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.     

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。     

Varp Is a Novel Rab32/38-binding Protein That Regulates Tyrp1 Trafficking in Melanocytes

Two small GTPase Rabs, Rab32 and Rab38, have recently been proposed to regulate trafficking of melanogenic enzymes to melanosomes in mammalian epidermal melanocytes; however, the exact molecular mechanism of Rab32/38-mediated transport of melanogenic enzymes has never been clarified, because no Rab32/38-specific effector has ever been identified. In this study, we screened for a Rab32/38-specific effector by a yeast two-hybrid assay using a guanosine triphosphate (GTP)-locked Rab32/38 as bait and found that VPS9-ankyrin-repeat protein (Varp)/Ankrd27, characterized previously as a guanine nucleotide exchange factor (GEF) for Rab21, functions as a specific Rab32/38-binding protein in mouse melanocyte cell line melan-a. Deletion analysis showed that the first ankyrin-repeat (ANKR1) domain functions as a GTP-dependent Rab32/38-binding domain, but that the N-terminal VPS9 domain (i.e., Rab21-GEF domain) does not. Small interfering RNA-mediated knockdown of endogenous Varp in melan-a cells caused a dramatic reduction in Tyrp1 (tyrosinase-related protein 1) signals from melanosomes but did not cause any reduction in Pmel17 signals. Furthermore, expression of the ANKR1 domain in melan-a cells also caused a dramatic reduction of Tyrp1 signals, whereas the VPS9 domain had no effect. Based on these findings, we propose that Varp functions as the Rab32/38 effector that controls trafficking of Tyrp1 in melanocytes.     

Molecular analysis of RANKL-independent cell fusion of osteoclast-like cells induced by TNF-alpha, lipopolysaccharide, or peptidoglycan

Focusing on the final step of osteoclastogenesis, we studied cell fusion from tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells into multinuclear cells. TRAP-positive mononuclear cells before generation of multinuclear cells by cell fusion were differentiated from RAW264.7 cells by treatment with receptor activator of nuclear factor kappa B ligand (RANKL), and then the cells were treated with lipopolysaccharide (LPS), followed by culturing for further 12 h. LPS-induced cell fusion even in the absence of RANKL. Similarly, tumor necrosis factor (TNF)-alpha and peptidoglycan (PGN) induced cell fusion, but M-CSF did not. The cell fusion induced by RANKL, TNF-alpha, and LPS was specifically blocked by osteoprotegerin (OPG), anti-TNF-alpha antibody, and polymyxin B, respectively. LPS- and PGN-induced cell fusion was partly inhibited by anti-TNF-alpha antibody but not by OPG. When TRAP-positive mononuclear cells fused to yield multinuclear cells, phosphorylation of Akt, Src, extracellular signal-regulated kinase (ERK), p38MAPK (p38), and c-Jun NH2-terminal kinase (JNK) was observed. The specific chemical inhibitors LY294002 (PI3K), PP2 (Src), U0126 (MAPK-ERK kinase (MEK)/ERK), and SP600125 (JNK) effectively suppressed cell fusion, although SB203580 (p38) did not. mRNA of nuclear factor of activated T-cells c1 (NFATc1) and dendritic cell-specific transmembrane protein (DC-STAMP) during the cell fusion was quantified, however, there was no obvious difference among the TRAP-positive mononuclear cells treated with or without M-CSF, RANKL, TNF-alpha, LPS, or PGN. Collectively, RANKL, TNF-alpha, LPS, and PGN induced cell fusion of osteoclasts through their own receptors. Subsequent activation of signaling pathways involving PI3K, Src, ERK, and JNK molecules was required for the cell fusion. Although DC-STAMP is considered to be a requisite for cell fusion of osteoclasts, cell fusion-inducing factors other than DC-STAMP might be necessary for the cell fusion.     

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.     

Angiotensin II mimics the hypoxic effect on regulating trophoblast proliferation and differentiation in human placental explant cultures

Regulation of cytotrophoblast differentiation toward extravillous trophoblasts (EVTs) is critical for establishing successful pregnancy. Previous studies have focused primarily on the factors promoting the differentiation, while inhibitory regulators except hypoxia have been less documented. In this study, to test our hypothesis that angiotensin II (Ang II) would inhibit EVT differentiation, we investigated the effects of Ang II on trophoblast outgrowth and the expression of molecules associated with the proliferation and invasion of trophoblasts using human first trimester villous explant cultures. Ang II increased EVT outgrowth and the number of cells in cell columns. Moreover, Ang II-treated explants exhibited increased Ki67 and integrin alpha5 immunoreactivity in EVTs as well as matrix metalloproteinase-2 activity in the conditioned media, and decreased alpha1 integrin immunoreactivity, which are compatible with the features of the proliferative phenotype EVTs. These effects of Ang II were similar to those of hypoxia (3% O(2)). Ang II stimulated the expression of hypoxia inducible factor-1alpha at both mRNA and protein levels, and also enhanced the expression of plasminogen activator inhibitor-1 (PAI-1). Data presented herein suggest a possible role for Ang II in impairing trophoblast differentiation toward an invasive phenotype, which might be associated with shallow invasion in preeclamptic placentas.