A “GC-rich” method for mammalian gene expression: A dominant role of non-coding DNA GC content in regulation of mammalian gene expression

High mammalian gene expression was obtained for more than twenty different proteins in different cell types by just a few laboratory scale stable gene transfections for each protein. The stable expression vectors were constructed by inserting a naturally-occurring 1.006 kb or a synthetic 0.733 kb DNA fragment (including intron) of extremely GC-rich at the 5′ or/and 3′ flanking regions of these protein genes or their gene promoters. This experiment is the first experimental evidence showing that a non-coding extremely GC-rich DNA fragment is a super “chromatin opening element” and plays an important role in mammalian gene expression. This experiment has further indicated that chromatin-based regulation of mammalian gene expression is at least partially embedded in DNA primary structure, namely DNA GC-content.     

A role for the AtMTP11 gene of Arabidopsis in manganese transport and tolerance

The Arabidopsis AtMTP family of genes encode proteins of the cation diffusion facilitator (CDF) family, with several members having roles in metal tolerances. Four of the 11 proteins in the family form a distinct cluster on a phylogenetic tree and are closely related to ShMTP8, a CDF identified in the tropical legume Stylosanthes hamata that is implicated in the transport of Mn(2+) into the vacuole as a tolerance mechanism. Of these four genes, AtMTP11 was the most highly expressed member of the Arabidopsis subgroup. When AtMTP11 was expressed in Saccharomyces cerevisiae, it conferred Mn(2+) tolerance and transported Mn(2+) by a proton-antiport mechanism. A mutant of Arabidopsis with a disrupted AtMTP11 gene (mtp11) was found to have increased sensitivity to Mn(2+) but not to Cu(2+) or Zn(2+). At a non-toxic but sufficient Mn(2+) supply (basal), the mutant accumulated more Mn(2+) than the wild type, but did not show any obvious deleterious effects on growth. When grown with Mn(2+) supplies that ranged from basal to toxic, the mutant accumulated Mn(2+) concentrations in shoots similar to those in wild-type plants, despite showing symptoms of Mn(2+) toxicity. AtMTP11 fused to green fluorescent protein co-localized with a reporter specific for pre-vacuolar compartments. These findings provide evidence for Mn(2+)-specific transport activity by AtMTP11, and implicate the pre-vacuolar compartments in both Mn(2+) tolerance and Mn(2+) homeostasis mechanisms of Arabidopsis.     

Rice SCAMP1 defines clathrin-coated, trans-golgi-located tubular-vesicular structures as an early endosome in tobacco BY-2 cells

We recently identified multivesicular bodies (MVBs) as prevacuolar compartments (PVCs) in the secretory and endocytic pathways to the lytic vacuole in tobacco (Nicotiana tabacum) BY-2 cells. Secretory carrier membrane proteins (SCAMPs) are post-Golgi, integral membrane proteins mediating endocytosis in animal cells. To define the endocytic pathway in plants, we cloned the rice (Oryza sativa) homolog of animal SCAMP1 and generated transgenic tobacco BY-2 cells expressing yellow fluorescent protein (YFP)-SCAMP1 or SCAMP1-YFP fusions. Confocal immunofluorescence and immunogold electron microscopy studies demonstrated that YFP-SCAMP1 fusions and native SCAMP1 localize to the plasma membrane and mobile structures in the cytoplasm of transgenic BY-2 cells. Drug treatments and confocal immunofluorescence studies demonstrated that the punctate cytosolic organelles labeled by YFP-SCAMP1 or SCAMP1 were distinct from the Golgi apparatus and PVCs. SCAMP1-labeled organelles may represent an early endosome because the internalized endocytic markers FM4-64 and AM4-64 reached these organelles before PVCs. In addition, wortmannin caused the redistribution of SCAMP1 from the early endosomes to PVCs, probably as a result of fusions between the two compartments. Immunogold electron microscopy with high-pressure frozen/freeze-substituted samples identified the SCAMP1-positive organelles as tubular-vesicular structures at the trans-Golgi with clathrin coats. These early endosomal compartments resemble the previously described partially coated reticulum and trans-Golgi network in plant cells.     

High-sensitivity TFA-free LC-MS for profiling histones

The analysis of proteins by RPLC commonly involves the use of TFA as an ion‐pairing agent, even though it forms adducts and suppresses sensitivity. The presence of adducts can complicate protein molecular weight assignment especially when protein isoforms coelute as in the case of histones. To mitigate the complicating effects of TFA adducts in protein LC‐MS, we have optimized TFA‐free methods for protein separation. Protein standards and histones were used to evaluate TFA‐free separations using capillary (0.3 mm id) and nanoscale (0.1 mm id) C₈ columns with the ion‐pairing agents, formic acid or acetic acid. The optimized method was then used to examine the applicability of the approach for histone characterization in human cancer cell lines and primary tumor cells from chronic lymphocytic leukemia patients.     

Plant RMR proteins: unique vacuolar sorting receptors that couple ligand sorting with membrane internalization

In receptor-mediated sorting of soluble protein ligands in the endomembrane system of eukaryotic cells, three completely different receptor proteins for mammalian (mannose 6-phosphate receptor), yeast (Vps10p) and plant cells (vacuolar sorting receptor; VSR) have in common the features of pH-dependent ligand binding and receptor recycling. In striking contrast, the plant receptor homology-transmembrane-RING-H2 (RMR) proteins serve as sorting receptors to a separate type of vacuole, the protein storage vacuole, but do not recycle, and their trafficking pathway results in their internalization into the destination vacuole. Even though plant RMR proteins share high sequence similarity with the best-characterized mammalian PA-TM-RING family proteins, these two families of proteins appear to play distinctly different roles in plant and animal cells. Thus, this minireview focuses on this unique sorting mechanism and traffic of RMR proteins via dense vesicles in various plant cell types.     

Long-term exposure to ambient air pollution and mortality due to cardiovascular disease and cerebrovascular disease in Shenyang, China

Background

The relationship between ambient air pollution exposure and mortality of cardiovascular and cerebrovascular diseases in human is controversial, and there is little information about how exposures to ambient air pollution contribution to the mortality of cardiovascular and cerebrovascular diseases among Chinese. The aim of the present study was to examine whether exposure to ambient-air pollution increases the risk for cardiovascular and cerebrovascular disease.

Methodology/Principal Findings

We conducted a retrospective cohort study among humans to examine the association between compound-air pollutants [particulate matter <10 µm in aerodynamic diameter (PM₁₀), sulfur dioxide (SO₂) and nitrogen dioxide (NO₂)] and mortality in Shenyang, China, using 12 years of data (1998–2009). Also, stratified analysis by sex, age, education, and income was conducted for cardiovascular and cerebrovascular mortality. The results showed that an increase of 10 µg/m³ in a year average concentration of PM₁₀ corresponds to 55% increase in the risk of a death cardiovascular disease (hazard ratio [HR], 1.55; 95% confidence interval [CI], 1.51 to 1.60) and 49% increase in cerebrovascular disease (HR, 1.49; 95% CI, 1.45 to 1.53), respectively. The corresponding figures of adjusted HR (95%CI) for a 10 µg/m³ increase in NO₂ was 2.46 (2.31 to 2.63) for cardiovascular mortality and 2.44 (2.27 to 2.62) for cerebrovascular mortality, respectively. The effects of air pollution were more evident in female that in male, and nonsmokers and residents with BMI<18.5 were more vulnerable to outdoor air pollution.

Conclusion/Significance

Long-term exposure to ambient air pollution is associated with the death of cardiovascular and cerebrovascular diseases among Chinese populations.