Improved protective efficacy of a chimeric Staphylococcus aureus vaccine candidate iron‐regulated surface determinant B (N126–P361)‐target of RNAIII activating protein in mice

The pathogen Staphylococcus aureus causes a wide range of serious infections, necessitating urgent development of a vaccine against this organism. However, currently developed vaccines are relatively ineffective because of the limited antigenic component that is contained in the vaccine formulations. To develop an effective S. aureus candidate vaccine, overlapping PCR was used to add the truncated immunodominant antigen iron‐regulated surface determinant B (IsdB)_{(N126–P361)} (tIsdB) to the N‐terminal of intact antigen target of RNAIII activating protein (TRAP) and thus construct a tIsdB‐TRAP chimera. The humoral and cellular immune responses against tIsdB‐TRAP were compared with those against single or combined formulations. tIsdB‐TRAP elicited significantly stronger humoral responses in mice (P < 0.05). As to cellular immune responses in mice, the tIsdB‐TRAP group resulted in a greater IL‐4 response than did other groups (P < 0.05). Greater amounts of IL‐2 and IFN‐γ were found in the tIsdB‐TRAP group. Mouse challenge also showed that tIsdB‐TRAP provided better protection against S. aureus than did the control groups. These results suggest that this chimeric protein may be a promising pathogen target for further vaccine development.     

Structural and functional analyses of catalytic domain of GH10 xylanase from Thermoanaerobacterium saccharolyticum JW/SL‐YS485

Xylanases are capable of decomposing xylans, the major components in plant cell wall, and releasing the constituent sugars for further applications. Because xylanase is widely used in various manufacturing processes, high specific activity, and thermostability are desirable. Here, the wild‐type and mutant (E146A and E251A) catalytic domain of xylanase from Thermoanaerobacterium saccharolyticum JW/SL‐YS485 (TsXylA) were expressed in Escherichia coli and purified subsequently. The recombinant protein showed optimal temperature and pH of 75°C and 6.5, respectively, and it remained fully active even after heat treatment at 75°C for 1 h. Furthermore, the crystal structures of apo‐form wild‐type TsXylA and the xylobiose‐, xylotriose‐, and xylotetraose‐bound E146A and E251A mutants were solved by X‐ray diffraction to high resolution (1.32–1.66 Å). The protein forms a classic (β/α)₈ folding of typical GH10 xylanases. The ligands in substrate‐binding groove as well as the interactions between sugars and active‐site residues were clearly elucidated by analyzing the complex structures. According to the structural analyses, TsXylA utilizes a double displacement catalytic machinery to carry out the enzymatic reactions. In conclusion, TsXylA is effective under industrially favored conditions, and our findings provide fundamental knowledge which may contribute to further enhancement of the enzyme performance through molecular engineering. Proteins 2013; 81:1256–1265. © 2013 Wiley Periodicals, Inc.     

The structure of a shellfish specific GST class glutathione S‐transferase from antarctic bivalve Laternula elliptica reveals novel active site architecture

Glutathione‐S‐transferases have been identified in all the living species examined so far, yet little is known about their function in marine organisms. In a previous report, the recently identified GST from Antarctic bivalve Laternula elliptica (LeGST) was classified into the rho class GST, but there are several unique features of LeGST that may justify reclassification, which could represent specific shellfish GSTs. Here, we determined the crystal structure of LeGST, which is a shellfish specific class of GST. The structural analysis showed that the relatively open and wide hydrophobic H‐site of the LeGST allows this GST to accommodate various substrates. These results suggest that the H‐site of LeGST may be the result of adaptation to their environments as sedentary organisms. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Multiple Factors from Bradykinin-Challenged Astrocytes Contribute to the Neuronal Apoptosis: Involvement of Astroglial ROS, MMP-9, and HO-1/CO System

Bradykinin (BK) has been shown to induce the expression of several inflammatory mediators, including reactive oxygen species (ROS) and matrix metalloproteinases (MMPs), in brain astrocytes. These mediators may contribute to neuronal dysfunction and death in various neurological disorders. However, the effects of multiple inflammatory mediators released from BK-challenged astrocytes on neuronal cells remain unclear. Here, we found that multiple factors were released from brain astrocytes (RBA-1) exposed to BK in the conditioned culture media (BK-CM), including ROS, MMP-9, and heme oxygenase-1 (HO-1)/carbon monoxide (CO), leading to neuronal cell (SK-N-SH) death. Exposure of SK-N-SH cells to BK-CM or H₂O₂ reduced cell viability and induced cell apoptosis which were attenuated by N-acetyl cysteine, indicating a role of ROS in these responses. The effect of BK-CM on cell viability and cell apoptosis was also reversed by immunoprecipitation of BK-CM with anti-MMP-9 antibody (MMP-9-IP-CM) or MMP2/9 inhibitor, suggesting the involvement of MMP-9 in BK-CM-mediated responses. Astroglial HO-1/CO in BK-CM induced cell apoptosis and reduced cell viability which was reversed by hemoglobin. Consistently, the involvement of CO in these cellular responses was revealed by incubation with a CO donor CO-RM2 which was reversed by hemoglobin. The role of HO-1 in BK-CM-induced responses was confirmed by overexpression of HO-1 in SK-N-SH infected with Adv-HO-1. BK-CM-induced cell apoptosis was due to the activation of caspase-3 and cleavage of PARP. Together, we demonstrate that BK-induced several neurotoxic factors, including ROS, MMP-9, and CO released from astrocytes, may induce neuronal death through a caspase-3-dependent apoptotic pathway.     

Overexpression of the Gossypium barbadense Actin-Depolymerizing Factor 1 Gene Mediates Biological Changes in Transgenic Tobacco

The function of a member of the actin-depolymerizing factor family from Gossypium barbadense, GbADF1, was investigated. Tobacco (Nicotiana tabacum) lines expressing GbADF1 were produced by Agrobacterium-mediated transformation. Southern and northern blot analyses showed that GbADF1 was successfully incorporated as a single copy into the tobacco genome and stably expressed in three lines of T₁ transgenic tobacco plants. Biological changes were detected in these transgenic lines, wherein GbADF1 transgenic seedlings exhibited shorter hypocotyls along with fewer root hairs than those of control plants. Moreover, guard cells of leaves of the transgenic plants were induced to close stomata, while flowering was delayed 5 days in T₁ lines compared to those of empty vector transgenic control plants. Segregation of GbADF1 in the T₂ generation fits the expected 3:1 ratio corresponding to a single dominant gene. Subsequently, GbADF1 was fused to the green fluorescent protein gene to generate a fusion expression vector. Transient expression analysis indicated that this fusion protein was localized in the nucleus and cytoskeleton of epidermal cells of onion. These results suggest that actin-depolymerizing factor 1 gene from G. barbadense plays an important role in the process of plant cell morphogenesis.     

Theoretical insight into the structural mechanism for the binding of vinblastine with tubulin

Vinblastine (VLB) is one of vinca alkaloids with high cytotoxicity toward cancer cells approved for clinical use. However, because of drug resistance, toxicity, and other side effects caused from the use of VLB, new vinca alkaloids with higher cytotoxicity toward cancer cells and other good qualities need to develop. One strategy is to further study and better understand the essence why VLB possesses the high cytotoxicity toward cancer cells. In present work, by using molecular simulation, molecular docking, density functional calculation, and the crystal structure of α,β-tubulin complex, we find two modes labeled in catharanthine moiety (CM) and vindoline moiety (VM) modes of VLB bound with the interface of α,β-tubulin to probe the essence why VLB has the high cytotoxicity toward cancer cells. In the CM mode, nine key residues B-Ser178, B-Asp179, B-Glu183, B-Tyr210, B-Asp226, C-Lys326, C-Asp327, C-Lys336, and C-Lys352 from the α,β-tubulin complex are determined as the active sites for the interaction of VLB with α,β-tubulin. Some of them such as B-Ser178, B-Glu183, B-Tyr210, B-Asp226, C-Lys326, C-Asp327, and C-Lys336 are newly identified as the active sites in present work. The affinity between VLB and the active pocket within the interface of α,β-tubulin is ?60.8 kJ mol^?1 in the CM mode. In the VM mode, that is a new mode established in present paper, nine similar key residues B-Lys176, B-Ser178, B-Asp179, B-Glu183, B-Tyr210, B-Asp226, C-Lys326, C-Asp327, and C-Lys336 from the α,β-tubulin complex are found as the active sites for the interaction with VLB. The difference is from one key residue C-Lys352 in the CM mode changed to the key residue B-Lys176 in the VM mode. The affinity between VLB and the active pocket within the interface of α,β-tubulin is ?96.3 kJ mol^?1 in the VM mode. Based on the results obtained in present work, and because VLB looks like two faces, composed of CM and VM both to have similar polar active groups, to interact with the active sites, we suggest double-faces sticking mechanism for the binding of VLB to the interface of α,β-tubulin. The double-faces sticking mechanism can be used to qualitatively explain high cytotoxicity toward cancer cells of vinca alkaloids including vinblastine, vincristine, vindestine, and vinorelbine approved for clinical use and vinflunine still in a phase III clinical trial. Furthermore, this mechanism will be applied to develop novel vinca alkaloids with much higher cytotoxicity toward cancer cells.     

Zinc Transporter 7 Induced by High Glucose Attenuates Epithelial-to-Mesenchymal Transition of Peritoneal Mesothelial Cells

Zinc (Zn) is an essential micronutrient and cytoprotectant involved in preventing many types of epithelial-to-mesenchymal transition (EMT)-driven fibrosis in vivo. The zinc-transporter family SLC30A (ZnT) is a pivotal factor in the regulation of Zn homeostasis. However, its function in EMT in peritoneal mesothelial cells (PMCs) remains unknown. This study explored the regulation of zinc transporters and the role they play in cell EMT, particularly in rat peritoneal mesothelial cells (RPMCs), surrounding glucose concentrations and the molecular mechanism involved. The effects of high glucose (HG) on zinc transporter gene expression were measured in RPMCs by real-time PCR. We explored ZnT7 (Slc30A7): the effect of ZnT7 over-expression and siRNA-mediated knock-down on HG-induced EMT was investigated as well as the underlying molecular mechanisms. Over-expression of ZnT7 resulted in significantly inhibited HG-induced EMT in RPMCs, while inhibition of ZnT7 expression using a considerable siRNA-mediated knock-down of RPMCs increased the levels of EMT. Furthermore, over-expression of ZnT7 is accompanied by down-regulation of TGF-β/Smad pathway, phospho-Smad3,4 expression levels. The finding suggests that the zinc-transporting system in RPMCs is influenced by the exposure to HG. The ZnT7 may account for the inhibition of HG-induced EMT in RPMCs, likely through targeting TGF-β/Smad signaling.     

Bioreactor studies predict whole microbial population dynamics in oil sands tailings ponds

Microorganisms in oil sands fluid fine tailings (FFT) are critical to biogeochemical elemental cycling as well as to the degradation of residual hydrocarbon constituents and subsequent methane and CO₂ production. Microbial activity enhances particulate matter sedimentation rates and the dewatering of FFT materials, allowing water to be recycled back into bitumen extraction. A bulk of this evidence comes from bioreactor studies and has implications for engineering and environmental management of the FFT ponds. Yet, it is largely uncertain whether such laboratory populations are representative of whole field scale microbial communities. By using population ecology tools, we compared whole microbial communities present in FFT bioreactors to reference populations existing in Syncrude's West In Pit (WIP) tailings pond. Bacteria were found to be persistent in a sulfidic zone in both the oxic and anoxic bioreactors at all occasions tested. In contrast to the WIP, archaea only became predominant in bioreactors after 300 days, at which point analysis of similarity (global R statistic p?<?0.5) revealed no significant dissimilarities between the populations present in either system. A whole community succession pattern from bacterial dominated prevalence to a new assemblage predominated by archaea was suggested. These results have implications for the stepwise development of microbial model systems for predictive management of field scale FFT basins.     

Pip介导铜绿假单胞菌吩嗪基因簇phz2的表达

[目的]为了确定铜绿假单胞菌调控因子Pip对两个不同吩嗪合成基因簇(phz1和phz2)的具体调控方式与可能的调控机制.[方法]根据基因比对结果,采用同源重组技术构建Pip调控因子缺失突变株PA-PG以及克隆ip基因作互补分析;再以已构建的吩嗪基因簇缺失突变株PA-Z1G和PA-Z2K为受体菌,构建突变株PA-PD-Z1G和PA-PG-Z2K,测定并比较野生株及相关突变株的吩嗪-1-羧酸和绿脓菌素的合成量,推定Pip对两个不同吩嗪合成基因簇的调控方式.[结果]在GA培养基中,突变株PA-PG的吩嗪-1-羧酸和绿脓菌素都比野生型明显减少;互补分析显示,突变株PA-PG的吩嗪-1-羧酸和绿脓菌素都显著提高并恢复到野生株PAO1水平;突变株PA-Z1G的吩嗪-1-羧酸和绿脓菌素合成量因Pip缺失而显著减少;而突变株PA-Z2K的吩嗪-1-羧酸和绿脓菌素合成量在Pip缺失后仍保持不变.[结论]初步推定,转录调控因子Pip对铜绿假单胞菌吩嗪合成代谢的确具有促进作用;Pip通过正向调控吩嗪基因簇phz2的合成功能实现对吩嗪合成代谢的调控.