Oligomeric Status and Nucleotide Binding Properties of the Plastid ATP/ADP Transporter 1: Toward a Molecular Understanding of the Transport Mechanism

Background: Chloroplast ATP/ADP transporters are essential to energy homeostasis in plant cells. However, their molecular mechanism remains poorly understood, primarily due to the difficulty of producing and purifying functional recombinant forms of these transporters. Methodology/Principal Findings: In this work, we describe an expression and purification protocol providing good yields and efficient solubilization of NTT1 protein from Arabidopsis thaliana. By biochemical and biophysical analyses, we identified the best detergent for solubilization and purification of functional proteins, LAPAO. Purified NTT1 was found to accumulate as two independent pools of well folded, stable monomers and dimers. ATP and ADP binding properties were determined, and Pi, a co-substrate of ADP, was confirmed to be essential for nucleotide steady-state transport. Nucleotide binding studies and analysis of NTT1 mutants lead us to suggest the existence of two distinct and probably inter-dependent binding sites. Finally, fusion and deletion experiments demonstrated that the C-terminus of NTT1 is not essential for multimerization, but probably plays a regulatory role, controlling the nucleotide exchange rate. Conclusions/Significance: Taken together, these data provide a comprehensive molecular characterization of a chloroplas

Antisense oligonucleotides and all-trans retinoic acid have a synergistic anti-tumor effect on oral squamous cell carcinoma

<p>Abstract</p> <p>Background</p> <p>Antisense oligonucleotides against hTR (As-ODN-hTR) have shown promising results as treatment strategies for various human malignancies. All-trans retinoic acid (ATRA) is a signalling molecule with important roles in differentiation and apoptosis. Biological responses to ATRA are currently used therapeutically in various human cancers. The aim of this study was to evaluate the anti-tumor effects of As-ODN-hTR combined with ATRA in vivo.</p> <p>Methods</p> <p>In situ human oral squamous cell carcinoma (OSCC) models were established by subcutaneous injection of Tca8113 cells. Mice were treated with sense oligonucleotides against hTR(S-ODN-hTR) alone, As-ODN-hTR alone, ATRA alone, As-ODN-hTR plus ATRA, or S-ODN-hTR plus ATRA. Tumor size and weight were assessed in the mice. Telomerase activity was detected by a TRAP assay, apoptotic cells were evaluated with a Tunel assay, the expression of apoptosis-related proteins (Bcl-2 and Bax) was evaluated by immunohistochemistry and ultrastructural morphological changes in the tumor specimen were examined.</p> <p>Results</p> <p>Both As-ODN-hTR and ATRA can significantly inhibit tumor growth in this OSCC xenograft solid-tumor model, and the combination of the two agents had a synergistic anti-tumorogenic effect. We also demonstrated that this anti-tumor effect correlated with inhibition of telomerase activity. Furthermore, significant increases in the number of apoptotic cells, typical apoptotic morphology and a downregulation of the anti-apoptotic protein, bcl-2 were observed in the treated tissues.</p> <p>Conclusion</p> <p>The combination of As-ODN-hTR and ATRA has a synergistic anti-tumor effect. This anti-tumor effect can be mainly attributed to apoptosis induced by a decrease in telomerase activity. Bcl-2 plays an important role in this process. Therefore, combining As-ODN-hTR and ATRA may be an approach for the treatment of human oral squamous cell carcinoma.</p

Alkaline-earth phosphonate MOFs with reversible hydration-dependent fluorescence

A new rigid tritopic phosphonic ligand, 2,4,6-tris(4-phosphonophenyl)pyridine (H6L), was synthesized and used to assemble isostructural barium (1) and strontium (2) phosphonate metal organic frameworks that exhibit fully reversible and selective water-dependent fluorescence red-shift at room temperature

Crystal Structure of Escherichia coli CusC, the Outer Membrane Component of a Heavy Metal Efflux Pump

Background: While copper has essential functions as an enzymatic co-factor, excess copper ions are toxic for cells, necessitating mechanisms for regulating its levels. The cusCBFA operon of E. coli encodes a four-component efflux pump dedicated to the extrusion of Cu(I) and Ag(I) ions. Methodology/Principal Findings: We have solved the X-ray crystal structure of CusC, the outer membrane component of the Cus heavy metal efflux pump, to 2.3 A ˚ resolution. The structure has the largest extracellular opening of any outer membrane factor (OMF) protein and suggests, for the first time, the presence of a tri-acylated N-terminal lipid anchor. Conclusions/Significance: The CusC protein does not have any obvious features that would make it specific for metal ions, suggesting that the narrow substrate specificity of the pump is provided by other components of the pump, most likely by the inner membrane component CusA

Roles of IP3R and RyR Ca2+ Channels in Endoplasmic Reticulum Stress and β-Cell Death

OBJECTIVE—Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of diabetes, but the roles of specific ER Ca2+ release channels in the ER stress–associated apoptosis pathway remain unknown. Here, we examined the effects of stimulating or inhibiting the ER-resident inositol trisphosphate receptors (IP3Rs) and the ryanodine receptors (RyRs) on the induction of β-cell ER stress and apoptosis

Selective Trihydroxylated Azepane Inhibitors of NagZ, a Glycosidase Involved in Pseudomonas Aeruginosa Resistance to β-lactam Antibiotics

The synthesis of a series of D-gluco-like configured 4,5,6-trihydroxyazepanes bearing a triazole, a sulfonamide or a fluorinated acetamide moiety at C-3 is described. These synthetic derivatives have been tested for their ability to selectively inhibit the muropeptide recycling glucosaminidase NagZ and to thereby increase sensitivity of Pseudomonas aeruginosa to β-lactams, a pathway with substantial therapeutic potential. While introduction of triazole and sulfamide groups failed to lead to glucosaminidase inhibitors, the NHCOCF3 analog proved to be a selective inhibitor of NagZ over other glucosaminidases including human OGA and lysosomal hexosaminidases HexA and B. The synthesis of a series of D-gluco-like configured 4,5,6-trihydroxyazepanes bearing a triazole, a sulfonamide or a fluorinated acetamide moiety at C-3 is described. These synthetic derivatives have been tested for their ability to selectively inhibit the muropeptide recycling glucosaminidase NagZ and to thereby increase sensitivity of Pseudomonas aeruginosa to β-lactams, a pathway with substantial therapeutic potential. While introduction of triazole and sulfamide groups failed to lead to glucosaminidase inhibitors, the NHCOCF3 analog proved to be a selective inhibitor of NagZ over other glucosaminidases including human OGA and lysosomal hexosaminidases HexA and B &nbsp

Mdm2-SNP309 polymorphism in prostate cancer: no evidence for association with increased risk or histopathological tumour characteristics

The search for inherited cancer susceptibility factors is a major focus of epidemiologic cancer studies. Analyses of single-nucleotide polymorphisms (SNP) in a variety of genes revealed a correlation between a specific allele variant and cancer predisposition. Human mouse double-minute 2 protein (Mdm2) is a cellular E3 ligase capable of ubiquitination and degradation of p53. Therefore, Mdm2 is a crucial factor of cell cycle control and cell survival. The Mdm2 promoter SNP309 was shown to increase Mdm2 expression and can, thereby, inhibit the p53 pathway. This SNP was found to be associated with increased risk and early onset of various malignancies. For prostate cancer no studies are reported to date. In a case–control study we determined the distribution of the Mdm2 SNP309 in 145 male subjects with prostate cancer and in 124 male controls without any malignancy using RFLP analysis. Cases and controls showed a similar distribution of the SNP (P=0.299). Genotype distribution showed neither an association with histopathological characteristics of the tumours nor with prognosis. Age at disease onset was also not modified by the SNP. This first study of the Mdm2 SNP309 in prostate cancer patients suggests no correlation between a certain allelic variant and an increased cancer risk

Enhancement of crystallization with nucleotide ligands identified by dye-ligand affinity chromatography

Ligands interacting with Mycobacterium tuberculosis recombinant proteins were identified through use of the ability of Cibacron Blue F3GA dye to interact with nucleoside/nucleotide binding proteins, and the effects of these ligands on crystallization were examined. Co-crystallization with ligands enhanced crystallization and enabled X-ray diffraction data to be collected to a resolution of at least 2.7 Å for 5 of 10 proteins tested. Additionally, clues about individual proteins’ functions were obtained from their interactions with each of a panel of ligands

From endoplasmic-reticulum stress to the inflammatory response

The endoplasmic reticulum is responsible for much of a cell's protein synthesis and folding, but it also has an important role in sensing cellular stress. Recently, it has been shown that the endoplasmic reticulum mediates a specific set of intracellular signalling pathways in response to the accumulation of unfolded or misfolded proteins, and these pathways are collectively known as the unfolded-protein response. New observations suggest that the unfolded-protein response can initiate inflammation, and the coupling of these responses in specialized cells and tissues is now thought to be fundamental in the pathogenesis of inflammatory diseases. The knowledge gained from this emerging field will aid in the development of therapies for modulating cellular stress and inflammation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62741/1/nature07203.pd