Construction of Aspergillus niger lipase mutants with oil-water interface independence

Based on previous bioinformational analytical results [Shu ZY, et al. Biotechnol Prog 2009;25:409-16], four A. niger lipase (ANL) mutants, ANL-Ser84Gly, ANL-Asp99Pro, ANL-Lys108Glu and ANL-EαH (obtained by replacing the lid domain of ANL with the corresponding domain from A. niger feruloyl esterase), were constructed to screen out ANL mutants with oil-water interface independence. ANL-S84G displayed a pronounced interfacial activation, while ANL-D99P and ANL-K108E displayed no interfacial activation. The specific activity of ANL-S84G towards p-nitrophenyl esters decreased from 29.8% to 76.5% compared with that of ANL, while the specific activity of ANL-D99P towards p-nitrophenyl palmitate increased 2.2-fold. The thermostability of ANL-K108E was almost unchanged, while the thermostability of ANL-S84G and ANL-D99P significantly decreased compared with that of ANL. The construction of oil-water interface-independent ANL mutants would help to further understand the mechanism of lipase interfacial activation.     

Characteristics of Cd uptake, translocation and accumulation in a novel Cd-accumulating tree, star fruit (Averrhoa carambola L., Oxalidaceae)

Cadmium (Cd) accumulation by terrestrial higher plants is an intriguing phenomenon that may be exploited for phytoextraction of Cd-contaminated soils. Characterizing the physiological processes responsible for elevated concentrations of Cd in shoots is a first step towards a comprehensive understanding of the mechanisms underlying Cd accumulation in plants and may eventually improve the efficiency of phytoextraction. Woody species that can accumulate Cd have been recently recommended as good candidates for phytoextraction of Cd-contaminated soils. However, little is known about the mechanisms of Cd accumulation by woody species. In an attempt to understand the physiological processes contributing to Cd accumulation in woody species, Cd uptake and translocation by a novel tropical Cd-accumulating tree, star fruit (Averrhoa carambola) were characterized and compared with those of a non-Cd-accumulating tree (Clausena lansium). Our results showed that A. carambola had higher Cd uptake and root-to-shoot translocation efficiencies than C. lansium, which might account for its greater Cd-accumulating capacity. Furthermore, Cd accumulation by A. carambola was not significantly affected by zinc (Zn), whereas Zn accumulation was greatly lowered by Cd. This phenomenon could not be fully explained by a simple competition between Cd²⁺ and Zn²⁺, implying the existence of a transport system with a preference for Cd over Zn. Collectively, our results indicate that A. carambola has noteworthy physiological traits associated with accumulation of Cd to high levels.     

Hybridization and asymmetric introgression between Cypripedium tibeticum and C. yunnanense in Shangrila County,Yunnan Province,China

Hybridization and introgression are thought to be important for speciation and adaptation in many plants. However, little is known about the hybridization and introgression among Cypripedium species. To investigate the evidence for hybridization and the pattern of introgression between Cypripedium yunnanense and C. tibeticum in Shangrila County, Yunnan Province, China, morphological characters and amplified fragment length polymorphism (AFLP) data for both the species and their putative hybrids were studied. Hand pollination was also performed to verify the crossability of the putative parents. Principal coordinate analysis based on morphological characters and the AFLP data suggested that the putative hybrids were true hybrids of these two Cypripedium species. Analysis with the NewHybrids software indicated that the putative hybrids were F1 generation individuals and backcrosses to C. yunnanese, but no F2 generation was found. Analysis with the Structure software demonstrated asymmetric introgression from C. tibeticum to C. yunnanense. We conclude that natural hybridization and introgression can occur between these two species and that in situ conservation of the parental species is required before fully assessing the evolutionary potential of hybrids.     

Biodegradation of Benazolin-Ethyl by Strain Methyloversatilis sp. cd-1 Isolated from Activated Sludge

Benazolin-ethyl has been used on a wide range of weeds present in various crops since 1964. Because benazolin-ethyl is a potential hazard to the environment and human health, it is important to remove this herbicide from the environment. However, to the best of our knowledge, no report is available in the literature regarding the microbial degradation of benazolin-ethyl by bacteria. In this study, one strain named cd-1, which is capable of degrading benazolin-ethyl, was isolated from benazolin-ethyl wastewater treatment pool. The isolate was identified as Methyloversatilis sp. according to its morphological, physiological, biochemical properties, and 16S rRNA gene sequences analysis. This strain utilizes benazolin-ethyl as the sole carbon source. and degrades 100?mg?l^?1 benazolin-ethyl to non-detectable level within 48?h. Three metabolites were identified as benazolin, 7-chloro-3-methylbenzo[d]thiazol-2(3H)-one, and 2-chloro-6-(methyleneamino)benzenethiol based on the MS/MS and GC/MS analyses. The first step involved in the degradation of benazolin-ethyl was the cleavage of the ester bond to form benazolin. Benazolin was subsequently subjected to demethylation for decomposition into 7-chloro-3-methylbenzo[d]thiazol-2(3H)-one and methanol. The last step was to form 2-chloro-6-(methyleneamino)benzenethiol.     

正常人长时数字记忆信息提取的神经基础的功能磁共振研究

本文旨在通过功能磁共振成像(functional magnetic resonance imaging,fMRI)技术研究正常人进行长时数字记忆信息提取的神经基础。选取22名右利手志愿者进行长时数字记忆任务实验,采用组块设计,记忆任务与对照任务交替进行,同时利用Siemens 1.5T超导型磁共振仪进行fMRI成像,采用SPM99软件进行数据分析,脑功能区定位在Talairach坐标中显示。结果显示被试者在进行长时数字记忆提取任务时,激活最显著的皮层是左侧额中回(Brodmann分区9区,BA9区),另外左额叶内侧回、左额下回、右额下回、扣带回、左顶下小叶、左顶上小叶、右顶上小叶、右颞中回、左枕舌回、左枕中回、右中脑、小脑、右尾状核尾部等结构也有激活,各大脑皮层的激活均呈现明显的左侧半球优势。根据上述结果推论,长时数字记忆由以左侧大脑半球为优势的各脑区共同参与完成,其中左侧额叶外侧面可能是信息提取的重要结构,而其它脑叶及其之间的广泛联系可能在数字信息的加工、处理和存储中起重要作用。     

Cloning and characterization of two ferritin subunit genes from bay scallop, <Emphasis Type="Italic">Argopecten irradians</Emphasis> (Lamarck 1819)

We have cloned two full-length cDNAs from two ferritin genes (Aifer1 and Aifer2) of the bay scallop, Argopecten irradians (Lamarck 1819). The cDNAs are 1,019 and 827 bp in length and encode proteins of 171 and 173 amino acids, respectively. The 5′ UTR of each contains a conserved iron response element (IRE) motif. Sequence analyses reveal that both proteins belong to the H-ferritin family with seven conserved amino acids in the ferroxidase center. Highest expression of Aifer1 is found in the mantle and adductor muscle, while that of Aifer2 is only in the latter tissue. These Aifer genes are differentially expressed following bacterial challenge of the scallop. The expression level of Aifer1 was acutely up-regulated (over 10 fold) at 6 h post-bacteria injection, whereas Aifer2 expression was not significantly changed by bacterial challenge. Both genes were effectively expressed in E. coli BL21 (DE3), producing proteins of similar molecular weight, approximately 23 kDa. Purified Aifer1 and Aifer2 proteins exhibited iron-chelating activity of 33.1% and 30.4%, respectively, at a concentration of 5 mg/ml. Cations, Mg²⁺, Zn²⁺ and Ca²⁺, depressed iron-chelating activity of both proteins. Additionally, the E. coli cells expressing recombinant Aifer1 and Aifer2 showed tolerance to H₂O₂, providing a direct evidence of the antioxidation function of ferritin. The results presented in this study suggest important roles of Aifer1 and Aifer2 in the regulation of iron homeostasis, immune response, and antioxidative stress in A. irradians.     

Dinuclear copper complexes of organic claw: potent inhibition of protein tyrosine phosphatases

Three dinuclear copper complexes of organic claw ligands (2,2′,2″,2?-(5-R-2-hydroxy-1,3-phenylene)bis(methylene)bis(azanetriyl)tetraacetic acid, R = methyl (H₅L1), chloro (H₅L2) and bromo (H₅L3)): [Cu₂NaL1(H₂O)₂] (1), [Cu₂HL2(H₂O)₂] (2), [Cu₂NaL3(H₂O)₂] (3), have been synthesized and characterized by elemental analyses, infrared spectra, thermo-gravimetric analyses, X-ray diffraction analysis, electrospray ionization mass spectra, pH-potentiometric titration, molar conductivity. Their inhibitory effects against human protein tyrosine phosphatase 1B (PTP1B), T cell protein tyrosine phosphatase (TCPTP), Megakaryocyte protein tyrosinephosphatase 2 (PTP-MEG2), srchomology phosphatase 1 (SHP-1) and srchomology phosphatase 2 (SHP-2) are evaluated in vitro. The three copper complexes exhibit potent and almost same inhibition against PTP1B and SHP-1 with IC₅₀ values ranging from 0.15 to 0.31 μM, about 2-fold stronger inhibition than against PTP-MEG2, 10-fold stronger inhibition than against TCPTP, but almost no inhibition against SHP-2. Kinetic analysis indicates that they are reversible competitive inhibitors of PTP1B. Molecular docking analyses confirm the inhibition model. Fluorescence titration studies suggest that the complexes bond to PTP1B with the formation of a 1:1 complex. The results demonstrate that copper complexes that are potent PTPs inhibitors but have different inhibitory effects over different PTPs, may be explored as new practical inhibitors towards individual PTP with some specificity.     

A study of the Distribution and Density of the VEGFR-2 Receptor on Glioma Microvascular Endothelial Cell Membranes

The purpose of the study was to examine the nanoscale distribution and density of the VEGFR-2 membrane receptor on the endothelial cell surface of glioma microvasculature. Immunofluorescence and atomic force microscopy combined with immunogold labeling techniques were used to characterize and determine the position of the glioma microvasculature endothelial cell surface receptor VEGFR-2. We aimed to indirectly detect the distribution of VEGFR-2 on the cell membrane at the nanoscale level and to analyze VEGFR-2 quantitatively. Immunofluorescence imaging showed a large amount of VEGFR-2 scattered across the endothelial cell surface; atomic force microscopy imaging also showed two globular structures of different sizes scattered across the endothelial cell surface. The difference between the average diameter of the small globular structure outside the cell surface (43.67 ± 5.02 nm) and that of IgG (44.61 ± 3.19 nm) was not statistically significant (P > 0.05). The three-dimensional morphologies of the small globular structure outside the cell surface and IgG were similar. The difference between the average diameter of the large globular structure outside the cell surface (74.19 ± 9.10 nm) and that of IgG-SpA-CG (74.54 ± 15.93 nm) was also not statistically significant (P > 0.05). The three-dimensional morphologies of this large globular structure outside the cell surface and IgG-SpA-CG were similar. The total density of these two globular structures within the unit area was 92 ± 19 particles μm(2). No globular structures were seen on the cell surface in the control group. The large globular structure on the surface of glioma microvascular endothelial cells was categorized as a VEGFR-2-IgG-SpA-CG immune complex, whereas the small globular structure was categorized as a VEGFR-2-IgG immune complex. The positions of the globular structures were the same as the positions of the VEGFR-2 molecules. A large amount of VEGFR-2 was scattered across glioma microvascular endothelial cell surfaces; the receptor density was about 92 per square micron.