Drosophila katanin is a microtubule depolymerase that regulates cortical-microtubule plus-end interactions and cell migration

Regulation of microtubule dynamics at the cell cortex is important for cell motility, morphogenesis and division. Here we show that the Drosophila katanin Dm-Kat60 functions to generate a dynamic cortical-microtubule interface in interphase cells. Dm-Kat60 concentrates at the cell cortex of S2 Drosophila cells during interphase, where it suppresses the polymerization of microtubule plus-ends, thereby preventing the formation of aberrantly dense cortical arrays. Dm-Kat60 also localizes at the leading edge of migratory D17 Drosophila cells and negatively regulates multiple parameters of their motility. Finally, in vitro, Dm-Kat60 severs and depolymerizes microtubules from their ends. On the basis of these data, we propose that Dm-Kat60 removes tubulin from microtubule lattice or microtubule ends that contact specific cortical sites to prevent stable and/or lateral attachments. The asymmetric distribution of such an activity could help generate regional variations in microtubule behaviours involved in cell migration.     

Yeast two hybrid analyses reveal novel binary interactions between human cytomegalovirus-encoded virion proteins

Human cytomegalovirus (HCMV) is the largest human herpesvirus and its virion contains many viral encoded proteins found in the capsid, tegument, and envelope. In this study, we carried out a yeast two-hybrid (YTH) analysis to study potential binary interactions among 56 HCMV-encoded virion proteins. We have tested more than 3,500 pairwise combinations for binary interactions in the YTH analysis, and identified 79 potential interactions that involve 37 proteins. Forty five of the 79 interactions were also identified in human cells expressing the viral proteins by co-immunoprecipitation (co-IP) experiments. To our knowledge, 58 of the 79 interactions revealed by YTH analysis, including those 24 that were also identified in co-IP experiments, have not been reported before. Novel potential interactions were found between viral capsid proteins and tegument proteins, between tegument proteins, between tegument proteins and envelope proteins, and between envelope proteins. Furthermore, both the YTH and co-IP experiments have identified 9, 7, and 5 interactions that were involved with UL25, UL24, and UL89, respectively, suggesting that these "hub" proteins may function as the organizing centers for connecting multiple virion proteins in the mature virion and for recruiting other virion proteins during virion maturation and assembly. Our study provides a framework to study potential interactions between HCMV proteins and investigate the roles of protein-protein interactions in HCMV virion formation or maturation process.     

Modified Mouse Embryonic Stem Cell based Assay for Quantifying Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/β-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/β-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.     

RAPD-based genetic diversities and correlation with morphological traits in Camellia (Theaceae) cultivars in China

Camellia is an economically important ornamental plant that has many uses, such as in beverages, foods and medicines. We examined 15 Camellia cultivars in Wenzhou, China, using RAPD markers and measurements of three traits (petal color, flower diameter, blooming period). PCR amplification with 15 random primers produced 1935 bands, observed at 88 amplification loci; 77% of the amplified loci were polymorphic, with a mean of 4.5 polymorphic loci per primer. The similarity coefficient ranged from 0.5419 to 0.7933 among the 15 samples; the lowest value was between Manao (C. reticulata) and Feibai FR (C. japonica), and the largest value was between Chidan (C. japonica) and Yuanyang FG (C. japonica). Cluster analysis divided the 15 cultivars into two groups at the similarity coefficient of 0.65. A correlation was found between RAPD markers and petal color in the first group. No correlation was found between RAPD markers and the other traits (flower diameter, blooming period). This study provides information useful for the identification, classification, phylogenesis, and breeding of Camellia cultivars.     

Genome dynamics and diversity of Shigella species, the etiologic agents of bacillary dysentery

The Shigella bacteria cause bacillary dysentery, which remains a significant threat to public health. The genus status and species classification appear no longer valid, as compelling evidence indicates that Shigella, as well as enteroinvasive Escherichia coli, are derived from multiple origins of E.coli and form a single pathovar. Nevertheless, Shigella dysenteriae serotype 1 causes deadly epidemics but Shigella boydii is restricted to the Indian subcontinent, while Shigella flexneri and Shigella sonnei are prevalent in developing and developed countries respectively. To begin to explain these distinctive epidemiological and pathological features at the genome level, we have carried out comparative genomics on four representative strains. Each of the Shigella genomes includes a virulence plasmid that encodes conserved primary virulence determinants. The Shigella chromosomes share most of their genes with that of E.coli K12 strain MG1655, but each has over 200 pseudogenes, 300 approximately 700 copies of insertion sequence (IS) elements, and numerous deletions, insertions, translocations and inversions. There is extensive diversity of putative virulence genes, mostly acquired via bacteriophage-mediated lateral gene transfer. Hence, via convergent evolution involving gain and loss of functions, through bacteriophage-mediated gene acquisition, IS-mediated DNA rearrangements and formation of pseudogenes, the Shigella spp. became highly specific human pathogens with variable epidemiological and pathological features.     

Spinal P2X receptor modulates reflex pressor response to activation of muscle afferents

Static contraction of skeletal muscle evokes increases in blood pressure and heart rate. Previous studies suggested that the dorsal horn of the spinal cord is the first synaptic site responsible for those cardiovascular responses. In this study, we examined the role of ATP-sensitive P2X receptors in the cardiovascular responses to contraction by microdialyzing the P2X receptor antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) into the L7 level of the dorsal horn of nine anesthetized cats. Contraction was elicited by electrical stimulation of the L7 and S1 ventral roots. Blockade of P2X receptor attenuated the contraction induced-pressor response [change in mean arterial pressure (delta MAP): 16 +/- 4 mmHg after 10 mM PPADS vs. 42 +/- 8 mmHg in control; P < 0.05]. In addition, the pressor response to muscle stretch was also blunted by PPADS (delta MAP: 27 +/- 5 mmHg after PPADS vs. 49 +/- 8 mmHg in control; P < 0.05). Finally, activation of P2X receptor by microdialyzing 0.5 mM alpha,beta-methylene into the dorsal horn significantly augmented the pressor response to contraction. This effect was antagonized by prior PPADS dialysis. These data demonstrate that blockade of P2X receptors in the dorsal horn attenuates the pressor response to activation of muscle afferents and that stimulation of P2X receptors enhances the reflex response, indicating that P2X receptors play a role in mediating the muscle pressor reflex at the first synaptic site of this reflex.     

Regulation of lipolytic activity by long-chain acyl-coenzyme A in islets and adipocytes

Intracellular lipolysis is a major pathway of lipid metabolism that has roles, not only in the provision of free fatty acids as energy substrate, but also in intracellular signal transduction. The latter is likely to be particularly important in the regulation of insulin secretion from islet beta-cells. The mechanisms by which lipolysis is regulated in different tissues is, therefore, of considerable interest. Here, the effects of long-chain acyl-CoA esters (LC-CoA) on lipase activity in islets and adipocytes were compared. Palmitoyl-CoA (Pal-CoA, 1-10 microM) stimulated lipase activity in islets from both normal and hormone-sensitive lipase (HSL)-null mice and in phosphatase-treated islets, indicating that the stimulatory effect was neither on HSL nor phosphorylation dependent. In contrast, we reproduced the previously published observations showing inhibition of HSL activity by LC-CoA in adipocytes. The inhibitory effect of LC-CoA on adipocyte HSL was dependent on phosphorylation and enhanced by acyl-CoA-binding protein (ACBP). In contrast, the stimulatory effect on islet lipase activity was blocked by ACBP, presumably due to binding and sequestration of LC-CoA. These data suggest the following intertissue relationship between islets and adipocytes with respect to fatty acid metabolism, LC-CoA signaling, and lipolysis. Elevated LC-CoA in islets stimulates lipolysis to generate a signal to increase insulin secretion, whereas elevated LC-CoA in adipocytes inhibits lipolysis. Together, these opposite actions of LC-CoA lower circulating fat by inhibiting its release from adipocytes and promoting fat storage via insulin action.     

High throughput screening of co-expressed gene pairs with controlled false discovery rate (FDR) and minimum acceptable strength (MAS).

Many exploratory microarray data analysis tools such as gene clustering and relevance networks rely on detecting pairwise gene co-expression. Traditional screening of pairwise co-expression either controls biological significance or statistical significance, but not both. The former approach does not provide stochastic error control, and the later approach screens many co-expressions with excessively low correlation. We have designed and implemented a statistically sound two-stage co-expression detection algorithm that controls both statistical significance (false discovery rate, FDR) and biological significance (minimum acceptable strength, MAS) of the discovered co-expressions. Based on estimation of pairwise gene correlation, the algorithm provides an initial co-expression discovery that controls only FDR, which is then followed by a second stage co-expression discovery which controls both FDR and MAS. It also computes and thresholds the set of FDR p-values for each correlation that satisfied the MAS criterion. Using simulated data, we validated asymptotic null distributions of the Pearson and Kendall correlation coefficients and the two-stage error-control procedure; we also compared our two-stage test procedure with another two-stage test procedure using the receiver operating characteristic (ROC) curve. We then used yeast galactose metabolism data to illustrate the advantage of our method for clustering genes and constructing a relevance network. The method has been implemented in an R package "GeneNT" that is freely available from the Comprehensive R Archive Network (CRAN): www.cran.r-project.org/.     

The structural view of bacterial translocation-specific chaperone SecB: implications for function

SecB is a molecular chaperone that functions in bacterial post-translational protein translocation pathway. It maintains newly synthesized precursor polypeptide chains in a translocation-competent state and guides them to the translocon via its high-affinity binding to the ligand as well as to the membrane-embedded ATPase SecA. Recent advances in elucidating the structures of SecB have enabled the examination of protein function in the structural context. Structures of SecB from both Haemophilus influenzae and Escherichia coli support the early two-subsite polypeptide-binding model. In addition, the detailed molecular interaction between SecB and SecA was revealed by a structure of SecB in complex with the C-terminal zinc-containing domain of SecA. These observations explain the dual role of SecB plays in the translocation pathway, as a molecular chaperone and a specific targeting factor. A model of SecB-SecA complex suggests that the binding of SecA to SecB changes the conformation of the polypeptide binding sites in the chaperone, enabling transfer of precursor polypeptides from SecB to SecA. Recent studies also show the presence of a second zinc-independent SecB binding site in SecA and the new interaction might contribute to the function of SecB.