Beta Transcript Levels Research Articles

Dynamics of cohesin proteins REC8, STAG3, SMC1 and SMC3 are consistent with a role in sister chromatid cohesion during meiosis in human oocytes

Sister chromatid cohesion is essential for ordered chromosome segregation at mitosis and meiosis. This is carried out by cohesin complexes, comprising four proteins, which seem to form a ring-like complex. Data from animal models suggest that loss of sister chromatid cohesion may be involved in age-related non-disjunction in human oocytes. Here, we describe the distribution of cohesins throughout meiosis in human oocytes. We used immunofluorescence in human oocytes at different meiotic stages to detect cohesin subunits REC8, STAG3, SMC1 beta and SMC3, [also synaptonemal complex (SC) protein 3 and shugoshin 1]. Samples from euploid fetuses and adult women were collected, and 51 metaphase I (MI) and 113 metaphase II (MII) oocytes analyzed. SMC1 beta transcript levels were quantified in 85 maturing germinal vesicle (GV) oocytes from 34 women aged 19-43 years by real-time PCR. At prophase I, cohesin subunits REC8, STAG3, SMC1 beta and SMC3 overlapped with the lateral element of the SC. Short cohesin fibers are observed in the oocyte nucleus during dictyate arrest. All four subunits are observed at centromeres and along chromosomal arms, except at chiasmata, at MI and are present at centromeric domains from anaphase I to MII. SMC1 beta transcripts were detected (with high inter-sample variability) in GV oocytes but no correlation between SMC1 beta mRNA levels and age was found. The dynamics of cohesins REC8, STAG3, SMC1 beta and SMC3 suggest their participation in sister chromatid cohesion throughout the whole meiotic process in human oocytes. Our data do not support the view that decreased levels of SMC1 beta gene expression in older women are involved in age-related non-disjunction.

Enhanced gastric IL-18 mRNA expression in Helicobacter pylori-infected children is associated with macrophage infiltration, IL-8, and IL-1β mRNA expression

To investigate IL-18 mRNA expression in the gastric mucosa in Helicobacter pylori-infected children and its association with macrophage infiltration, IL-8, and IL-1 beta mRNA expression. From 39 children, blood samples were taken for IL-1 beta gene polymorphism analysis and antral biopsies were obtained for histology (including macrophage immunostaining), culture and semiquantitative analysis of IL-18, IL-8, IL-1 beta, and CD14 mRNA expression by reverse transcription-PCR (RT-PCR). RT-PCR was used for H. pylori ureA and cagA mRNA detection in gastric tissue. H. pylori-infected patients had significantly higher IL-18, IL-8, and IL-1 beta transcript levels and macrophage numbers in the antral mucosa than H. pylori-negative children. IL-1 beta-511/31 gene polymorphism had no impact on gastric IL-1 beta mRNA levels. IL-18 mRNA expression correlated with mRNA expression of IL-8 and IL-1 beta, and transcript levels of all three cytokines were associated with macrophage infiltration and CD14 mRNA expression in the gastric tissue. Significant correlation was also observed between macrophage numbers and histological parameters of gastritis. These results suggest that interleukin(IL)-18 and macrophages may have an important function in gastric inflammatory response to H. pylori infection in children. IL-18, and possibly CD14 receptor signalling pathway, may be involved in macrophage activation and subsequent IL-8 and IL-1 beta release.

V-Src-mediated Down-regulation of SSeCKS Metastasis Suppressor Gene Promoter by the Recruitment of HDAC1 into a USF1-Sp1-Sp3 Complex

SSeCKS (Src-suppressed C kinase substrate), also called gravin/AKAP12, is a large scaffolding protein with metastasis suppressor activity. Two major isoforms of SSeCKS are expressed in most cell and tissue types under the control of two independent promoters, designated alpha and beta, separated by 68 kb. SSeCKS transcript and protein levels are severely decreased in Src- and Ras-transformed fibroblasts and in many epithelial tumors. By dissecting its promoters with progressive deletion analysis, we identified the sequence between -106 and -49 in the alpha proximal promoter as the minimal v-Src-responsive element, which contains E- and GC-boxes bound by USF1 and Sp1/Sp3, respectively. Both E- and GC-boxes are crucial for v-Src-responsive and basal promoter activities. v-Src does not alter USF1 binding levels at the E-box, but it increases Sp1/Sp3 binding to the GC-box despite no change in their cellular protein abundance. SSeCKS alpha and beta transcript levels in v-Src/3T3 cells can be restored by treatment with the histone deacetylase inhibitor, trichostatin A, but not with the DNA demethylation agent, 5-azacytidine. Chromatin changes are found only on the alpha promoter even though the beta proximal promoter contains a similar E- and GC-box arrangement. Recruitment of HDAC1 is necessary and sufficient to cause repression of alpha proximal promoter activity, and the addition of Sp1 and/or Sp3 potentiates the repression. Our data suggest that suppression of the beta promoter is facilitated by Src-induced changes in the alpha promoter chromatinization mediated by a USF1-Sp1-Sp3 complex.

PPAR /RXR Heterodimers Are Involved in Human CG Synthesis and Human Trophoblast Differentiation

Recent studies performed with null mice suggested a role of either RXR alpha or PPAR gamma in murine placental development. We report here that both PPAR gamma and RXR alpha are strongly expressed in human villous cytotrophoblasts and syncytiotrophoblasts. Moreover, specific ligands for RXRs or PPAR gamma (but not for PPAR alpha or PPAR delta) increase both human CG beta transcript levels and the secretion of human CG and its free beta-subunit. When combined, these ligands have an additive effect on human CG secretion. Pan-RXR and PPAR gamma ligands also have an additive effect on the synthesis of other syncytiotrophoblast hormones such as human placental lactogen, human placental GH, and leptin. Therefore, in human placenta, PPAR gamma/RXR alpha heterodimers are functional units during cytotrophoblast differentiation into the syncytiotrophoblast in vitro. Elements located in the regulatory region of the human CG beta gene (beta 5) were found to bind RXR alpha and PPAR gamma from human cytotrophoblast nuclear extracts, suggesting that PPAR gamma/RXR alpha heterodimers directly regulate human CG beta transcription. Altogether, these data show that PPAR gamma/RXR alpha heterodimers play an important role in human placental development.

All-trans retinoic acid stimulates and maintains neurite outgrowth in nerve growth factor-supported developing chick embryonic sympathetic neurons.

In explanted embryonic chick sympathetic neurons, all-trans retinoic acid (RA) as well as nerve growth factor (NGF) were found to be required for neuronal survival and neurite outgrowth at early stages of development (day 6.5-7) in agreement with previous work (Rodriguez-Tébar and Rohrer [1991] Development 112:813-820). The dependence of neurons on all-trans RA for survival diminished at later stages of development. However, all-trans RA was found to be needed at all stages of development in order to maximize neurite outgrowth. Further, removal of all-trans RA from the cultures led to a rapid degeneration of the formed neurites, demonstrating the essentiality of all-trans RA for both the development of neurites, and for the maintenance of existing neurites in cultured embryonic sympathetic neurons. The mechanism whereby all-trans RA exerts its effects on embryonic sympathetic neurons may involve activation of the nuclear retinoic acid and retinoid-X receptor (RAR and RXR) families. The results of Northern blot analyses and/or reverse transcriptase-polymerase chain reaction (RT-PCR) studies show that embryonic sympathetic ganglia express RAR beta, RAR gamma and RXR gamma mRNAs. RXR gamma mRNA is expressed at highest levels in immature neurons that are not yet responsive to NGF (day 6.5-7) and message levels decline with increasing developmental age. In contrast, RAR beta transcript levels are barely detectable at day 6.5-7, and increase approximately 4-fold in ganglia from embryos at day 8.5-9 and decline thereafter. RT-PCR studies show that RAR gamma mRNA is expressed both early (day 6.5-7) and late (day 15) in ganglionic development. Transcripts for the NGF receptors, p75NGFR and p140trk were also examined. The appearance of a single 2.7 kb p140trk transcript coincides with the time when RAR beta mRNA is maximally expressed, raising the possibility that NGF receptors may be targets of retinoid action. Evidence is also presented that all-trans RA may enhance neurite outgrowth by mechanisms other than simply inducing NGF-responsiveness of neurons.

Relative V beta transcript levels in thymus and peripheral lymphoid tissues from various mouse strains. Inverse correlation of I-E and Mls expression with relative abundance of several V beta transcripts in peripheral lymphoid tissues.

We have measured the relative levels of transcripts for 15 of the 22 known V beta gene segments. The level of transcripts for the highest and lowest expressed V beta gene segment differed by greater than 20-fold in the thymus and an even larger difference was observed in the periphery. The levels of expressions were unrelated to the order of the V beta genes on the chromosome. For most of the V beta gene segments, the relative transcript levels were the same in the thymus and periphery, suggesting that thymic selection in general does not act solely upon the V beta gene segment. One V beta gene segment in the BALB and B10 mice strains was an exception to this rule. V beta 5.2 expression in the periphery of BALB and B10 mice inversely correlated with the expression of the MHC class II molecule I-E. Five V beta gene segments had reduced transcript levels in the periphery of Mls-1a mice compared with their thymic levels or to the levels found in Mls-1b mice. The peripheral level of V beta 3 transcripts vary with MHC and Mls-2 haplotypes. The observation that certain V beta transcript levels are reduced in the periphery when compared with the thymus favors the hypothesis that self tolerance at the T cell level results in the elimination of self-reactive T cells, rather than paralysis by a block at some post-transcriptional step. Finally, the wide variability of V beta gene segment expression in the thymus suggests mechanisms exist to import an early bias to the repertoire. Whether this bias results from differential V beta segment rearrangement rates, differential V beta expression rates, or events occurring after TCR-alpha/beta expression on immature/nonmature thymocyte cell surfaces is yet to be determined.

Genes encoding the T-cell receptor alpha and beta subunits are transcribed in an ordered manner during intrathymic ontogeny.

To further characterize sequential events involved in activation of genes encoding the T-cell receptor (a complex of T3 molecules and a disulfide-linked heterodimer designated Ti, T3-Ti) for antigen and the major histocompatibility complex during intrathymic ontogeny, cDNA probes specific for Ti alpha and Ti beta subunits were used for transcriptional analysis. Ti beta transcript levels were minimal in stage I thymocytes, maximal in stage II thymocytes, and intermediate in state III thymocytes. In contrast, Ti alpha transcriptional activity was virtually undetectable in stage I, was low in stage II, and achieved high levels only in the stage III compartment. Analysis of tumor populations derived from individual stages of thymic differentiation confirmed these observations and demonstrated that clonal stage I-II cells often express Ti beta RNA in the absence of Ti alpha RNA. The latter was not a consequence of functional Ti alpha-subunit isotypy because each of 13 interleukin 2-dependent T-cell clones, including inducer, suppressor, and cytotoxic T cells, contain transcripts that hybridize with the Ti alpha probe. From these data it is concluded that Ti beta gene activation precedes Ti alpha gene activation. Moreover, the high level of Ti beta mRNA preceding Ti alpha mRNA expression implies that Ti beta mRNA and/or its protein products may regulate Ti alpha gene transcription.