环境水体中GⅡ型诺如病毒浓缩研究

自行设计一种适合野外现场采样的病毒采集浓缩仪,采用一种新型阳离子膜NanoCeram,进行环境水体中GⅡ型诺如病毒浓缩研究。通过预实验,考察了预处理膜、不同的二次浓缩方法、不同洗脱液对病毒回收率的影响,随后优化整个浓缩过程并确定最佳的病毒浓缩方法。结果显示,预处理膜对病毒回收影响较大,PEG-NaCl沉淀、硅藻土吸附这两种二次浓缩方法的效果相当,选择0.15mol/L Na2HPO4作为硅藻土洗脱液。确定了NanoCeram阳离子膜的病毒回收率为3.02%。最后对北京市丰台区洋桥生活污水进行现场采样,成功检测到GⅡ型诺如病毒,证明该方法有效可行,适合于环境水体中诺如病毒的浓缩分离和检测。     

Repair mechanisms of bone marrow mesenchymal stem cells in myocardial infarction

The prognosis of patients with myocardial infarction (MI) and resultant chronic heart failure remains extremely poor despite advances in optimal medical therapy and interventional procedures. Animal experiments and clinical trials using adult stem cell therapy following MI have shown a global improvement of myocardial function. Bone marrow-derived mesenchymal stem cells (MSCs) hold promise for cardiac repair following MI, due to their multilineage, self-renewal and proliferation potential. In addition, MSCs can be easily isolated, expanded in culture, and have immunoprivileged properties to the host tissue. Experimental studies and clinical trials have revealed that MSCs not only differentiate into cardiomyocytes and vascular cells, but also secrete amounts of growth factors and cytokines which may mediate endogenous regeneration via activation of resident cardiac stem cells and other stem cells, as well as induce neovascularization, anti-inflammation, anti-apoptosis, anti-remodelling and cardiac contractility in a paracrine manner. It has also been postulated that the anti-arrhythmic and cardiac nerve sprouting potential of MSCs may contribute to their beneficial effects in cardiac repair. Most molecular and cellular mechanisms involved in the MSC-based therapy after MI are still unclear at present. This article reviews the potential repair mechanisms of MSCs in the setting of MI.     

Terpenoid and phenolic metabolites from the fungus xylaria sp. associated with termite nests

Three new sesquiterpene acids, xylaric acids A-C (1-3, resp.), and a new tetralone (=3,4-dihydronaphthalen-1(2H)-one) derivative, 4, along with nine known compounds, xylaric acid D (5), hydroheptelidic acid (6), gliocladic acid (7), chlorine heptelidic acid (8), trichoderonic acid A (9), 16-(α-D-mannopyranosyloxy)isopimar-7-en-19-oic acid (10), 16-(α-D-glucopyranosyloxy)isopimar-7-en-19-oic acid (11), 5-carboxymellein (12), and naphthalen-1,8-diol 1-O-α-D-glucopyranoside (13) have been isolated from the solid culture of the ascomycete fungus Xylaria sp. associated with termite nest. The structures of these compounds were elucidated primarily by NMR experiments. The absolute configurations of compounds 1-3 and 5-9 were determined by combination of X-ray data and CD spectral analysis. The absolute configuration of 4 was assigned by Snatzke's method. Compounds 8 and 11 showed slight cytotoxicities against two cell lines A549 and SGC7901.     

Purification and characterization of a highly selective sucrose isomerase from Erwinia rhapontici NX-5

A highly selective sucrose isomerase (SIase) was purified to homogeneity from the cell-free extract of Erwinia rhapontici NX-5 with a recovery of 27.7% and a fold purification of 213.6. The purified SIase showed a high specific activity of 427.1 U mg⁻¹ with molecular weight of 65.6 kDa. The K _m for sucrose was 222 mM while V _max was 546 U mg⁻¹. The optimum pH and temperature for SIase activity were 6.0 and 30 °C, respectively. The purified SIase was stable in the temperature range of 10–40 °C and retained 65% of the enzyme activity after 2 weeks’ storage at 30 °C. The SIase activity was enhanced by Mg²⁺ and Mn²⁺, inhibited by Ca²⁺, Cu²⁺, Zn²⁺, and Co²⁺, completely inhibited by Hg²⁺ and Ag²⁺. The purified SIase was strongly inhibited by SDS, while partially inhibited by dimethylformamide, tetrahydrofuran, and PMSF. Additionally, glucose and fructose acted as competitive inhibitors for purified SIase.     

Enhanced welan gum production using a two-stage agitation speed control strategy in Alcaligenes sp. CGMCC2428

Batch fermentative production of welan gum by Alcaligenes sp. CGMCC2428 was investigated under various oxygen supply conditions using regulating agitation speed. Based on a three kinetic parameters analysis that includes specific cell growth rate (μ), specific glucose consumption rate (q _s), and specific welan formation rate (q _p), a two-stage agitation speed control strategy was proposed to achieve high concentration, high yield, and high viscosity of welan. During the first 22 h, the agitation speed in 7.5 L fermenter was controlled at 800 rpm to maintain high μ for cell growth. The agitation was then reduced step-wise to 600 rpm to maintain a changing profile with stable dissolved oxygen levels and obtain high qp for high welan accumulation. Finally, the maximum concentration of welan was reached at 26.3 ± 0.89 g L⁻¹ with a yield of 0.53 ± 0.003 g g⁻¹ and the welan gum viscosity of 3.05 ± 0.10 Pa s, which increased by an average of 15.4, 15.2, and 20.1% over the best results controlled by constant agitation speeds.     

响应面法优化重组大肠杆菌产蔗糖异构酶发酵培养基

通过碳氮源的不同浓度对重组大肠杆菌E.coil BL21(DE3)发酵产蔗糖异构酶(SIase)的影响,并借助于数学分析软件Design Expert,结合Plackett-Burman试验设计和中心复合试验设计分析法,对蔗糖异构酶的产生菌进行了发酵培养基的优化研究。实验表明,最佳培养基组分为甘蔗糖蜜10.65 g/L,玉米浆22.22 g/L,NaCl 7.57 g/L ,MgSO₄·7H₂O 0.52 g/L, KH₂PO₄ 4.46g/L,优化后的蔗糖异构酶活力达到29.1U/ml,比LB培养基培养重组大肠杆菌(15U/ml),蔗糖异构酶活力提高了94%,与原始菌大黄欧文菌NX-5相比提高了21.4倍(1.3U/ml)。     

Dissimilarity in the folding of human cytosolic creatine kinase isoenzymes

Creatine kinase (CK, EC 2.7.3.2) plays a key role in the energy homeostasis of excitable cells. The cytosolic human CK isoenzymes exist as homodimers (HMCK and HBCK) or a heterodimer (MBCK) formed by the muscle CK subunit (M) and/or brain CK subunit (B) with highly conserved three-dimensional structures composed of a small N-terminal domain (NTD) and a large C-terminal domain (CTD). The isoforms of CK provide a novel system to investigate the sequence/structural determinants of multimeric/multidomain protein folding. In this research, the role of NTD and CTD as well as the domain interactions in CK folding was investigated by comparing the equilibrium and kinetic folding parameters of HMCK, HBCK, MBCK and two domain-swapped chimeric forms (BnMc and MnBc). Spectroscopic results indicated that the five proteins had distinct structural features depending on the domain organizations. MBCK BnMc had the smallest CD signals and the lowest stability against guanidine chloride-induced denaturation. During the biphasic kinetic refolding, three proteins (HMCK, BnMc and MnBc), which contained either the NTD or CTD of the M subunit and similar microenvironments of the Trp fluorophores, refolded about 10-fold faster than HBCK for both the fast and slow phase. The fast folding of these three proteins led to an accumulation of the aggregation-prone intermediate and slowed down the reactivation rate thereby during the kinetic refolding. Our results suggested that the intra- and inter-subunit domain interactions modified the behavior of kinetic refolding. The alternation of domain interactions based on isoenzymes also provides a valuable strategy to improve the properties of multidomain enzymes in biotechnology.     

Dynamics of Th17 cells and their role in Schistosoma japonicum infection in C57BL/6 mice

Background

The current knowledge of immunological responses to schistosomiasis, a major tropical helminthic disease, is insufficient, and a better understanding of these responses would support vaccine development or therapies to control granuloma-associated immunopathology. CD4⁺ T cells play critical roles in both host immune responses against parasitic infection and immunopathology in schistosomiasis. The induction of T helper (Th)1, Th2 and T regulatory (Treg) cells and their roles in schistosome infections are well-illustrated. However, little in vivo data are available on the dynamics of Th17 cells, another important CD4⁺ T cell subset, after Schistosoma japonicum infection or whether these cells and their defining IL-17 cytokine mediate host protective responses early in infection.

Methodology

Levels of Th17 and the other three CD4⁺ T cell subpopulations and the cytokines related to induction or repression of Th17 cell generation in different stages of S. japonicum infection were observed. Contrary to reported in vitro studies, our results showed that the Th17 cells were induced along with the Th1, Th2, Treg cells and the IFN-γ and IL-4 cytokines in S. japonicum infected mice. The results also suggested that S. japonicum egg antigens but not adult worm antigens preferentially induced Th17 cell generation. Furthermore, decreasing IL-17 with a neutralizing anti-IL-17 monoclonal antibody (mAb) increased schistosome-specific antibody levels and partial protection against S. japonicum infection in mice.

Conclusions

Our study is the first to report the dynamics of Th17 cells during S. japonicum infection and indicate that Th17 cell differentiation results from the integrated impact of inducing and suppressive factors promoted by the parasite. Importantly, our findings suggest that lower IL-17 levels may result in favorable host protective responses. This study significantly contributes to the understanding of immunity to schistosomiasis and may aid in developing interventions to protect hosts from infection or restrain immunopathology.     

CCN2 expression and localization in melanoma cells

The matricellular protein connective tissue growth factor (CTGF, CCN2) is overexpressed in several forms of cancer and may represent a novel target in anti-cancer therapy. However, whether CCN2 is expressed in melanoma cells is unknown. The highly metastatic murine melanoma cell line B16(F10) was used for our studies. Real time polymerase chain reaction analysis was used to detect mRNA expression of CCN1, CCN2, CCN3 and CCN4 in Western blot and immunofluorescence analyses were used to detect CCN2 protein. Inhibitors of signal transduction cascades were used to probe the mechanism underlying CCN2 expression in B16(F10) cells. CCN2 was expressed in B16(F10) cells, and was reduced by the FAK/src inhibitor PP2 and the MEK/ERK inhibitor U0126 indicating that CCN2 acts downstream of these pathways in B16(F10) murine melanoma cells. Expression of CCN1, CCN3 and CCN4 was not reduced by PP2 or U0126; in fact, expression of CCN4 mRNA was elevated by PP2 or U0126 treatment. To our surprise, CCN2 protein was detected in the nuclei of B16(F10) cells, and was undetectable in the cytoplasm. CCN2 was expressed in B16(F10) melanoma cells, adding to the list of cancer cells in which CCN2 is expressed. Of the CCN family members tested, only CCN2 is downstream of the highly oncogenic MEK/ERK pathway. CCN2 should be further evaluated for a possible role in melanoma growth and progression.     

Introgression of chromosome 3Ns from Psathyrostachys huashanica into wheat specifying resistance to stripe rust

Wheat stripe rust is a destructive disease in the cool and humid wheat-growing areas of the world. Finding diverse sources of stripe rust resistance is critical for increasing genetic diversity of resistance for wheat breeding programs. Stripe rust resistance was identified in the alien species Psathyrostachys huashanica, and a wheat-P. huashanica amphiploid line (PHW-SA) with stripe rust resistance was reported previously. In this study, a P. huashanica 3Ns monosomic addition line (PW11) with superior resistance to stripe rust was developed, which was derived from the cross between PHW-SA and wheat J-11. We evaluated the alien introgressions PW11-2, PW11-5 and PW11-8 which were derived from line PW11 for reaction to new Pst race CYR32, and used molecular and cytogenetic tools to characterize these lines. The introgressions were remarkably resistant to CYR32, suggesting that the resistance to stripe rust of the introgressions thus was controlled by gene(s) located on P. huashanica chromosome 3Ns. All derived lines were cytologically stable in term of meiotic chromosome behavior. Two 3Ns chromosomes of P. huashanica were detected in the disomic addition line PW11-2. Chromosomes 1B of substitution line PW11-5 had been replaced by a pair of P. huashanica 3Ns chromosomes. In PW11-8, a small terminal segment from P. huashanica chromosome arm 3NsS was translocated to the terminal region of wheat chromosomes 3BL. Thus, this translocated chromosome is designated T3BL-3NsS. These conclusions were further confirmed by SSR analyses. Two 3Ns-specific markers Xgwm181 and Xgwm161 will be useful to rapidly identify and trace the translocated fragments. These introgressions, which had significant characteristics of resistance to stripe rust, could be utilized as novel germplasms for wheat breeding.