Picoliter-scale protein microarrays by laser direct write

We demonstrate the accurate picoliter-scale dispensing of active proteins using a novel laser transfer technique. Droplets of protein solution are dispensed onto functionalized glass slides and into plastic microwells, activating as small as 50-microm diameter areas on these surfaces. Protein microarrays fabricated by laser transfer were assayed using standard fluorescent labeling techniques to demonstrate successful protein and antigen binding. These results indicate that laser transfer does not damage the active site of the dispensed protein and that this technique can be used to successfully fabricate a functioning protein microarray. Also, as a result of the efficient nature of the process, material usage is reduced by two to four orders of magnitude compared to conventional pin dispensing methods for protein spotting.     

Ginkgo biloba extract EGb 761 increases stress resistance and extends life span of Caenorhabditis elegans.

EGb 761, a standardized extract of Ginkgo biloba leaves, has been used in clinical trials for its beneficial effects on brain functions. In mammals, EGb 761 has been shown to enhance cognition, stress resistance, and longevity, but its molecular and cellular mechanisms are not known. In the present investigation, we used the model organism Caenorhabditis elegans to evaluate pharmacological effects of EGb 761 on aging. We tested the theory that EGb 761 augments the natural antioxidant system of C elegans, and thus increases stress resistance and longevity. We found that treatment of the wild-type worms with EGb 761 extended their median life span by 8%. Amongst several purified components of EGb 761, the flavonoid tamarixetin showed the most dramatic effect: it extended the median life span by 25%. Furthermore, EGb 761 increased the wild type's resistance to acute oxidative and thermal stress by 33% and 25%, respectively. Treatment of the prematurely aging mutant worms mev-1 with EGb 761 increased their resistance to acute oxidative and thermal stress by 33% and 11%, respectively. It appears that oxidative stress, a major determinant of life span, as well as other types of stress, can be successfully counteracted by the Ginlkgo biloba extract EGb 761.     

Consumption of flea faeces and eggs by larvae of the cat flea,Ctenocephalides felis

The effects of the consumption of flea faeces and non-viable eggs on larval development in the cat flea Ctenocephalides felis (Bouché) (Siphonaptera: Pulicidae) were investigated. Only 13.3% of larvae developed into adults when fed a diet of male or female flea faeces alone; however, 90% of larvae developed into adults when fed on flea faeces supplemented with non-viable flea eggs. When fed with non-viable eggs alone, larvae did not develop into adults. Nevertheless, non-viable eggs may provide critical supplemental nutrients, lacking in flea faeces and required for larval development. None of the larvae fed on flea faeces or non-viable eggs alone formed a cocoon. A diet of flea faeces alone significantly extended the second as well as third larval stadia compared to larvae fed on diets containing non-viable eggs. It is suggested that the cannibalism of fertile eggs may limit population growth in the cat flea.     

Design, synthesis, and biological activity of novel factor Xa inhibitors: 4-aryloxy substituents of 2,6-diphenoxypyridines

A novel series of triaryloxypyridines have been designed to inhibit factor Xa, a serine protease strategically located in the coagulation cascade. Inhibitor 5e has a K(I) against factor Xa of 0.12nM and is greater than 8000- and 2000-fold selective over two related serine proteases, thrombin and trypsin, respectively. The 4-position of the central pyridine has been identified as a site that tolerates various substitutions without deleterious effects on potency and selectivity. This suggests that the 4-position of the pyridine ring is an ideal site for chemical modifications to identify inhibitors with improved pharmacokinetic characteristics. This investigation has resulted in inhibitor 5d, which has an oral availability of 6% in dogs. The synthesis, in vitro activity, and in vivo profile of this class of inhibitors is outlined.     

Effect of shear stress on cultivation of Bacillus thuringiensis for thuringiensin production

Cultivation of Bacillus thuringiensis for thuringiensin production is a mixed-growth-associated system. Cultivation conditions should be different during the cell growth stage and production stage. In this study, agitation speed and aeration rate were varied during the exponential growth phase and stationary phase in order to investigate the effect of shear stress via agitation on cultivation of B. thuringiensis for thuringiensin production. It was found that shear stress had a significant effect on thuringiensin production during the stationary phase. By decreasing the agitation speed during the stationary phase, product formation was increased up to 43%.     

Inheritance and expression of the cry1Ab gene in Bt ( Bacillus thuringiensis) transgenic rice

The inheritance and expression patterns of the cry1Ab gene were studied in the progenies derived from different Bt (Bacillus thuringiensis) transgenic japonica rice lines under field conditions. Both Mendelian and distorted segregation ratios were observed in some selfed and crossed F₂ populations. Crosses between japonica intra-subspecies had no significant effect on the segregation ratios of the cry1Ab gene, but crossing between japonica and indica inter-subspecies led to distorted segregation of the cry1Ab gene in the F₂ population. Field-release experiments indicated that the cry1Ab gene was stably transmitted in an intact manner via successive sexual generations, and the concentration of the Cry1Ab protein was kept quantitatively stable up to the R₆ generation. The cry1Ab gene, driven by the maize ubiquitin promoter, displayed certain kinds of spatial and temporal expression patterns under field conditions. The content of the Cry1Ab protein varied in different tissues of the main stems, the primary tillers and the secondary tillers. Higher levels of the Cry1Ab protein were found in the stems, leaves and leaf sheaths than in the roots, while the lowest level was detected in grains at the maturation stage. The content of the Cry1Ab protein in the leaves peaked at the booting stage and was lowest at the heading stage. Furthermore, the Cry1Ab content of cry1Ab expression in different tissues of transgenic rice varied individually with temperature. Received: 17 April 2001 / Accepted: 7 May 2001     

Regulation of the mitogen-activated protein kinase signaling pathway by SHP2.

Gab1-SHP2 association is required for Erk mitogen-activated protein kinase activation by several growth factors. Gab1-SHP2 interaction activates SHP2. However, an activated SHP2 still needs to associate with Gab1 to mediate Erk activation. It was unclear whether SHP2 is required to dephosphorylate a negative phosphorylation site on Gab1 or whether SHP2 needs the Gab1 pleckstrin homology (PH) domain to target it to the plasma membrane. We found that expression of a fusion protein consisting of the Gab1 PH domain and an active SHP2 (Gab1PH-SHP2DeltaN) induced constitutive Mek1 and Erk2 activation. Linking the active SHP2DeltaN to the PDK1 PH domain or the FRS2beta myristoylation sequence also induced Mek1 activation. Mek1 activation by Gab1PH-SHP2DeltaN was inhibited by an Src inhibitor and by Csk. Significantly, Gab1PH-SHP2DeltaN induced Src activation. Gab1PH-SHP2DeltaN expression activated Ras, and the Gab1PH-SHP2DeltaN-induced Mek1 activation was blocked by RasN17. These findings suggest that Gab1PH-SHP2DeltaN activated a signaling step upstream of Src and Ras. The SHP2 tyrosine phosphatase activity is essential for the function of the fusion protein. Together, these data show that the Gab1 sequence, besides the PH domain and SHP2 binding sites, is dispensable for Erk activation, suggesting that the primary role of Gab1 association with an activated SHP2 is to target it to the membrane.     

His-tag对胆固醇氧化酶基因在大肠杆菌中表达的影响

为考察组氨酸标签(His-tag)对Brevibacterium sp.DGCDC-82中胆固醇氧化酶基因(ChoAb)在大肠杆菌中表达的影响,将PCR扩增后得到的结构基因与pET28a(+)连接,构建重组质粒pETChoAb(不带His-tag),pETChoAbn(His-tag位于N端)和pETChoAbc(His-tag位于C端)并在大肠杆菌中进行表达.对重组酶进行酶活检测,结果表明His-tag位于ChoAb的C端和N端,COD单位体积酶活由未带标签时的1.72 U/mL分别提高到4.03 U/mL和11.36 U/mL.利用软件Quantity One对SDS-PAGE电泳条带进行灰度分析,结果显示与不带His-tag的COD相比,His-tag位于ChoAb的C端和N端,COD表达量由8.8％增加到16.4％与72.3％.同时菌体浓度分别提高了1.2倍和3.2倍.作为纯化标签,该研究结果对His-tag用于诊断用酶COD的分离纯化可以提供一定的理论指导.     

Spatial associations of tree species in a subtropical evergreen broad-leaved forest

Aims The spatial segregation hypothesis and the low-frequency hypothesis are two important proposed mechanisms that delay or prevent competitive exclusion in ecosystems. Because tree species interact with their neighbors, the importance of these potential processes can be investigated by analyzing the spatial structures of tree species.Methods The distribution of the adults of 27 common tree species in a fully mapped 5-ha subtropical forest plot in Baishanzu, eastern China, was analyzed to investigate the community-level intra- and interspecific spatial association patterns. We first tested for the overall spatial pattern in the 5- to 40-m neighborhoods and classified first-order bivariate associations with a diametric scheme based on Ripley's K and nearest-neighbor statistic (G -function). Then heterogeneous Poisson null models were used to distinguish second-order interactions from overall spatial associations (including first-order effects). Finally, we analyzed correlations between the existence of species interactions and some attributes of the species involved.Important findings Partial overlap and segregation increased with scale, whereas mixing decreased. Nearly 70% of the species pairs occurred less than expected at random, and only 3.4% of the species pairs were well mixed; 11.0% of all species pairs showed significant small-scale interactions, which was a greater frequency than expected by chance if species are abundant or prefer the same habitat, but less frequent than expected if species are highly aggregated. This suggests that both spatial segregation and low frequency of species facilitate species coexistence by reducing the opportunity that trees of two species encounter each other. The study also revealed that positive interactions were more prevalent than negative interactions in the forest, which indicates that positive interactions may have important effects on forest species assemblies.