Messenger RNA half-life measurements in mammalian cells.

The recognition of the importance of mRNA turnover in regulating eukaryotic gene expression has mandated the development of reliable, rigorous, and user-friendly methods to accurately measure changes in mRNA stability in mammalian cells. Frequently, mRNA stability is studied indirectly by analyzing the steady-state level of mRNA in the cytoplasm; in this case, changes in mRNA abundance are assumed to reflect only mRNA degradation, an assumption that is not always correct. Although direct measurements of mRNA decay rate can be performed with kinetic labeling techniques and transcriptional inhibitors, these techniques often introduce significant changes in cell physiology. Furthermore, many critical mechanistic issues as to deadenylation kinetics, decay intermediates, and precursor-product relationships cannot be readily addressed by these methods. In light of these concerns, we have previously reported transcriptional pulsing methods based on the c-fos serum-inducible promoter and the tetracycline-regulated (Tet-off) promoter systems to better explain mechanisms of mRNA turnover in mammalian cells. In this chapter, we describe and discuss in detail different protocols that use these two transcriptional pulsing methods. The information described here also provides guidelines to help develop optimal protocols for studying mammalian mRNA turnover in different cell types under a wide range of physiologic conditions

BTG/TOB factors impact deadenylases.

BTG/TOB factors are a family of antiproliferative proteins whose expression is altered in numerous cancers. They have been implicated in cell differentiation, development and apoptosis. Although proposed to affect transcriptional regulation, these factors interact with CAF1, a subunit of the main eukaryotic deadenylase, and with poly(A)-binding-proteins, strongly suggesting a role in post-transcriptional regulation of gene expression. The recent determination of the structures of BTG2, TOB1 N-terminal domain (TOB1N138) and TOB1N138-CAF1 complexes support a role for BTG/TOB proteins in mRNA deadenylation, a function corroborated by recently published functional characterizations. We highlight molecular mechanisms by which BTG/TOB proteins influence deadenylation and discuss the need for a better understanding of BTG/TOB physiological functions

Deadenylation is prerequisite for P-body formation and mRNA decay in mammalian cells

Deadenylation is the major step triggering mammalian mRNA decay. One consequence of deadenylation is the formation of nontranslatable messenger RNA (mRNA) protein complexes (messenger ribonucleoproteins [mRNPs]). Nontranslatable mRNPs may accumulate in P-bodies, which contain factors involved in translation repression, decapping, and 5′-to-3′ degradation. We demonstrate that deadenylation is required for mammalian P-body formation and mRNA decay. We identify Pan2, Pan3, and Caf1 deadenylases as new P-body components and show that Pan3 helps recruit Pan2, Ccr4, and Caf1 to P-bodies. Pan3 knockdown causes a reduction of P-bodies and has differential effects on mRNA decay. Knocking down Caf1 or overexpressing a Caf1 catalytically inactive mutant impairs deadenylation and mRNA decay. P-bodies are not detected when deadenylation is blocked and are restored when the blockage is released. When deadenylation is impaired, P-body formation is not restorable, even when mRNAs exit the translating pool. These results support a dynamic interplay among deadenylation, mRNP remodeling, and P-body formation in selective decay of mammalian mRNA

Coordinated changes in mRNA turnover, translation, and RNA processing bodies in bronchial epithelial cells following inflammatory stimulation.

Bronchial epithelial cells play a pivotal role in airway inflammation, but little is known about posttranscriptional regulation of mediator gene expression during the inflammatory response in these cells. Here, we show that activation of human bronchial epithelial BEAS-2B cells by proinflammatory cytokines interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-alpha) leads to an increase in the mRNA stability of the key chemokines monocyte chemotactic protein 1 and IL-8, an elevation of the global translation rate, an increase in the levels of several proteins critical for translation, and a reduction of microRNA-mediated translational repression. Moreover, using the BEAS-2B cell system and a mouse model, we found that RNA processing bodies (P bodies), cytoplasmic domains linked to storage and/or degradation of translationally silenced mRNAs, are significantly reduced in activated bronchial epithelial cells, suggesting a physiological role for P bodies in airway inflammation. Our study reveals an orchestrated change among posttranscriptional mechanisms, which help sustain high levels of inflammatory mediator production in bronchial epithelium during the pathogenesis of inflammatory airway diseases

The correlation of blue shift of photoluminescence and morphology of silicon nanoporous

Porous silicon with diameters ranging from 6.41 to 7.12 nm were synthesized via electrochemical etching by varied anodization current density in ethanoic solutions containing aqueous hydrofluoric acid up to 65mA/cm2.The luminescence properties of the nanoporous at room temperature were analyzed via photoluminescence spectroscopy. Photoluminescence PL spectra exhibit a broad emission band in the range of 360-700 nm photon energy. The PL spectrum has a blue shift in varied anodization current density; the blue shift incremented as the existing of anodization although the intensity decreased. The current blue shift is owning to alteration of silicon nanocrystal structure at the superficies. The superficial morphology of the PS layers consists of unified and orderly distribution of nanocrystalline Si structures, have high porosity around (93.75%) and high thickness 39.52 µm

Elevated Aspartate and Alanine Aminotransferase Levels and Natural Death among Patients with Methamphetamine Dependence

Background: Methamphetamine is one of the fastest growing illicit drugs worldwide, causing multiple organ damage and excessive natural deaths. The authors aimed to identify potential laboratory indices and clinical characteristics associated with natural death through a two-phase study. Methods: Methamphetamine-dependent patients (n = 1,254) admitted to a psychiatric center in Taiwan between 1990 and 2007 were linked with a national mortality database for causes of death. Forty-eight subjects died of natural causes, and were defined as the case subjects. A time-efficient sex-and age-matched nested case-control study derived from the cohort was conducted first to explore the potential factors associated with natural death through a time-consuming standardized review of medical records. Then the identified potential factors were evaluated in the whole cohort to validate the findings. Results: In phase I, several potential factors associated with natural death were identified, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), comorbid alcohol use disorder, and the prescription of antipsychotic drugs. In phase II, these factors were confirmed in the whole cohort using survival analysis. For the characteristics at the latest hospital admission, Cox proportional hazards models showed that the adjusted hazard ratios for natural death were 6.75 (p<0.001) in the group with markedly elevated AST (>80 U/L) and 2.66 (p<0.05) in the group with mildly elevated AST (40-80 U/L), with reference to the control group (>40 U/L). As for ALT, the adjusted hazard ratios were 5.41 (p<0.001), and 1.44 (p>0.05). Comorbid alcohol use disorder was associated with an increased risk of natural death, whereas administration of antipsychotic drugs was not associated with lowered risk. Conclusions: This study highlights the necessity of intensive follow-up for those with elevated AST and ALT levels and comorbid alcohol use disorder for preventing excessive natural deaths

Geochemistry of soil gas in the seismic fault zone produced by the Wenchuan Ms 8.0 earthquake, southwestern China

The spatio-temporal variations of soil gas in the seismic fault zone produced by the 12 May 2008 Wenchuan Ms 8.0 earthquake were investigated based on the field measurements of soil gas concentrations after the main shock. Concentrations of He, H2, CO2, CH4, O2, N2, Rn, and Hg in soil gas were measured in the field at eight short profiles across the seismic rupture zone in June and December 2008 and July 2009. Soil-gas concentrations of more than 800 sampling sites were obtained. The data showed that the magnitudes of the He and H2 anomalies of three surveys declined significantly with decreasing strength of the aftershocks with time. The maximum concentrations of He and H2 (40 and 279.4 ppm, respectively) were found in three replicates at the south part of the rupture zone close to the epicenter. The spatio-temporal variations of CO2, Rn, and Hg concentrations differed obviously between the north and south parts of the fault zone. The maximum He and H2 concentrations in Jun 2008 occurred near the parts of the rupture zone where vertical displacements were larger. The anomalies of He, H2, CO2, Rn, and Hg concentrations could be related to the variation in the regional stress field and the aftershock activity

HuD stimulates translation via eIF4A.

In this issue of Molecular Cell, Fukao et al. (2009) report that HuD upregulates mRNA translation through direct interaction with eIF4A in the 5\u27 cap-binding complex, revealing a posttranscriptional role for HuD in neuronal development and plasticity