptor complex. We further present evidence that the ligand of the ecdysone receptor preferentially drives Ultraspiracle from a homodimer into a heterodimer. This mechanism might contribute additionally to a tight control of target gene expression.     

The nuclear receptor gene nhr-25 plays multiple roles in the Caenorhabditis elegans heterochronic gene network to control the larva-to-adult transition

Developmental timing in the nematode Caenorhabditis elegans is controlled by heterochronic genes, mutations in which cause changes in the relative timing of developmental events. One of the heterochronic genes, let-7, encodes a microRNA that is highly evolutionarily conserved, suggesting that similar genetic pathways control developmental timing across phyla. Here we report that the nuclear receptor nhr-25, which belongs to the evolutionarily conserved fushi tarazu-factor 1/nuclear receptor NR5A subfamily, interacts with heterochronic genes that regulate the larva-to-adult transition in C. elegans. We identified nhr-25 as a regulator of apl-1, a homolog of the Alzheimer's amyloid precursor protein-like gene that is downstream of let-7 family microRNAs. NHR-25 controls not only apl-1 expression but also regulates developmental progression in the larva-to-adult transition. NHR-25 negatively regulates the expression of the adult-specific collagen gene col-19 in lateral epidermal seam cells. In contrast, NHR-25 positively regulates the larva-to-adult transition for other timed events in seam cells, such as cell fusion, cell division and alae formation. The genetic relationships between nhr-25 and other heterochronic genes are strikingly varied among several adult developmental events. We propose that nhr-25 has multiple roles in both promoting and inhibiting the C. elegans heterochronic gene pathway controlling adult differentiation programs.     

Schistosoma mansoni infection reduces severity of collagen-induced arthritis via down-regulation of pro-inflammatory mediators

Various experimental and epidemiological studies have demonstrated that helminth infections affect outcomes of allergic or autoimmune disorders. Here, we examined the effects of Schistosoma mansoni infection on mouse collagen-induced arthritis, one of the most widely used animal models for rheumatoid arthritis. Male DBA/1 mice were infected with S. mansoni 2 weeks prior to being immunized with type II collagen (IIC). Cytokine mRNA expression in mouse paws, cytokine production by ConA-stimulated spleen cells, and anti-IIC antibodies were evaluated in addition to the severity of arthritis. S. mansoni infection significantly reduced the severity of arthritis. Anti-IIC IgG and IgG2a levels were lower in infected than uninfected mice. With regard to cytokine producing potentials in the infected mice, the down-regulation of Th1 (IFNγ) and pro-inflammatory cytokines (TNFα and IL-17A), and up-regulation of Th2 (IL-4) and an anti-inflammatory cytokine (IL-10) were observed. In addition, real-time PCR revealed that the augmentation of pro-inflammatory mediators such as IL-1β, IL-6 and receptor activator of NFκB in inflamed paws was abrogated by S. mansoni infection. In conclusion, schistosome infection reduced the severity of autoimmune arthritis via systemic and local suppression of pro-inflammatory mediators, suggesting the potential of parasite-derived materials as therapeutic agents against rheumatoid arthritis.     

Association of IL-17A and IL-17F single nucleotide polymorphisms with susceptibility to osteoarthritis in a Korean population

The damage incurred in osteoarthritis (OA) is mediated by a variety of cytokines, growth factors and inflammatory mediators. The importance of the interleukin-17 (IL-17) family in inflammatory and autoimmune disease is becoming increasingly apparent. Microsatellite association mapping reveals a primary osteoarthritis susceptibility locus on chromosome 6p12.3-q13. IL-17A and IL-17F genes that resided on chromosome 6p12.3-q13 are believed to play an important role in the primary OA susceptibility. We investigated the allele and genotype of IL-17A G-197A and IL-17F T7488C in 302 OA patients and 300 healthy subjects as controls. We employed a PCR-SSCP assay to identify the genotypes IL-17A G-197A and IL-17F T7488C. For IL-17A G-197A, there were significant differences in frequencies of genotype and allele of IL-17A G-197A between OA patients and controls (both p < 0.0001). For IL-17F T7488C, there were no significant differences in the allele frequency and genotype distribution for IL-17F T7488C between OA patients and controls (p = 0.938 and p = 0.1735, respectively). In conclusion, current study showed that polymorphism of IL-17A G-197A may be closely associated with susceptibility to the development of OA in the Korean population. However, there was no relationship between IL-17F T7488C polymorphism and OA susceptibility.     

Histamine signalling in Schistosoma mansoni: Immunolocalisation and characterisation of a new histamine-responsive receptor (SmGPR-2)

In parasitic platyhelminthes, including Schistosoma mansoni, biogenic amines play several important roles in the control of motility, metabolism and reproduction. A bioinformatics analysis of the S. mansoni genome identified approximately 16 full-length G protein-coupled receptors (GPCRs) that share significant homology with aminergic receptors from other species. Six of these sequences are structurally related to SmGPR-1 (formerly SmGPCR), a previously described histamine receptor of S. mansoni, and constitute a new clade of amine-like GPCRs. Here we report the cloning of a second member of this clade, named SmGPR-2. The full-length receptor cDNA was expressed in Saccharomyces cerevisiae and shown to be activated by histamine and 1-methylhistamine, whereas other common biogenic amines had no significant effect. Antagonist assays showed that SmGPR-2 was inhibited by classical biogenic amine antagonists but the pharmacological profile was unlike those of known mammalian histamine receptors. Confocal immunolocalisation studies revealed that SmGPR-2 was expressed in the nervous system and was particularly enriched in the subtegumental neuronal plexus of adult S. mansoni and larvae. The ligand, histamine, was found to be widely distributed, mainly in the peripheral nervous system including the subtegumental plexus where the receptor is also expressed. Finally, SmGPR-2 was shown to be developmentally regulated at the RNA level. Quantitative PCR studies showed it was up-regulated in the parasitic stages compared with cercaria and expressed at the highest level in young schistosomula. The widespread distribution of histamine and the presence of at least two receptors in S. mansoni suggest that this transmitter is an important neuroactive substance in schistosomes.