Association between phosphodiesterase 4D gene and ischaemic stroke.

BACKGROUND: An association between the phosphodiesterase 4D (PDE4D) gene and risk of ischaemic stroke in an Icelandic population has been suggested by the deCODE group. METHODS: A case-control study of 151 hospitalised patients with first-ever ischaemic stroke and 164 randomly selected age-matched and sex-matched community controls was conducted. PDE4D genotypes for the six single-nucleotide polymorphisms (SNPs) previously reported to be independently associated with stroke were determined, common haplotypes were inferred using the expectation-maximisation algorithm, and SNP and haplotype associations with stroke were examined. A meta-analysis of published studies examining the association between PDE4D and stroke was also carried out. RESULTS: Our study of Australian patients with stroke showed an independent association between ischaemic stroke and PDE4D SNP 89 (CC: odds ratio (OR) 5.55, 95% confidence interval (CI) 1.02 to 30.19; CA: OR 1.68, 95% CI 0.96 to 2.96; AA: OR 1 (reference)), SNP 87 (CC: OR 2.13, 95% CI 1.08 to 4.20; TC: OR 1.64, 95% CI 0.89 to 3.00; TT: OR 1 (reference)) and SNP 83 (TT: OR 2.16, 95% CI 1.08 to 4.32; TC: OR 1.37, 95% CI 0.77 to 2.43; CC: OR 1 (reference)), and between ischaemic stroke and PDE4D haplotypes at SNP 89-87-83 (A-C-C: OR 2.13, 95% CI 1.15 to 3.96; C-C-T: OR 2.25, 95% CI 1.29 to 3.92), but no association between ischaemic stroke and PDE4D SNP 56, SNP 45 or SNP 41, or with PDE4D haplotypes at SNP 56-45-41. A meta-analysis of nine case-control studies (including our current results) of 3808 stroke cases and 4377 controls confirmed a significant association between stroke and PDE SNP 87 (pooled p = 0.002), SNP 83 (0.003) and SNP 41 (0.003). However, there was statistical heterogeneity (p < 0.1) among the studies in the direction of association for each of the individual SNPs tested. CONCLUSIONS: Our results and the pooled analyses from all the studies indicate a strong association between PDE4D and ischaemic stroke. This strengthens the evidence that PDE4D plays a key part in the pathogenesis of ischaemic stroke. Heterogeneity among the studies in the direction of association between individual SNPs and stroke suggests that the SNPs tested are in linkage disequilibrium with the causal allele(s).

Posttranscriptional regulation of thyrotropin beta-subunit messenger ribonucleic acid by thyroid hormone in murine thyrotrope tumor cells: a conserved mechanism across species.

Thyroid hormone (T3) negatively regulates TSH beta-subunit (TSHbeta) messenger RNA (mRNA) gene expression in whole rat pituitary, in part at the level of mRNA stability. However, the regulation of TSHbeta mRNA turnover by T3 in pure populations of thyrotropes and in other species is unknown. To further investigate this, we used murine thyrotropic TtT97 tumor cells. Using primary cultures of TtT97 cells, T3 down-regulated TSHbeta mRNA to approximately 35% of the control level by 8 h. Actinomycin D chase revealed that T3 destabilized TSHbeta mRNA, reducing the half-life from approximately 24 to 7 h, and was accompanied by a decrease in TSHbeta mRNA size. Ribonuclease H analysis revealed that this T3-induced decrease in size was due to a shortening of poly(A) tail from approximately 160 to approximately 30 nucleotides and was specific for TSHbeta mRNA. Cycloheximide mimicked the poly(A) tail effect observed with T3. In the absence of T3, actinomycin D deadenylated TSHbeta mRNA without inducing rapid decay. We conclude that T3 reduces the steady state half-life of TSHbeta mRNA in murine TtT97 thyrotropic tumor cells accompanied by a reduction in poly(A) tail length. However, in the absence of T3, deadenylation alone is not sufficient to induce TSHbeta mRNA decay. Together with the high degree of sequence conservation in the 3'-untranslated region of murine and rat TSHbeta mRNA sequences and the similarities of the T3 effect, these data provide the first evidence for a highly conserved posttranscriptional mechanism operative across species. We propose a model in which T3 coordinately regulates shortening of the poly(A) tail and the activity of a transacting RNA-binding protein and/or an exonuclease to accelerate TSHbeta mRNA turnover.

Collagen platelet receptor polymorphisms integrin alpha2beta1 C807T and GPVI Q317L and risk of ischemic stroke.

Several polymorphisms of integrin alpha2beta1 and glycoprotein (GP) VI that may modify platelet-collagen interactions or subsequent signaling have been described. We conducted a case-control study involving 180 stroke patients and 172 controls to determine whether the alpha2 C807T and GPVI Q317L polymorphisms were associated with an increased risk of ischemic stroke. We found no statistically significant differences in the distribution of alpha2 C807T and GPVI Q317L in patients and controls overall or after stratification by etiological subtype. The GPVI 317QQ genotype was found to be over-represented in a subgroup of patients >/=60 years compared to corresponding controls. However, this association did not remain significant after adjustment for other cardiovascular risk factors. Our results do not support a role for the integrin alpha2 C807T and GPVI Q317L polymorphisms in the development of first-ever ischemic stroke. However, larger studies are required to confirm this.

Hormonal regulation of mRNA stability and RNA-protein interactions in the pituitary.

Regulating gene expression from DNA to protein is a complex multistage process with multiple control mechanisms. Transcriptional regulation has been considered the major control point of protein production in eukaryotic cells; however, there is growing evidence of pivotal posttranscriptional regulation for many genes. This has prompted extensive investigations to elucidate the mechanisms controlling RNA processing, mRNA nuclear export and localization, mRNA stability and turnover, in addition to translational rates and posttranslational events. The regulation of mRNA stability has emerged as a critical control step in determining the cellular mRNA level, with individual mRNAs displaying a wide range of stability that has been linked to discrete sequence elements and specific RNA-protein interactions. This review will focus on current knowledge of the determinants of mRNA stability and RNA-protein interactions in the pituitary. This field is rapidly expanding with the identification of regulated cis-acting stability-modifying elements within many mRNAs, and the cloning and characterization of trans-acting proteins that specifically bind to their cognate cis elements. We will present evidence for regulation of multiple pituitary genes at the level of mRNA stability and some examples of the emerging data characterizing RNA-protein interactions.

Optimized RNA gel-shift and UV cross-linking assays for characterization of cytoplasmic RNA-protein interactions.

Considerable interest has recently focused on defining the mechanisms involved in the regulation of gene expression at the level of mRNA stability and translational efficiency. However, the assays used to directly investigate interactions between RNA and cytoplasmic proteins have been difficult to establish, and methods are not widely available. Here, we describe a robust method for RNA electrophoretic mobility shift and UV cross-linking assays that allows rapid detection of cytoplasmic RNA-protein interactions. For added convenience to new investigators, these assays use mini-gels with an electrophoresis time of 15-20 min, enabling a high throughput of samples. The method works successfully with many different probes and cytoplasmic extracts from a variety of cell lines. Furthermore, we provide a system to optimize characterization of the RNA-protein complex and troubleshoot most assay difficulties.

Expression and immune recognition of stress proteins in sarcoidosis and other chronic interstitial lung diseases.

Stress proteins (SP) are major immunogens in a number of microbial infections and have been implicated in some autoimmune diseases. The aetiology of sarcoidosis, a non-caseating granulomatous disease, remains unknown, but mycobacteria as well as autoimmunity have been considered. In the present study, patients diagnosed with sarcoidosis and other interstitial lung diseases (ILD), as well as healthy volunteers were studied to determine: (i) the level of expression of SP in alveolar macrophages and blood monocytes; (ii) the serum levels of antibodies specific for mycobacterial SP65 and SP70; and (iii) the reactivity of peripheral blood and alveolar lymphocytes to mycobacterial SP65. Our results suggest that SP are expressed constitutively at high levels in alveolar macrophages, retrieved by bronchoalveolar lavage, from all individuals regardless of health status. In contrast, freshly isolated blood monocytes express low levels of SP, which are, however, readily upregulated following exposure to IFN-gamma and TNF-alpha. Lymphocyte reactivity and presence of antibodies against mycobacterial SP may reflect the current state of in vivo inflammation rather than the cause of inflammation.