Automated identification and quantification of protein phosphorylation sites by LC/MS on a hybrid triple quadrupole linear ion trap mass spectrometer

Complete phosphorylation mapping of protein kinases was successfully undertaken using an automated LC/MS/MS approach. This method uses the direct combination of triple quadrupole and ion trapping capabilities in a hybrid triple quadrupole linear ion trap to selectively identify and sequence phosphorylated peptides. In particular, the use of a precursor ion scan of m/z -79 in negative ion mode followed by an ion trap high resolution scan (an enhanced resolution scan) and a high sensitivity MS/MS scan (enhanced product ion scan) in positive mode is a very effective method for identifying phosphorylation sites in proteins at low femtomole levels. Coupling of this methodology with a stable isotope N-terminal labeling strategy using iTRAQtrade mark reagents enabled phosphorylation mapping and relative protein phosphorylation levels to be determined between the active and inactive forms of the protein kinase MAPKAPK-1 in the same LC/MS run.     

流式细胞术研究细胞凋亡的方法与技术

细胞发生凋亡时,会伴随着一系列形态学、生物化学及分子生物学性质的变化,包括细胞皱缩,核染色质凝聚,细胞膜通透性改变,Caspases激活,线粒体跨膜电位降低,膜磷酯酰丝氨酸外化,胞质Ca2+浓度升高,DNA片段化及含量变化等特点.应用流式细胞术进行细胞凋亡的研究,对于探讨胚胎发育、衰老以及研究肿瘤的发生、发展和转化等病理生理过程和病毒感染及免疫等具有十分重要的意义.本文就细胞凋亡的特征、基于细胞膜功能的流式细胞术检测方法和基于细胞器功能的流式细胞术检测方法等关键性问题进行了阐述.     

白音华矿区土壤重金属含量的空间异质性

为探讨开矿对白音华矿区土壤重金属空间分布的影响,本研究以内蒙古西乌珠穆沁旗白音华煤矿区周边土壤为对象,分析了距离矿区8 km内的重金属Cu、Cr、Pb和Mn含量的空间异质性.结果表明:土壤重金属Cu、Cr、Pb和Mn的平均含量分别为12.7、32.6、29.9和201.3 mg ? kg-1,其变异系数分别为26.8％...     

Clinical Burkholderia pseudomallei isolates from north Queensland carry diverse bimABm genes that are associated with central nervous system disease and are phylogenomically distinct from other Australian strains

BackgroundBurkholderia pseudomallei is an environmental gram-negative bacterium that causes the disease melioidosis and is endemic in many countries of the Asia-Pacific region. In Australia, the mortality rate remains high at approximately 10%, despite curative antibiotic treatment being available. The bacterium is almost exclusively found in the endemic region, which spans the tropical Northern Territory and North Queensland, with clusters occasionally present in more temperate climates. Despite being endemic to North Queensland, these infections remain understudied compared to those of the Northern Territory.Methodology/Principal findingsThis study aimed to assess the prevalence of central nervous system (CNS) disease associated variant bimA_Bm, identify circulating antimicrobial resistance mutations and genetically distinct strains from Queensland, via comparative genomics. From 76 clinical isolates, we identified the bimA_Bm variant in 20 (26.3%) isolates and in 9 (45%) of the isolates with documented CNS infection (n = 18). Explorative analysis suggests a significant association between isolates carrying the bimA_Bm variant and CNS disease (OR 2.8, 95% CI 1.3–6.0, P = 0.009) compared with isolates carrying the wildtype bimA_Bp. Furthermore, 50% of isolates were identified as novel multi-locus sequence types, while the bimA_Bm variant was more commonly identified in isolates with novel sequence types, compared to those with previously described. Additionally, mutations associated with acquired antimicrobial resistance were only identified in 14.5% of all genomes.Conclusions/SignificanceThe findings of this research have provided clinically relevant genomic data of B. pseudomallei in Queensland and suggest that the bimA_Bm variant may enable risk stratification for the development CNS complications and be a potential therapeutic target.     

MO25alpha/beta interact with STRADalpha/beta enhancing their ability to bind, activate and localize LKB1 in the cytoplasm

Mutations in the LKB1 protein kinase result in the inherited Peutz Jeghers cancer syndrome. LKB1 has been implicated in regulating cell proliferation and polarity although little is known about how this enzyme is regulated. We recently showed that LKB1 is activated through its interaction with STRADalpha, a catalytically deficient pseudokinase. Here we show that endogenous LKB1-STRADalpha complex is associated with a protein of unknown function, termed MO25alpha, through the interaction of MO25alpha with the last three residues of STRADalpha. MO25alpha and STRADalpha anchor LKB1 in the cytoplasm, excluding it from the nucleus. Moreover, MO25alpha enhances the formation of the LKB1-STRADalpha complex in vivo, stimulating the catalytic activity of LKB1 approximately 10-fold. We demonstrate that the related STRADbeta and MO25beta isoforms are also able to stabilize LKB1 in an active complex and that it is possible to isolate complexes of LKB1 bound to STRAD and MO25 isoforms, in which the subunits are present in equimolar amounts. Our results indicate that MO25 may function as a scaffolding component of the LKB1-STRAD complex and plays a crucial role in regulating LKB1 activity and cellular localization.     

Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD

The LKB1 gene encodes a serine/threonine kinase mutated in Peutz-Jeghers cancer syndrome. Despite several proposed models for LKB1 function in development and in tumour suppression, the detailed molecular action of LKB1 remains undefined. Here, we report the identification and characterization of an LKB1-specific adaptor protein and substrate, STRAD (STe20 Related ADaptor). STRAD consists of a STE20- like kinase domain, but lacks several residues that are indispensable for intrinsic catalytic activity. Endogenous LKB1 and STRAD form a complex in which STRAD activates LKB1, resulting in phosphorylation of both partners. STRAD determines the subcellular localization of wild-type, but not mutant LKB1, translocating it from nucleus to cytoplasm. One LKB1 mutation previously identified in a Peutz-Jeghers family that does not compromise its kinase activity is shown here to interfere with LKB1 binding to STRAD, and hence with STRAD-dependent regulation. Removal of endogenous STRAD by siRNA abrogates the LKB1-induced G(1) arrest. Our results imply that STRAD plays a key role in regulating the tumour suppressor activities of LKB1.     

14-3-3s regulate fructose-2,6-bisphosphate levels by binding to PKB-phosphorylated cardiac fructose-2,6-bisphosphate kinase/phosphatase

The cardiac isoform of 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase (PFK-2), regulator of the glycolysis-stimulating fructose-2,6-bisphosphate, was among human HeLa cell proteins that were eluted from a 14-3-3 affinity column using the phosphopeptide ARAApSAPA. Tryptic mass fingerprinting and phospho-specific antibodies showed that Ser466 and Ser483 of 14-3-3-affinity-purified PFK-2 were phosphorylated. 14-3-3 binding was abolished by selectively dephosphorylating Ser483, and 14-3-3 binding was restored when both Ser466 and Ser483 were phosphorylated with PKB, but not when Ser466 alone was phosphorylated by AMPK. Furthermore, the phosphopeptide RNYpS(483)VGS blocked binding of PFK-2 to 14-3-3s. These data indicate that 14-3-3s bind to phosphorylated Ser483. When HeLa cells expressing HA-tagged PFK-2 were co-transfected with active PKB or stimulated with IGF-1, HA-PFK-2 was phosphorylated and bound to 14-3-3s. The response to IGF-1 was abolished by PI 3-kinase inhibitors. In addition, IGF-1 promoted the binding of endogenous PFK-2 to 14-3-3s. When cells were transduced with penetratin-linked AARAApSAPA, we found that this reagent bound specifically to 14-3-3s, blocked the IGF-1-induced binding of HA-PFK-2 to 14-3-3s, and completely inhibited the IGF-1-induced increase in cellular fructose-2,6-bisphosphate. These findings suggest that PKB-dependent binding of 14-3-3s to phospho-Ser483 of cardiac PFK-2 mediates the stimulation of glycolysis by growth factor.     

Preliminary linkage map of the turkey (Meleagris gallopavo) based on microsatellite markers

The turkey is an agriculturally important species for which, until now, there is no published genetic linkage map based on microsatellite markers--still the markers most used in the chicken and other farm animals. In order to increase the number of markers on a turkey genetic linkage map we decided to map new microsatellite sequences obtained from a GT-enriched turkey genomic library. In different chicken populations more than 35-55% of microsatellites are polymorphic. In the turkey populations tested here, 43% of all turkey primers tested were found to be polymorphic, in both commercial and wild type turkeys. Twenty linkage groups (including the Z chromosome) containing 74 markers have been established, along with 37 other unassigned markers. This map will lay the foundations for further genetic mapping and the identification of genes and quantitative trait loci in this economically important species. Genome comparisons, based on genetic maps, with related species such as the chicken would then also be possible. All primer information, polymerase chain reaction (PCR) conditions, allele sizes and genetic linkage maps can be viewed at http://roslin.thearkdb.org/. The DNA is also available on request through the Roslin Institute.