The Germ Cell Nuclear Proteins hnRNP G-T and RBMY Activate a Testis-Specific Exon

The human testis has almost as high a frequency of alternative splicing events as brain. While not as extensively studied as brain, a few candidate testis-specific splicing regulator proteins have been identified, including the nuclear RNA binding proteins RBMY and hnRNP G-T, which are germ cell-specific versions of the somatically expressed hnRNP G protein and are highly conserved in mammals. The splicing activator protein Tra2β is also highly expressed in the testis and physically interacts with these hnRNP G family proteins. In this study, we identified a novel testis-specific cassette exon TLE4-T within intron 6 of the human transducing-like enhancer of split 4 (TLE4) gene which makes a more transcriptionally repressive TLE4 protein isoform. TLE4-T splicing is normally repressed in somatic cells because of a weak 5′ splice site and surrounding splicing-repressive intronic regions. TLE4-T RNA pulls down Tra2β and hnRNP G proteins which activate its inclusion. The germ cell-specific RBMY and hnRNP G-T proteins were more efficient in stimulating TLE4-T incorporation than somatically expressed hnRNP G protein. Tra2b bound moderately to TLE4-T RNA, but more strongly to upstream sites to potently activate an alternative 3′ splice site normally weakly selected in the testis. Co-expression of Tra2β with either hnRNP G-T or RBMY re-established the normal testis physiological splicing pattern of this exon. Although they can directly bind pre-mRNA sequences around the TLE4-T exon, RBMY and hnRNP G-T function as efficient germ cell-specific splicing co-activators of TLE4-T. Our study indicates a delicate balance between the activity of positive and negative splicing regulators combinatorially controls physiological splicing inclusion of exon TLE4-T and leads to modulation of signalling pathways in the testis. In addition, we identified a high-affinity binding site for hnRNP G-T protein, showing it is also a sequence-specific RNA binding protein

Rapid Chromosome Evolution in Recently Formed Polyploids in Tragopogon (Asteraceae)

Polyploidy, frequently termed "whole genome duplication", is a major force in the evolution of many eukaryotes. Indeed, most angiosperm species have undergone at least one round of polyploidy in their evolutionary history. Despite enormous progress in our understanding of many aspects of polyploidy, we essentially have no information about the role of chromosome divergence in the establishment of young polyploid populations. Here we investigate synthetic lines and natural populations of two recently and recurrently formed allotetraploids Tragopogon mirus and T. miscellus (formed within the past 80 years) to assess the role of aberrant meiosis in generating chromosomal/genomic diversity. That diversity is likely important in the formation, establishment and survival of polyploid populations and species.Applications of fluorescence in situ hybridisation (FISH) to natural populations of T. mirus and T. miscellus suggest that chromosomal rearrangements and other chromosomal changes are common in both allotetraploids. We detected extensive chromosomal polymorphism between individuals and populations, including (i) plants monosomic and trisomic for particular chromosomes (perhaps indicating compensatory trisomy), (ii) intergenomic translocations and (iii) variable sizes and expression patterns of individual ribosomal DNA (rDNA) loci. We even observed karyotypic variation among sibling plants. Significantly, translocations, chromosome loss, and meiotic irregularities, including quadrivalent formation, were observed in synthetic (S(0) and S(1) generations) polyploid lines. Our results not only provide a mechanism for chromosomal variation in natural populations, but also indicate that chromosomal changes occur rapidly following polyploidisation.These data shed new light on previous analyses of genome and transcriptome structures in de novo and establishing polyploid species. Crucially our results highlight the necessity of studying karyotypes in young (<150 years old) polyploid species and synthetic polyploids that resemble natural species. The data also provide insight into the mechanisms that perturb inheritance patterns of genetic markers in synthetic polyploids and populations of young natural polyploid species

Elevated Stress-Hemoconcentration in Major Depression Is Normalized by Antidepressant Treatment: Secondary Analysis from a Randomized, Double-Blind Clinical Trial and Relevance to Cardiovascular Disease Risk

Major depressive disorder (MDD) is an independent risk factor for cardiovascular disease (CVD); the presence of MDD symptoms in patients with CVD is associated with a higher incidence of cardiac complications following acute myocardial infarction (MI). Stress-hemoconcentration, a result of psychological stress that might be a risk factor for the pathogenesis of CVD, has been studied in stress-challenge paradigms but has not been systematically studied in MDD.Secondary analysis of stress hemoconcentration was performed on data from controls and subjects with mild to moderate MDD participating in an ongoing pharmacogenetic study of antidepressant treatment response to desipramine or fluoxetine. Hematologic and hemorheologic measures of stress-hemoconcentration included blood cell counts, hematocrit, hemoglobin, total serum protein, and albumin, and whole blood viscosity.Subjects with mild to moderate MDD had significantly increased hemorheologic measures of stress-hemoconcentration and blood viscosity when compared to controls; these measures were correlated with depression severity. Measures of stress-hemoconcentration improved significantly after 8 weeks of antidepressant treatment. Improvements in white blood cell count, red blood cell measures and plasma volume were correlated with decreased severity of depression.Our secondary data analyses support that stress-hemoconcentration, possibly caused by decrements in plasma volume during psychological stress, is present in Mexican-American subjects with mild to moderate MDD at non-challenged baseline conditions. We also found that after antidepressant treatment hemorheologic measures of stress-hemoconcentration are improved and are correlated with improvement of depressive symptoms. These findings suggest that antidepressant treatment may have a positive impact in CVD by ameliorating increased blood viscosity. Physicians should be aware of the potential impact of measures of hemoconcentration and consider the implications for cardiovascular risk in depressed patients

Adaptor SKAP-55 Binds p21ras Activating Exchange Factor RasGRP1 and Negatively Regulates the p21ras-ERK Pathway in T-Cells

While the adaptor SKAP-55 mediates LFA-1 adhesion on T-cells, it is not known whether the adaptor regulates other aspects of signaling. SKAP-55 could potentially act as a node to coordinate the modulation of adhesion with downstream signaling. In this regard, the GTPase p21ras and the extracellular signal-regulated kinase (ERK) pathway play central roles in T-cell function. In this study, we report that SKAP-55 has opposing effects on adhesion and the activation of the p21ras -ERK pathway in T-cells. SKAP-55 deficient primary T-cells showed a defect in LFA-1 adhesion concurrent with the hyper-activation of the ERK pathway relative to wild-type cells. RNAi knock down (KD) of SKAP-55 in T-cell lines also showed an increase in p21ras activation, while over-expression of SKAP-55 inhibited activation of ERK and its transcriptional target ELK. Three observations implicated the p21ras activating exchange factor RasGRP1 in the process. Firstly, SKAP-55 bound to RasGRP1 via its C-terminus, while secondly, the loss of binding abrogated SKAP-55 inhibition of ERK and ELK activation. Thirdly, SKAP-55−/− primary T-cells showed an increased presence of RasGRP1 in the trans-Golgi network (TGN) following TCR activation, the site where p21ras becomes activated. Our findings indicate that SKAP-55 has a dual role in regulating p21ras-ERK pathway via RasGRP1, as a possible mechanism to restrict activation during T-cell adhesion

Receptor and secreted targets of Wnt-1/β-catenin signalling in mouse mammary epithelial cells

BACKGROUND: Deregulation of the Wnt/ β-catenin signal transduction pathway has been implicated in the pathogenesis of tumours in the mammary gland, colon and other tissues. Mutations in components of this pathway result in β-catenin stabilization and accumulation, and the aberrant modulation of β-catenin/TCF target genes. Such alterations in the cellular transcriptional profile are believed to underlie the pathogenesis of these cancers. We have sought to identify novel target genes of this pathway in mouse mammary epithelial cells. METHODS: Gene expression microarray analysis of mouse mammary epithelial cells inducibly expressing a constitutively active mutant of β-catenin was used to identify target genes of this pathway. RESULTS: The differential expression in response to ΔNβ-catenin for five putative target genes, Autotaxin, Extracellular Matrix Protein 1 (Ecm1), CD14, Hypoxia-inducible gene 2 (Hig2) and Receptor Activity Modifying Protein 3 (RAMP3), was independently validated by northern blotting. Each of these genes encodes either a receptor or a secreted protein, modulation of which may underlie the interactions between Wnt/β-catenin tumour cells and between the tumour and its microenvironment. One of these genes, Hig2, previously shown to be induced by both hypoxia and glucose deprivation in human cervical carcinoma cells, was strongly repressed upon ΔNβ-catenin induction. The predicted N-terminus of Hig2 contains a putative signal peptide suggesting it might be secreted. Consistent with this, a Hig2-EGFP fusion protein was able to enter the secretory pathway and was detected in conditioned medium. Mutation of critical residues in the putative signal sequence abolished its secretion. The expression of human HIG2 was examined in a panel of human tumours and was found to be significantly downregulated in kidney tumours compared to normal adjacent tissue. CONCLUSIONS: HIG2 represents a novel non-cell autonomous target of the Wnt pathway which is potentially involved in human cancer

Expression of the H+-ATPase AHA10 proton pump is associated with citric acid accumulation in lemon juice sac cells.

The sour taste of lemons (Citrus limon (L.) Burm.) is determined by the amount of citric acid in vacuoles of juice sac cells. Faris is a “sweet” lemon variety since it accumulates low levels of citric acid. The University of California Riverside Citrus Variety Collection includes a Faris tree that produces sweet (Faris non-acid; FNA) and sour fruit (Faris acid; FA) on different branches; it is apparently a graft chimera with layer L1 derived from Millsweet limetta and layer L2 from a standard lemon. The transcription profiles of Faris sweet lemon were compared with Faris acid lemon and Frost Lisbon (L), which is a standard sour lemon genetically indistinguishable from Faris in prior work with SSR markers. Analysis of microarray data revealed that the transcriptomes of the two sour lemon genotypes were nearly identical. In contrast, the transcriptome of Faris sweet lemon was very different from those of both sour lemons. Among about 1,000 FNAspecific, presumably pH-related genes, the homolog of Arabidopsis H+-ATPase proton pump AHA10 was not expressed in FNA, but highly expressed in FA and L. Since Arabidopsis AHA10 is involved in biosynthesis and acidification of vacuoles, the lack of expression of the AHA10 citrus homolog represents a very conspicuous molecular feature of the FNA sweet phenotype. In addition, high expression of several 2-oxoglutarate degradationrelated genes in FNA suggests activation of the GABA shunt and degradation of valine and tyrosine as components of the mechanism that reduces the level of citric acid in sweet lemon