Molecular characterization and expression pattern of dmrt1 in the immature Chinese sturgeon Acipenser sinensis

In this study, the cDNA of dmrt1 gene from the Chinese sturgeon Acipenser sinensis was isolated and its expression pattern was characterized in different tissues of immature A. sinensis. By real‐time quantitative PCR (qrtPCR) analysis, the A. sinensis dmrt1 mRNA was detected mainly in gonad and with a higher level in the testis than the ovary, especially in 3 and 4 year‐old samples. This indicated that the dmrt1 expression exhibited gradual testis specificity with development. The subcellular localization analysis indicated that the Dmrt1 protein exists only in germ cells and not in somatic cells. These results suggest that A. sinensis dmrt1 might be a highly specific sex differentiation gene for testis development and spermatogenesis.     

Helper component‐proteinase enhances the activity of 1‐deoxy‐D‐xylulose‐5‐phosphate synthase and promotes the biosynthesis of plastidic isoprenoids in Potato virus Y‐infected tobacco

Virus‐infected plants show strong morphological and physiological alterations. Many physiological processes in chloroplast are affected, including the plastidic isoprenoid biosynthetic pathway [the 2C‐methyl‐D‐erythritol‐4‐phosphate (MEP) pathway]; indeed, isoprenoid contents have been demonstrated to be altered in virus‐infected plants. In this study, we found that the levels of photosynthetic pigments and abscisic acid (ABA) were altered in Potato virus Y (PVY)‐infected tobacco. Using yeast two‐hybrid assays, we demonstrated an interaction between virus protein PVY helper component‐proteinase (HC‐Pro) and tobacco chloroplast protein 1‐deoxy‐D‐xylulose‐5‐phosphate synthase (NtDXS). This interaction was confirmed using bimolecular fluorescence complementation (BiFC) assays and pull‐down assays. The Transket_pyr domain (residues 394–561) of NtDXS was required for interaction with HC‐Pro, while the N‐terminal region of HC‐Pro (residues 1–97) was necessary for interaction with NtDXS. Using in vitro enzyme activity assays, PVY HC‐Pro was found to promote the synthase activity of NtDXS. We observed increases in photosynthetic pigment contents and ABA levels in transgenic plants with HC‐Pro accumulating in the chloroplasts. During virus infection, the enhancement of plastidic isoprenoid biosynthesis was attributed to the enhancement of DXS activity by HC‐Pro. Our study reveals a new role of HC‐Pro in the host plant metabolic system and will contribute to the study of host–virus relationships.     

Role of Deacetylase Activity of N-Deacetylase/N-Sulfotransferase 1 in Forming N-Sulfated Domain in Heparan Sulfate

Heparan sulfate (HS) is a highly sulfated polysaccharide that plays important physiological roles. The biosynthesis of HS involves a series of enzymes, including glycosyltransferases (or HS polymerase), epimerase, and sulfotransferases. N-Deacetylase/N-Sulfotransferase isoform 1 (NDST-1) is a critical enzyme in this pathway. NDST-1, a bifunctional enzyme, displays N-deacetylase and N-sulfotransferase activities to convert an N-acetylated glucosamine residue to an N-sulfo glucosamine residue. Here, we report the cooperative effects between N-deacetylase and N-sulfotransferase activities. Using baculovirus expression in insect cells, we obtained three recombinant proteins: full-length NDST-1 and the individual N-deacetylase and N-sulfotransferase domains. Structurally defined oligosaccharide substrates were synthesized to test the substrate specificities of the enzymes. We discovered that N-deacetylation is the limiting step and that interplay between the N-sulfotransferase and N-deacetylase accelerates the reaction. Furthermore, combining the individually expressed N-deacetylase and N-sulfotransferase domains produced different sulfation patterns when compared with that made by the NDST-1 enzyme. Our data demonstrate the essential role of domain cooperation within NDST-1 in producing HS with specific domain structures.     

A high-throughput screen measuring ubiquitination of p53 by human mdm2

Tumor suppressor p53 is typically maintained at low levels in normal cells. In response to cellular stresses, such as DNA damage, p53 is stabilized and can stimulate responses leading to cell cycle arrest or apoptosis. Corresponding to its central role in preventing propagation of damaged cells, mutation or deletion of p53 is found in nearly 50% of all human tumors. Mdm2 (mouse-d-minute 2) and its human ortholog (hmdm2 or hdm2) catalyze the ubiquitination of p53, targeting it for degradation via the proteosome. Thus, the activity of mdm2 is inversely correlated with p53 levels. Based on this, inhibition of human mdm2 activity by a small-molecule therapeutic will lead to net stabilization of p53 and be the basis for development of a novel cancer therapeutic. Previous high-throughput screening assays of mdm2 measured the autoubiquitination activity of mdm2, which occurs in the absence of an acceptor substrate such as p53. The major drawback to this approach is that inhibitors of mdm2 autoubiquitination may lead to a net stabilization of mdm2 and thus have the opposite effect of inhibitors that interfere with p53 ubiquitination. The authors describe the development, validation, and execution of a high-throughput screening measuring the ubiquitination of p53 by mdm2, with p53 labeled with europium and the other substrate (Ub-UbcH5b) labeled with a Cy5 on the ubiquitin. After confirming that known inhibitors are detected with this assay, it was successfully automated and used to query >600,000 compounds from the GlaxoSmithKline collection for mdm2 inhibitors.     

Death don't have no mercy and neither does calcium: Arabidopsis CYCLIC NUCLEOTIDE GATED CHANNEL2 and innate immunity

Plant innate immune response to pathogen infection includes an elegant signaling pathway leading to reactive oxygen species generation and resulting hypersensitive response (HR); localized programmed cell death in tissue surrounding the initial infection site limits pathogen spread. A veritable symphony of cytosolic signaling molecules (including Ca(2+), nitric oxide [NO], cyclic nucleotides, and calmodulin) have been suggested as early components of HR signaling. However, specific interactions among these cytosolic secondary messengers and their roles in the signal cascade are still unclear. Here, we report some aspects of how plants translate perception of a pathogen into a signal cascade leading to an innate immune response. We show that Arabidopsis thaliana CYCLIC NUCLEOTIDE GATED CHANNEL2 (CNGC2/DND1) conducts Ca(2+) into cells and provide a model linking this Ca(2+) current to downstream NO production. NO is a critical signaling molecule invoking plant innate immune response to pathogens. Plants without functional CNGC2 lack this cell membrane Ca(2+) current and do not display HR; providing the mutant with NO complements this phenotype. The bacterial pathogen-associated molecular pattern elicitor lipopolysaccharide activates a CNGC Ca(2+) current, which may be linked to NO generation due to buildup of cytosolic Ca(2+)/calmodulin.     

Polymorphism of heart fatty acid-binding protein gene associatied with fatness traits in the chicken

Fatty acid-binding proteins (FABP) belong to a superfamily of lipid binding proteins that exhibit a high affinity for long chain fatty acids and appear to function in metabolism and intracellular transportation of lipids. The current study was designed to investigate the effects of heart (H)-FABP gene on chicken growth and body composition traits. The Northeast Agricultural University divergent broiler lines for abdominal fat and a broiler X silkie F2 population were used in this study. Body weight and body composition traits were measured in the populations. Primers were designed according to the chicken H-FABP gene sequence. Polymorphisms between parental lines were detected by DNA sequencing. PCR-RFLP and PCR-fragment length polymorphism methods were developed to genotype the populations. The results showed that the H-FABP gene polymorphisms in the two populations were associated with abdominal fat percentage. It implied that H-FABP gene can be a candidate locus or linked to a major gene(s) that affects abdominal fat content in the chicken.     

MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment

The cytokine macrophage migration inhibitory factor (MIF) plays a critical role in inflammatory diseases and atherogenesis. We identify the chemokine receptors CXCR2 and CXCR4 as functional receptors for MIF. MIF triggered G(alphai)- and integrin-dependent arrest and chemotaxis of monocytes and T cells, rapid integrin activation and calcium influx through CXCR2 or CXCR4. MIF competed with cognate ligands for CXCR4 and CXCR2 binding, and directly bound to CXCR2. CXCR2 and CD74 formed a receptor complex, and monocyte arrest elicited by MIF in inflamed or atherosclerotic arteries involved both CXCR2 and CD74. In vivo, Mif deficiency impaired monocyte adhesion to the arterial wall in atherosclerosis-prone mice, and MIF-induced leukocyte recruitment required Il8rb (which encodes Cxcr2). Blockade of Mif but not of canonical ligands of Cxcr2 or Cxcr4 in mice with advanced atherosclerosis led to plaque regression and reduced monocyte and T-cell content in plaques. By activating both CXCR2 and CXCR4, MIF displays chemokine-like functions and acts as a major regulator of inflammatory cell recruitment and atherogenesis. Targeting MIF in individuals with manifest atherosclerosis can potentially be used to treat this condition.     

The physiological response of marine diatoms to ocean acidification: differential roles of seawater pCO2 and pH

Although increasing the pCO₂ for diatoms will presumably down‐regulate the CO₂‐concentrating mechanism (CCM) to save energy for growth, different species have been reported to respond differently to ocean acidification (OA). To better understand their growth responses to OA, we acclimated the diatoms Thalassiosira pseudonana, Phaeodactylum tricornutum, and Chaetoceros muelleri to ambient (pCO₂ 400 μatm, pH 8.1), carbonated (pCO₂ 800 μatm, pH 8.1), acidified (pCO₂ 400 μatm, pH 7.8), and OA (pCO₂ 800 μatm, pH 7.8) conditions and investigated how seawater pCO₂ and pH affect their CCMs, photosynthesis, and respiration both individually and jointly. In all three diatoms, carbonation down‐regulated the CCMs, while acidification increased both the photosynthetic carbon fixation rate and the fraction of CO₂ as the inorganic carbon source. The positive OA effect on photosynthetic carbon fixation was more pronounced in C. muelleri, which had a relatively lower photosynthetic affinity for CO₂, than in either T. pseudonana or P. tricornutum. In response to OA, T. pseudonana increased respiration for active disposal of H⁺ to maintain its intracellular pH, whereas P. tricornutum and C. muelleri retained their respiration rate but lowered the intracellular pH to maintain the cross‐membrane electrochemical gradient for H⁺ efflux. As the net result of changes in photosynthesis and respiration, growth enhancement to OA of the three diatoms followed the order of C. muelleri > P. tricornutum > T. pseudonana. This study demonstrates that elucidating the separate and joint impacts of increased pCO₂ and decreased pH aids the mechanistic understanding of OA effects on diatoms in the future, acidified oceans.