应用纤维素结合域标签构建谷氨酸棒状杆菌表达系统

为优化谷氨酸棒状杆菌表达系统的纯化工艺,合成里氏木霉的CBD基因,将其与谷氨酸棒状杆菌分泌表达载体pXMJ19-sp连接,构建以CBD为纯化标签的重组载体pXMJ 19-sp-CBD.在该载体中插入GFP基因并转化至谷氨酸棒状杆菌,可获得分泌表达融合蛋白GFP-CBD的重组菌.该菌经IPTG诱导后的发酵液在紫外灯下显示强烈的绿色荧光,重组蛋白的分泌表达量达200 mg/L.利用CBD标签对纤维素柱的可逆性吸附,可直接对谷氨酸棒状杆菌分泌到培养基中的重组蛋白进行纯化,从而简化工艺和降低成本,为工业化大生产奠定基础.     

A Model for the Acrosome Reaction in Mammalian Sperm

The acrosome reaction is a complex, calcium-dependent reaction that results in an exocytotic event required for successful fertilization of the egg. It has long been thought that the acrosome reaction occurs upon sperm binding to the zona pellucida, a viscoelastic layer surrounding the oocyte. Recent studies have suggested that the reaction may even occur before the sperm encounters the zona, perhaps mediated by progesterone or some other agonist. It has been particularly difficult to understand differences between progesterone-induced and zona-induced reactions experimentally and whether one substance is the more biologically relevant trigger. Until this present work, there has been little effort to mathematically model the acrosome reaction in sperm as a whole. Instead, attention has been paid to modeling portions of the pathways involved in other cell types. Here we present a base model for the acrosome reaction which characterizes the known biochemical reactions and behaviors of the system. Our model allows us to analyze several pathways that may act as a stabilizing mechanism for avoiding sustained oscillatory calcium responses often observed in other cell types. Such an oscillatory regime might otherwise prevent acrosomal exocytosis and therefore inhibit fertilization. Results indicate that the acrosome reaction may rely upon multiple redundant mechanisms to avoid entering an oscillatory state and instead maintain a high resting level of calcium, known to be required for successful acrosomal exocytosis and, ultimately, fertilization of the oocyte.     

Well-to-wheel GHG emissions and mitigation potential from light-duty vehicles in Macau

Purpose

The rapid growth of vehicle sales and usage has highlighted the need for greenhouse gas (GHG) emission reduction in Macau, a special administrative region (SAR) of China. As the most primary vehicle type, light-duty vehicles (LDV, including light-duty gasoline vehicles (LDGVs) and light-duty diesel vehicles (LDDVs)) play a key role in promoting the GHG reduction and development of green transportation system in Macau.

Methods

This study, on the basis of real-world tested and statistical data, firstly performed a streamlined life-cycle assessment (SLCA) on LDVs, to evaluate the potential GHG emissions and reduction through shifting to hybrid electric vehicles (HEVs) and electric vehicles (EVs).

Results and discussion

The results show that the mean GHG emissions from the LDGVs, LDDVs, and HEVs per 100 km were 25.16, 20.30, and 15.00 kg CO₂ eq, respectively. Under the current electricity mix in Macau, EVs with the emissions of 12.39 kg CO₂ eq/100 km can achieve a significant GHG emission reduction of LDVs in Macau. The total GHG emissions from LDVs increased from 124.99 to 247.82 thousand metric tons over the periods 2001–2014, with a 5.42% annual growth rate. A scenario analysis indicated that the development of HEVs and EVs—especially EVs—has the potential to control the GHG emissions from LDVs. Under the electricity mix of natural gas (NG) and solar energy (SE), the GHG emissions from EVs would drop by about 22 and 28%, respectively, by 2030.

Conclusions

This study develops a useful approach to evaluate the potential GHG emissions and its reduction strategies in Macau. All the obtained results could be useful for decision makers, providing robust support for drawing up an appropriate plan for improving green transportation systems in Macau.

     

Factors Affecting the Tailing of Blunt End DNA with Fluorescent Pyrimidine dNTPs

The transferase activity of non-proofreading DNA polymerases is a well-known phenomenon that has been utilized in cloning and sequencing applications. The non-templated addition of modified nucleotides at DNA blunt ends is a potentially useful feature of DNA polymerases that can be used for selective transformation of DNA 3′ ends. In this paper, we characterized the tailing reaction at perfectly matched and mismatched duplex ends with Cy3- and Cy5-modified pyrimidine nucleotides. It was shown that the best DNA tailing substrate does not have a perfect Watson–Crick base pair at the end. Mismatched duplexes with a 3′ dC were the most efficient in the Taq DNA polymerase-catalysed tailing reaction with a Cy5-modified dUTP. We further demonstrated that the arrangement of the dye residue relative to the nucleobase notably affects the outcome of the tailing reaction. A comparative study of labelled deoxycytidine and deoxyuridine nucleotides showed higher efficiency for dUTP derivatives. The non-templated addition of modified nucleotides by Taq polymerase at a duplex blunt end was generally complicated by the pyrophosphorolysis and 5′ exonuclease activity of the enzyme.     

Structural analysis of <Emphasis Type="Italic">N</Emphasis>-/<Emphasis Type="Italic">O</Emphasis>-glycans assembled on proteins in yeasts

Protein glycosylation, the most universal and diverse post-translational modification, can affect protein secretion, stability, and immunogenicity. The structures of glycans attached to proteins are quite diverse among different organisms and even within yeast species. In yeast, protein glycosylation plays key roles in the quality control of secretory proteins, and particularly in maintaining cell wall integrity. Moreover, in pathogenic yeasts, glycans assembled on cell-surface glycoproteins can mediate their interactions with host cells. Thus, a comprehensive understanding of protein glycosylation in various yeast species and defining glycan structure characteristics can provide useful information for their biotechnological and clinical implications. Yeast-specific glycans are a target for glyco-engineering; implementing human-type glycosylation pathways in yeast can aid the production of recombinant glycoproteins with therapeutic potential. The virulenceassociated glycans of pathogenic yeasts could be exploited as novel targets for antifungal agents. Nowadays, several glycomics techniques facilitate the generation of species-and strain-specific glycome profiles and the delineation of modified glycan structures in mutant and engineered yeast cells. Here, we present the protocols employed in our laboratory to investigate the N-and O-glycan chains released from purified glycoproteins or cell wall mannoproteins in several yeast species.     

Responses of two kidney bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>) cultivars to the combined stress of sulfur deficiency and cadmium toxicity

Plants suffer from combined stress of sulfur deficiency and cadmium toxicity in some agricultural lands. However, little is known about the reaction in plants, such as responses in antioxidant enzymes and non-protein thiol compounds, to such combined stress. Therefore, in this study, four treatments, S-sufficiency (T_S?Cd), S-deficiency (T_?S?Cd), Cd stress (T_S+Cd) and combined stress of S-deficiency and Cd stress (T_?S+Cd), were set up to investigate (1) the effects of sulfur deficiency or sulfur sufficiency on Cd toxicity to kidney bean cultivar seedlings and the related mechanisms, and (2) the responses of two kidney bean cultivars to combined stress of S-deficiency and Cd-tolerance. The results showed significant increases in hydrogen peroxide (H₂O₂) and malondialdehyde contents and significant increases in antioxidant enzyme (superoxide dismutase, catalase, peroxidase, and glutathione S-transferase) activities and non-protein thiol compounds (non-protein thiols, reduced glutathione, phytochelatins) synthesis in the plants in T_S+Cd and T_?S+Cd. On the tissue level, higher proportion of Cd was found to be immobilized/deposited in roots, while on the sub-cell level, higher proportion of Cd was located in cell walls and vacuole fractions with lower in cell organelles. Taken together, the results indicated that Cd detoxification was achieved by the two kidney bean cultivars through antioxidant enzyme activation, non-protein thiol compound synthesis and sub-cellular compartmentalization. In addition, the results indicated that sufficient S supply helped to relieve Cd toxicity, which is of special significance for remediation or utilization of Cd-contaminated soils as S is a plant essential nutrient.     

Antifungal Drug Resistance: Clinical Relevance and Impact of Antifungal Drug Use

Antifungal drug resistance significantly impacts treatment outcomes in patients with invasive fungal infections (IFIs). Although primary (intrinsic) resistance may occur independent of previous therapy, prior concomitant antifungal exposure increases the risk for secondary (acquired) resistance and subsequent colonization or infection with less-susceptible pathogens. Among various pathogen-antifungal combinations, this effect has been best studied clinically with azole exposure and the risk of Candida spp. with reduced susceptibility. The rapid development of secondary resistance to flucytosine in Candida spp. has limited its use as monotherapy. Secondary resistance to amphotericin B is infrequent. In contrast, secondary resistance in Aspergillus spp. is less of a concern. Recent reports of secondary resistance in patients receiving fluconazole for cryptococcal infections may justify susceptibility testing in the setting of prior therapy or treatment failure. Despite numerous patient-focused, drug-focused, and disease-focused strategies to improve treatment outcomes, clinical resistance (manifesting as treatment failures despite adequate antifungal therapy) continues to be problematic in patients with serious IFIs.     

Factors affecting immobilization of heavy metals by purple nonsulfur bacteria isolated from contaminated shrimp ponds

In order to remove heavy metals (HMs) from contaminated shrimp pond at the highest concentrations found of; 0.75 mg/l Cd²⁺, 62.63 mg/l Pb²⁺, 34.60 mg/l Cu²⁺ and 58.50 mg/l Zn²⁺, two strains of purple nonsulfur bacteria isolated from shrimp ponds (NW16 and KMS24) were investigated for their ability to immobilize HMs in 3% NaCl in both microaerobic-light and aerobic-dark conditions. Based on metabolic inhibition and metabolic-dependent studies, it was concluded that both strains removed HMs using biosorption and also bioaccumulation. The efficiency of removal by both strains with both incubating conditions tested was in the order of lead (Pb) > copper (Cu) > zinc (Zn) > cadmium (Cd). Optimal conditions for removal of HMs by strain NW16 were; cells in the log phase at 4.5 mg DCW/ml, pH 6.0, and 30°C for 30 min. With microaerobic-light conditions, the relative percent removal of HMs was: Pb, 83; Cu, 59; Zn, 39; Cd, 23 and slightly more with the aerobic-dark conditions (Pb, 90; Cu, 69; Zn, 46; Cd, 28). Cells in the log phase at 5.0 mg DCW/ml, pH 5.5, and 35°C for 45 min were optimal conditions for strain KMS24 and there were no significant differences for the removal percentages of HMs with either incubating conditions (averages: Pb, 96; Cu, 75; Zn, 46; Cd, 30). The presence of Ca²⁺ and Mg²⁺ significantly decreased the removal capacity of HMs for both strains.     

人参茎叶皂甙对高胆固醇饮食大鼠再灌注性心律失常和脂质过氧化的影响(英文)

研究人参茎叶皂甙(GSL)对高胆固醇饮食大鼠心肌再灌注性心律失常(RPA_r)和脂质过氧化的影响。方法:将胆固醇乳剂用灌胃法饲养大鼠14d,建立高脂血症模型,各组大鼠进行心肌缺血再灌注实验,观察高脂血症和GSL对大鼠心肌缺血再灌注2h后血丙二醇(MDA),超氧化物歧化酶(SOD)和一氧化氮(NO)水平的影响和对再灌注性心律失常发生率的影响。结果显示:(1)用胆固醇乳剂饲养大鼠14d,成功建立高脂血症模型。同时给予GSL14d有明显降脂作用。(2)高脂血症状态下,心肌缺血再灌注2h后,血MDA升高(p<0.01),SOD降低(p<0.01)和NO(p<0.05)降低,再灌注10min内RPAr的发生率增高。(3)GSL组再灌注后2h的血MDA降低,而SOD和NO水平显著升高;使RPAr发生率大为降低,无VF发生。实验显示高脂血症加重心肌缺血再灌注损伤和提高RPAr发生率及动物死亡率,GSL可减少高脂饮食大鼠脂质过氧化和诱导体内NO生成而减轻缺血再灌注心肌损伤,降低缺血再灌注性心律失常发生率。