Characterisation of the Mouse Vasoactive Intestinal Peptide Receptor Type 2 Gene, Vipr2, and Identification of a Polymorphic LINE-1-like Sequence That Confers Altered Promoter Activity

The VPAC(2) receptor is a seven transmembrane spanning G protein-coupled receptor for two neuropeptides, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). It has a distinct tissue-specific, developmental and inducible expression that underlies an important neuroendocrine role. Here, we report the characterisation of the gene that encodes the mouse VPAC(2) receptor (Vipr2), localisation of the transcriptional start site and functional analysis of the promoter region. The Vipr2 gene contains 12 introns within its protein-coding region and spans 68.6 kb. Comparison of the 5′ untranslated region sequences for cloned 5′-RACE products amplified from different tissues showed they all were contained within the same exon, with the longest extending 111 bp upstream of the ATG start site. Functional analysis of the 3.2-kb 5′-flanking region using sequentially deleted sequences cloned into a luciferase gene reporter vector revealed that this region is active as a promoter in mouse AtT20 D16:16 and rat GH4C1 cell lines. The core promoter is located within a 180-bp GC-rich region proximal to the ATG start codon and contains potential binding sites for Sp1 and AP2, but no TATA-box. Further upstream, in two out of three mice strains examined, we have discovered a 496-bp polymorphic DNA sequence that bears a significant identity to mouse LINE-1 DNA. Comparison of the promoter activity between luciferase reporter gene constructs derived from the BALB/c (which contains this sequence) and C57BL/6J (which lacks this sequence) Vipr2 promoter regions has shown three-fold difference in luciferase gene activity when expressed in mouse AtT20 D16:16 and αT3-1 cells, but not when expressed in the rat GH4C1 cells or in COS 7 cells. Our results suggest that the mouse Vipr2 gene may be differentially active in different mouse strains, depending on the presence of this LINE-1-like sequence in the promoter region

Genetic deficiency of aldose reductase counteracts the development of diabetic nephropathy in C57BL/6 mice

National Science Foundation of China [30770490]; 973 Program of China [2009CB941601]; Science Planning Program of Fujian Province [2009J1010]; Natural Science Foundation of Fujian Province [2009J01180]; Fujian Provincial Department of Science and TechnoloThe aim of the study was to investigate the effects of genetic deficiency of aldose reductase in mice on the development of key endpoints of diabetic nephropathy. A line of Ar (also known as Akr1b3)-knockout (KO) mice, a line of Ar-bitransgenic mice and control C57BL/6 mice were used in the study. The KO and bitransgenic mice were deficient for Ar in the renal glomeruli and all other tissues, with the exception of, in the bitransgenic mice, a human AR cDNA knockin-transgene that directed collecting-tubule epithelial-cell-specific AR expression. Diabetes was induced in 8-week-old male mice with streptozotocin. Mice were further maintained for 17 weeks then killed. A number of serum and urinary variables were determined for these 25-week-old mice. Periodic acid-Schiff staining, western blots, immunohistochemistry and protein kinase C (PKC) activity assays were performed for histological analyses, and to determine the levels of collagen IV and TGF-beta 1 and PKC activities in renal cortical tissues. Diabetes-induced extracellular matrix accumulation and collagen IV overproduction were completely prevented in diabetic Ar-KO and bitransgenic mice. Ar deficiency also completely or partially prevented diabetes-induced activation of renal cortical PKC, TGF-beta 1 and glomerular hypertrophy. Loss of Ar results in a 43% reduction in urine albumin excretion in the diabetic Ar-KO mice and a 48% reduction in the diabetic bitransgenic mice (p < 0.01). Genetic deficiency of Ar significantly ameliorated development of key endpoints linked with early diabetic nephropathy in vivo. Robust and specific inhibition of aldose reductase might be an effective strategy for the prevention and treatment of diabetic nephropathy

Characterization of Yeast Extracellular Vesicles: Evidence for the Participation of Different Pathways of Cellular Traffic in Vesicle Biogenesis

Background: Extracellular vesicles in yeast cells are involved in the molecular traffic across the cell wall. In yeast pathogens, these vesicles have been implicated in the transport of proteins, lipids, polysaccharide and pigments to the extracellular space. Cellular pathways required for the biogenesis of yeast extracellular vesicles are largely unknown. Methodology/Principal Findings: We characterized extracellular vesicle production in wild type (WT) and mutant strains of the model yeast Saccharomyces cerevisiae using transmission electron microscopy in combination with light scattering analysis, lipid extraction and proteomics. WT cells and mutants with defective expression of Sec4p, a secretory vesicleassociated Rab GTPase essential for Golgi-derived exocytosis, or Snf7p, which is involved in multivesicular body (MVB) formation, were analyzed in parallel. Bilayered vesicles with diameters at the 100–300 nm range were found in extracellular fractions from yeast cultures. Proteomic analysis of vesicular fractions from the cells aforementioned and additional mutants with defects in conventional secretion pathways (sec1-1, fusion of Golgi-derived exocytic vesicles with the plasm

Evolutionary Conservation of the Functional Modularity of Primate and Murine LINE-1 Elements

LINE-1 (L1) retroelements emerged in mammalian genomes over 80 million years ago with a few dominant subfamilies amplifying over discrete time periods that led to distinct human and mouse L1 lineages. We evaluated the functional conservation of L1 sequences by comparing retrotransposition rates of chimeric human-rodent L1 constructs to their parental L1 counterparts. Although amino acid conservation varies from ∼35% to 63% for the L1 ORF1p and ORF2p, most human and mouse L1 sequences can be functionally exchanged. Replacing either ORF1 or ORF2 to create chimeric human-mouse L1 elements did not adversely affect retrotransposition. The mouse ORF2p retains retrotransposition-competency to support both Alu and L1 mobilization when any of the domain sequences we evaluated were substituted with human counterparts. However, the substitution of portions of the mouse cys-domain into the human ORF2p reduces both L1 retrotransposition and Alu trans-mobilization by 200–1000 fold. The observed loss of ORF2p function is independent of the endonuclease or reverse transcriptase activities of ORF2p and RNA interaction required for reverse transcription. In addition, the loss of function is physically separate from the cysteine-rich motif sequence previously shown to be required for RNP formation. Our data suggest an additional role of the less characterized carboxy-terminus of the L1 ORF2 protein by demonstrating that this domain, in addition to mediating RNP interaction(s), provides an independent and required function for the retroelement amplification process. Our experiments show a functional modularity of most of the LINE sequences. However, divergent evolution of interactions within L1 has led to non-reciprocal incompatibilities between human and mouse ORF2 cys-domain sequences

Competitive and Cooperative Interactions Mediate RNA Transfer from Herpesvirus Saimiri ORF57 to the Mammalian Export Adaptor ALYREF

The essential herpesvirus adaptor protein HVS ORF57, which has homologs in all other herpesviruses, promotes viral mRNA export by utilizing the cellular mRNA export machinery. ORF57 protein specifically recognizes viral mRNA transcripts, and binds to proteins of the cellular transcription-export (TREX) complex, in particular ALYREF. This interaction introduces viral mRNA to the NXF1 pathway, subsequently directing it to the nuclear pore for export to the cytoplasm. Here we have used a range of techniques to reveal the sites for direct contact between RNA and ORF57 in the absence and presence of ALYREF. A binding site within ORF57 was characterized which recognizes specific viral mRNA motifs. When ALYREF is present, part of this ORF57 RNA binding site, composed of an a-helix, binds preferentially to ALYREF. This competitively displaces viral RNA from the a-helix, but contact with RNA is still maintained by a flanking region. At the same time, the flexible N-terminal domain of ALYREF comes into contact with the viral RNA, which becomes engaged in an extensive network of synergistic interactions with both ALYREF and ORF57. Transfer of RNA to ALYREF in the ternary complex, and involvement of individual ORF57 residues in RNA recognition, were confirmed by UV cross-linking and mutagenesis. The atomic-resolution structure of the ORF57-ALYREF interface was determined, which noticeably differed from the homologous ICP27-ALYREF structure. Together, the data provides the first site-specific description of how viral mRNA is locked by a herpes viral adaptor protein in complex with cellular ALYREF, giving herpesvirus access to the cellular mRNA export machinery. The NMR strategy used may be more generally applicable to the study of fuzzy protein-protein-RNA complexes which involve flexible polypeptide regions

Genome-Wide Screen of Three Herpesviruses for Protein Subcellular Localization and Alteration of PML Nuclear Bodies

Herpesviruses are large, ubiquitous DNA viruses with complex host interactions, yet many of the proteins encoded by these viruses have not been functionally characterized. As a first step in functional characterization, we determined the subcellular localization of 234 epitope-tagged proteins from herpes simplex virus, cytomegalovirus, and Epstein–Barr virus. Twenty-four of the 93 proteins with nuclear localization formed subnuclear structures. Twelve of these localized to the nucleolus, and five at least partially localized with promyelocytic leukemia (PML) bodies, which are known to suppress viral lytic infection. In addition, two proteins disrupted Cajal bodies, and 19 of the nuclear proteins significantly decreased the number of PML bodies per cell, including six that were shown to be SUMO-modified. These results have provided the first functional insights into over 120 previously unstudied proteins and suggest that herpesviruses employ multiple strategies for manipulating nuclear bodies that control key cellular processes

Genome-wide analyses identify common variants associated with macular telangiectasia type 2

Idiopathic juxtafoveal retinal telangiectasis type 2 (macular telangiectasia type 2; MacTel) is a rare neurovascular degenerative retinal disease. To identify genetic susceptibility loci for MacTel, we performed a genome-wide association study (GWAS) with 476 cases and 1,733 controls of European ancestry. Genome-wide significant associations (P < 5 × 10−8) were identified at three independent loci (rs73171800 at 5q14.3, P = 7.74 × 10−17; rs715 at 2q34, P = 9.97 × 10−14; rs477992 at 1p12, P = 2.60 × 10−12) and then replicated (P < 0.01) in an independent cohort of 172 cases and 1,134 controls. The 5q14.3 locus is known to associate with variation in retinal vascular diameter, and the 2q34 and 1p12 loci have been implicated in the glycine/serine metabolic pathway. We subsequently found significant differences in blood serum levels of glycine (P = 4.04 × 10−6) and serine (P = 2.48 × 10−4) between MacTel cases and controls

Climate simulations for 1880-2003 with GISS modelE

We carry out climate simulations for 1880-2003 with GISS modelE driven by ten measured or estimated climate forcings. An ensemble of climate model runs is carried out for each forcing acting individually and for all forcing mechanisms acting together. We compare side-by-side simulated climate change for each forcing, all forcings, observations, unforced variability among model ensemble members, and, if available, observed variability. Discrepancies between observations and simulations with all forcings are due to model deficiencies, inaccurate or incomplete forcings, and imperfect observations. Although there are notable discrepancies between model and observations, the fidelity is sufficient to encourage use of the model for simulations of future climate change. By using a fixed well-documented model and accurately defining the 1880-2003 forcings, we aim to provide a benchmark against which the effect of improvements in the model, climate forcings, and observations can be tested. Principal model deficiencies include unrealistically weak tropical El Nino-like variability and a poor distribution of sea ice, with too much sea ice in the Northern Hemisphere and too little in the Southern Hemisphere. The greatest uncertainties in the forcings are the temporal and spatial variations of anthropogenic aerosols and their indirect effects on clouds.Comment: 44 pages; 19 figures; Final text accepted by Climate Dynamic

The role of the mitochondria and the endoplasmic reticulum contact sites in the development of the immune responses

Abstract Mitochondria and endoplasmic reticulum (ER) contact sites (MERCs) are dynamic modules enriched in subset of lipids and specialized proteins that determine their structure and functions. The MERCs regulate lipid transfer, autophagosome formation, mitochondrial fission, Ca2+ homeostasis and apoptosis. Since these functions are essential for cell biology, it is therefore not surprising that MERCs also play a critical role in organ physiology among which the immune system stands by its critical host defense function. This defense system must discriminate and tolerate host cells and beneficial commensal microorganisms while eliminating pathogenic ones in order to preserve normal homeostasis. To meet this goal, the immune system has two lines of defense. First, the fast acting but unspecific innate immune system relies on anatomical physical barriers and subsets of hematopoietically derived cells expressing germline-encoded receptors called pattern recognition receptors (PRR) recognizing conserved motifs on the pathogens. Second, the slower but very specific adaptive immune response is added to complement innate immunity. Adaptive immunity relies on another set of specialized cells, the lymphocytes, harboring receptors requiring somatic recombination to be expressed. Both innate and adaptive immune cells must be activated to phagocytose and process pathogens, migrate, proliferate, release soluble factors and destroy infected cells. Some of these functions are strongly dependent on lipid transfer, autophagosome formation, mitochondrial fission, and Ca2+ flux; this indicates that MERCs could regulate immunity