重组人MASP2蛋白在大肠杆菌中的表达和纯化

目的:获得酶原形式的重组人甘露聚糖结合凝集素相关丝氨酸蛋白酶2(MASP2)。方法:在大肠杆菌中诱导表达重组人MASP2全长蛋白,包涵体裂解后,经复性、透析、浓缩、考马斯亮蓝染色、SDS-PAGE及Western印迹,鉴定纯化结果及酶活性。结果:复性后的MASP2蛋白经考马斯亮蓝染色未见杂带。自激活实验表明,当MASP2浓度在1μmool/L以下时,无论在4℃还是37℃,都能较稳定地保持酶原形式;蛋白浓度为3.5μmool/L时只能在4℃保持稳定,37℃发生自激活;蛋白浓度达到12μmool/L后,在4℃时已不能稳定存在。结论:获得了较纯的重组人MASP2蛋白,且具有自激活活性。     

Evidence for a SAL1-PAP chloroplast retrograde pathway that functions in drought and high light signaling in Arabidopsis

Compartmentation of the eukaryotic cell requires a complex set of subcellular messages, including multiple retrograde signals from the chloroplast and mitochondria to the nucleus, to regulate gene expression. Here, we propose that one such signal is a phosphonucleotide (3'-phosphoadenosine 5'-phosphate [PAP]), which accumulates in Arabidopsis thaliana in response to drought and high light (HL) stress and that the enzyme SAL1 regulates its levels by dephosphorylating PAP to AMP. SAL1 accumulates in chloroplasts and mitochondria but not in the cytosol. sal1 mutants accumulate 20-fold more PAP without a marked change in inositol phosphate levels, demonstrating that PAP is a primary in vivo substrate. Significantly, transgenic targeting of SAL1 to either the nucleus or chloroplast of sal1 mutants lowers the total PAP levels and expression of the HL-inducible ASCORBATE PEROXIDASE2 gene. This indicates that PAP must be able to move between cellular compartments. The mode of action for PAP could be inhibition of 5' to 3' exoribonucleases (XRNs), as SAL1 and the nuclear XRNs modulate the expression of a similar subset of HL and drought-inducible genes, sal1 mutants accumulate XRN substrates, and PAP can inhibit yeast (Saccharomyces cerevisiae) XRNs. We propose a SAL1-PAP retrograde pathway that can alter nuclear gene expression during HL and drought stress.     

变性高效液相色谱技术对创伤弧菌检测的研究

应用PCR结合变性高效液相色谱技术对创伤弧菌进行检测,建立创伤弧菌快速准确的检测新方法。经过DHPLC分析条件优化,在DHPLC非变性温度下分析创伤弧菌特异性PCR扩增产物。同时进行方法特异性、灵敏度、重复性实验。实验结果表明所建立的创伤弧菌PCR-DHPLC检测方法特异性强、灵敏度高、重现性好、结果稳定可靠、检测时间短,检测低限可达到124 CFU/mL,是创伤弧菌快速检测的新技术。     

胸内正压对正常人左室功能影响及其力学原理

目的：探讨胸内正压对正常人左室射血及充盈的影响及其力学原理。方法：超声心动图观测30例正常人初始时与标准乏氏动作张力期10s时左室舒张末容积（LVEDV）、左室收缩末容积（LVESV）、每搏量（SV）、射血分值（EF）、流入道血流速度（E峰、A峰）、E／A值、二尖瓣环舒张早期运动速度（e）及舒张早期充盈压（E/e）的变化。结果：与初始时比较,标准乏氏动作张力期LVEDV、LVESV及SV减低而心率（陬）增快（P均〈0．001）,EF值增加,但无统计学意义（P〉0．05）;E峰与E／A值减低（P均〈O．05）;e没有变化（P〉0．05）．E／e值减低（P〈O．05）。结论：胸内正压对左室游离壁的力学作用促进了左室收缩运动而阻碍了左室舒张运动,会引起EF值增加,E峰及E／A值减低;2,胸内正压降低了肺静脉系统与心脏的跨壁压力,增加了血流阻力也是导致肺静脉系统与左室血液回流减少．E峰减低．E／e值减低的一个原因。     

Nitrogen stable isotopes indicate differences in nitrogen cycling between two contrasting Jamaican montane forests

Background and aims

The aim of this study is to enhance our knowledge of nitrogen (N) cycling and N acquisition in tropical montane forests through analysis of stable N isotopes (δ¹⁵N).

Methods

Leaves from eight common tree species, leaf litter, soils from three depths and roots were sampled from two contrasting montane forest types in Jamaica (mull ridge and mor ridge) and were analysed for δ¹⁵N.

Results

All foliar δ¹⁵N values were negative and varied among the tree species but were significantly more negative in the mor ridge forest (by about 2 ‰). δ¹⁵N of soils and roots were also more negative in mor ridge forests by about 3 ‰. Foliar δ¹⁵N values were closer to that of soil ammonium than soil nitrate suggesting that trees in these forests may have a preference for ammonium; this may explain the high losses of nitrate from similar tropical montane forests. There was no correlation between the rankings of foliar δ¹⁵N in the two forest types suggesting a changing uptake ratio of different N forms between forest types.

Conclusions

These results indicate that N is found at low concentrations in this ecosystem and that there is a tighter N cycle in the mor ridge forest, confirmed by reduced nitrogen availability and lower rates of nitrification. Overall, soil or root δ¹⁵N values are more useful in assessing ecosystem N cycling patterns as different tree species showed differences in foliar δ¹⁵N between the two forest types.     

Snapshots during the catalytic cycle of a histidine acid phytase reveal an induced-fit structural mechanism

Highly engineered phytases, which sequentially hydrolyze the hexakisphosphate ester of inositol known as phytic acid, are routinely added to the feeds of monogastric animals to improve phosphate bioavailability. New phytases are sought as starting points to further optimize the rate and extent of dephosphorylation of phytate in the animal digestive tract. Multiple inositol polyphosphate phosphatases (MINPPs) are clade 2 histidine phosphatases (HP2P) able to carry out the stepwise hydrolysis of phytate. MINPPs are not restricted by a strong positional specificity making them attractive targets for development as feed enzymes. Here, we describe the characterization of a MINPP from the Gram-positive bacterium Bifidobacterium longum (BlMINPP). BlMINPP has a typical HP2P-fold but, unusually, possesses a large α-domain polypeptide insertion relative to other MINPPs. This insertion, termed the U-loop, spans the active site and contributes to substrate specificity pockets underpopulated in other HP2Ps. Mutagenesis of U-loop residues reveals its contribution to enzyme kinetics and thermostability. Moreover, four crystal structures of the protein along the catalytic cycle capture, for the first time in an HP2P, a large ligand-driven α-domain motion essential to allow substrate access to the active site. This motion recruits residues both downstream of a molecular hinge and on the U-loop to participate in specificity subsites, and mutagenesis identified a mobile lysine residue as a key determinant of positional specificity of the enzyme. Taken together, these data provide important new insights to the factors determining stability, substrate recognition, and the structural mechanism of hydrolysis in this industrially important group of enzymes.     

Energy Crop and Biotechnology for Biofuel Production

Selection of energy crops is the first priority for large-scale biofuel production in China. As a major topic, it was extensively discussed in the Second International Symposium on Bioen-     

Arabidopsis thaliana inositol 1,3,4-trisphosphate 5/6-kinase 4 (AtITPK4) is an outlier to a family of ATP-grasp fold proteins from Arabidopsis

The Arabidopsis genome encodes a family of inositol 1,3,4-trisphosphate 5/6-kinases which form a subgroup of a larger group of ATP-grasp fold proteins. An analysis of the inositol 1,3,4-trisphosphate 5/6-kinase family might, ultimately, be best rewarded by detailed comparison of related enzymes in a single genome. The enzyme encoded by At2G43980, AtITPK4; is an outlier to its family. At2G43980 is expressed in male and female organs of young and mature flowers. AtITPK4 differs from other family members in that it does not display inositol 3,4,5,6-tetrakisphosphate 1-kinase activity; rather, it displays inositol 1,4,5,6-tetrakisphosphate and inositol 1,3,4,5-tetrakisphosphate isomerase activity.     

Chronic stress and its effects on adrenal cortex apoptosis in pregnant rats

The model of chronic intermittent stress by immobilization during pregnancy may produce alterations in the mechanisms that maintain adrenal gland homeostasis. In earlier investigations using this model, significant variations in plasma prolactin and corticosterone levels, and adrenal gland weights were observed. We hypothesized that chronic stress causes changes in apoptosis in the adrenal glands of pregnant rats. We identified and quantified apoptotic cells in the adrenal cortex and examined their ultrastructural characteristics using transmission electron microscopy. Adrenal glands of pregnant rats at gestation days 12, 17 and 21 were studied for control and experimental (stressed) rats. Immunolabelling techniques, stereological analysis and image quantification of adrenal gland sections were combined to determine differences in apoptosis in the different cell populations of the adrenal cortex. The apoptotic index of the experimental rats showed a significant reduction at gestation day 17, while at days 12 and 21 there were no differences from controls. Moreover, the apoptotic index of the reticular zones in control and experimental animals showed a significant increase compared to the glomerular and fascicular zones at the three gestation times studied. Chronic stress by immobilization reduced the caspase-dependent apoptotic index at gestation day 17, which may be related to variations in plasma concentrations of estrogens and prolactin.     

尼古丁在帕金森病中的神经保护作用

帕金森病(Parkinson’s disease,PD)是常见的中老年神经退行性疾病。研究表明,尼古丁具有抵抗黑质多巴胺神经元损伤的作用,其通过烟碱型乙酰胆碱受体(nicotinic acetylcholine receptor,nAChR)途径与非受体途径抑制帕金森病的发生与发展。本文就尼古丁在帕金森病中的神经保护作用以及保护机制的相关研究进行综述。