Isolation and purification of deoxyribonucleosides from 90% 13C-enriched DNA of algal cells and their characterization by 1H and 13C NMR.

13C-enriched deoxyribonucleosides have been isolated from the DNA of Algal cells grown in an atmosphere of 90% 13C-labelled carbon dioxide. The 13C enriched DNA was quantitatively hydrolysed with DNase I, snake venom phosphodiesterase I and alkaline phosphatase of intestinal mucosa. The resulting deoxyribonucleosides were separated by preparative reversed-phase high pressure liquid chromatography in 60 minutes with detection by ultraviolet absorption at 254 nm. The final products were obtained in milligram quantities in high purity and in high yield. The 1H resonances of the base and sugar protons of these deoxyribonucleosides appear as well resolved multiplets in the 600 MHz NMR spectrum, due to the extensive 1H-13C couplings. Similarly, the 13C resonances of these deoxyribonucleosides appear as multiplets in the 75.5 MHz 13C NMR spectrum, due to 13C-13C couplings. The 1H-13C and 13C-13C coupling constants were also measured and tabulated. The isotopic enrichment of 13C these deoxyribonucleosides was obtained by integration of the 1H and/or 13C NMR spectra. It was found that the enrichment varied from carbon to carbon and species to species in the range of 70-89%, suggesting differential uptake and assimilation of 90% 13CO2 during metabolism pathways. This protocol provides experimentally useful quantities of 13C-enriched deoxyribonucleosides, which may be incorporated into site-specifically labeled oligonucleotides by chemical synthesis.     

Geometry-Dependent Surface Plasmonic Properties and Dielectric Sensitivity of Bimetallic Au@Pd Nanorods

Plasmonics - Bimetallic nanoparticles have attracted increasing interest because of their unique optical, electronic, magnetic, and catalytic properties which are different from that of their...     

Pleiotropic effects of the twin-arginine translocation system on biofilm formation, colonization, and virulence in Vibrio cholerae

Background  

The Twin-arginine translocation (Tat) system serves to translocate folded proteins, including periplasmic enzymes that bind redox cofactors in bacteria. The Tat system is also a determinant of virulence in some pathogenic bacteria, related to pleiotropic effects including growth, motility, and the secretion of some virulent factors. The contribution of the Tat pathway to Vibrio cholerae has not been explored. Here we investigated the functionality of the Tat system in V. cholerae, the etiologic agent of cholera.     

Elevated levels of fibrinogen-derived endogenous citrullinated peptides in synovial fluid of rheumatoid arthritis patients

Introduction

Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammation of the joints and the presence of autoantibodies directed against proteins containing the non-standard arginine-derived amino acid citrulline. The protein fibrinogen, which has an essential role in blood clotting, is one of the most prominent citrullinated autoantigens in RA, particularly because it can be found in the inflamed tissue of affected joints. Here, we set out to analyze the presence of citrullinated endogenous peptides in the synovial fluid of RA and arthritic control patients.

Methods

Endogenous peptides were isolated from the synovial fluid of RA patients and controls by filtration and solid phase extraction. The peptides were identified and quantified using high-resolution liquid chromatography-mass spectrometry.

Results

Our data reveal that the synovial fluid of RA patients contains soluble endogenous peptides, derived from fibrinogen, containing significant amounts of citrulline residues and, in some cases, also phosphorylated serine. Several citrullinated peptides are found to be more abundantly present in the synovial fluid of RA patients compared to patients suffering from other inflammatory diseases affecting the joints.

Conclusions

The increased presence of citrullinated peptides in RA patients points toward a possible specific role of these peptides in the immune response at the basis of the recognition of citrullinated peptides and proteins by RA patient autoantibodies.     

碳离子辐照对菘蓝药性品质和分子水平的诱变效应

以野生菘蓝种子为材料,以不同剂量的碳离子(12 C6+)进行辐照处理(辐照剂量分别为30Gy、60Gy、90Gy、120Gy),分析12 C6+辐照对菘蓝种子萌发、幼苗生长、主要药用成分含量及其基因和蛋白质多态性变化的影响,为菘蓝品质育种、分子生物学研究和重离子辐照诱变的应用提供依据。结果显示:(1)12 C6+辐照处理后菘蓝的成苗率和根鲜重均随辐照剂量增加而逐步显著降低,其中30Gy处理对菘蓝生长抑制程度最小,但处理后菘蓝根中的主要药效成分4(3H)喹唑酮和靛玉红的含量增加幅度最大且最高,分别为野生型的2.2倍和2.3倍。(2)SRAP分子标记分析表明,菘蓝基因组的变异度随着辐照强度的增强而增大,其中30Gy处理的突变体与野生型相比有33.59%的多态性变异。(3)SDS-PAGE考马斯亮蓝染色和磷酸化染色分析表明,菘蓝的总蛋白和磷酸化蛋白表达水平均随辐照剂量变化出现了不同程度的改变,但并不与辐照强度呈正相关,说明植物在防御重离子辐照伤害时存在补偿机制。研究发现,30Gy的12 C6+辐照是菘蓝诱变的最佳剂量,能够显著提高其有效成分的含量,为优质菘蓝诱变育种奠定了基础。     

A genetic variant in osteoprotegerin is associated with progression of joint destruction in rheumatoid arthritis

Introduction

Progression of joint destruction in rheumatoid arthritis (RA) is partly heritably; 45 to 58% of the variance in joint destruction is estimated to be explained by genetic factors. The binding of RANKL (Receptor Activator for Nuclear Factor κ B Ligand) to RANK results in the activation of TRAF6 (tumor necrosis factor (TNF) receptor associated factor-6), and osteoclast formation ultimately leading to enhanced bone resorption. This bone resorption is inhibited by osteoprotegerin (OPG) which prevents RANKL-RANK interactions. The OPG/RANK/RANKL/TRAF6 pathway plays an important role in bone remodeling. Therefore, we investigated whether genetic variants in OPG, RANK, RANKL and TRAF6 are associated with the rate of joint destruction in RA.

Methods

1,418 patients with 4,885 X-rays of hands and feet derived from four independent data-sets were studied. In each data-set the relative increase of the progression rate per year in the presence of a genotype was assessed. First, explorative analyses were performed on 600 RA-patients from Leiden. 109 SNPs, tagging OPG, RANK, RANKL and TRAF6, were tested. Single nucleotide polymorphisms (SNPs) significantly associated in phase-1 were genotyped in data-sets from Groningen (Netherlands), Sheffield (United Kingdom) and Lund (Switzerland). Data were summarized in an inverse weighted variance meta-analysis. Bonferonni correction for multiple testing was applied.

Results

We found that 33 SNPs were significantly associated with the rate of joint destruction in phase-1. In phase-2, six SNPs in OPG and four SNPs in RANK were associated with progression of joint destruction with P-value <0.05. In the meta-analyses of all four data-sets, RA-patients with the minor allele of OPG-rs1485305 expressed higher rates of joint destruction compared to patients without these risk variants (P = 2.35x10⁻⁴). This variant was also significant after Bonferroni correction.

Conclusions

These results indicate that a genetic variant in OPG is associated with a more severe rate of joint destruction in RA.     

Structural specificity in a FGF7-affinity purified heparin octasaccharide required for formation of a complex with FGF7 and FGFR2IIIb

Variations in sulfation of heparan sulfate (HS) affect interaction with FGF, FGFR, and FGF-HS-FGFR signaling complexes. Whether structurally distinct HS motifs are at play is unclear. Here we used stabilized recombinant FGF7 as a bioaffinity matrix to purify size-defined heparin oligosaccharides. We show that only 0.2%-4% of 6 to 14 unit oligosaccharides, respectively, have high affinity for FGF7 based on resistance to salt above 0.6M NaCl. The high affinity fractions exhibit highest specific activity for interaction with FGFR2IIIb and formation of complexes of FGF7-HS-FGFR2IIIb. The majority fractions with moderate (0.30-0.6M NaCl), low (0.14-0.30M NaCl) or no affinity at 0.14M NaCl for FGF7 supported no complex formation. The high affinity octasaccharide mixture exhibited predominantly 7- and 8-sulfated components (7,8-S-OctaF7) and formed FGF7-HS-FGFR2IIIb complexes with highest specific activity. Deduced disaccharide analysis indicated that 7,8-S-OctaF7 comprised of DeltaHexA2SGlcN6S in a 2:1 ratio to a trisulfated and a variable unsulfated or monosulfated disaccharide. The inactive octasaccharides with moderate affinity for FGF7 were much more heterogenous and highly sulfated with major components containing 11 or 12 sulfates comprised of predominantly trisulfated disaccharides. This suggests that a rare undersulfated motif in which sulfate groups are specifically distributed has highest affinity for FGF7. The same motif also exhibits structural requirements for high affinity binding to dimers of FGFR2IIIb prior to binding FGF7 to form FGF7-HS-FGFR2IIIb complexes. In contrast, the majority of more highly sulfated HS motifs likely play FGFR-independent roles in stability and control of access of FGF7 to FGFR2IIIb in the tissue matrix.     

3-[6-(2-Dimethylamino-1-imidazol-1-yl-butyl)-naphthalen-2-yloxy]-2,2-dimethyl-propionic acid as a highly potent and selective retinoic acid metabolic blocking agent

3-[6-(2-Dimethylamino-1-imidazol-1-yl-butyl)-naphthalen-2-yloxy]-2,2-dimethyl-propionic acid and analogs were designed and synthesized as highly potent and selective CYP26 inhibitors, serving as retinoic acid metabolic blocking agents (RAMBAs), with demonstrated in vivo efficacy to increase the half-life of exogenous atRA.     

Anti-proliferative effects of evodiamine on human prostate cancer cell lines DU145 and PC3

Prostate carcinoma is one of the most common malignant tumors and has become a more common cancer in men. Previous studies demonstrated that evodiamine (EVO) exhibited anti-tumor activities on several cancers, but its effects on androgen-independent prostate cancer are unclear. In the present study, the action mechanisms of EVO on the growth of androgen-independent prostate cancer cells (DU145 and PC3 cells) were explored. EVO dramatically inhibited the growth and elevated cytotoxicity of DU145 and PC3 cells. The flow cytometric analysis of EVO-treated cells indicated a block of G2/M phase and an elevated level of DNA fragmentation. The G2/M arrest was accompanied by elevated Cdc2 kinase activity, an increase in expression of cyclin B1 and phosphorylated Cdc2 (Thr 161), and a decrease in expression of phosphorylated Cdc2 (Tyr 15), Myt-1, and interphase Cdc25C. TUNEL examination showed that EVO-induced apoptosis was observed at 72 h. EVO elevated the activities of caspase 3, 8, and 9 in DU145 cells, while in PC3 cells only the activities of caspase 3 and 9 were elevated. EVO also triggered the processing of caspase 3 and 9 in both DU145 and PC3 cells. We demonstrate that roscovitine treatment result in the reversion of G2/M arrest in response to EVO in both DU145 and PC3. However, inhibitory effect of roscovitine on EVO-induced apoptosis could only be observed in DU145 rather than PC3. In DU145, G2/M arrest might be a signal for initiation of EVO-triggered apoptosis. Whereas EVO-triggered PC3 apoptosis might be independent of G2/M arrest. These results suggested that EVO inhibited the growth of prostate cancer cell lines, DU145 and PC3, through an accumulation at G2/M phase and an induction of apoptosis.     

Menadione increases hepatic tight-junctional permeability. Its effect can be decreased by butylated hydroxytoluene and verapamil.

Infusion of menadione at two different doses [2.7 mg and 5.5 mg in 100 microliters of dimethyl sulphoxide (DMSO)] into perfused rat livers for 30 min caused no or a 6-fold increase respectively in junctional permeability to horseradish peroxidase as compared with controls receiving 100 microliters of DMSO alone. The total glutathione (GSH) contents in these livers measured at the end of the experiments were 115% and 53%, compared with the controls. The free-radical scavenger butylated hydroxytoluene (BHT) (final concn. 5 microM) protected against the GSH depletion caused by the higher dose of menadione and partially decreased the menadione-induced increase in junctional permeability. Verapamil, a Ca2(+)-channel blocker which was added into the perfusion medium (final concn. 40 microM) 10 min before the infusion of 5.5 mg of menadione, completely abolished the effect of menadione on junctional permeability. Menadione exposure therefore increases tight-junctional permeability in the liver; this may involve a depletion of GSH and a subsequent increase in intracellular Ca2+.