Rhizobium meliloti nodulation genes: identification of nodDABC gene products, purification of nodA protein, and expression of nodA in Rhizobium meliloti.

A set of conserved, or common, bacterial nodulation (nod) loci is required for host plant infection by Rhizobium meliloti and other Rhizobium species. Four such genes, nodDABC, have been indicated in R. meliloti 1021 by genetic analysis and DNA sequencing. An essential step toward understanding the function of these genes is to characterize their protein products. We used in vitro and maxicell Escherichia coli expression systems, together with gel electrophoresis and autoradiography, to detect proteins encoded by nodDABC. We facilitated expression of genes on these DNA fragments by inserting them downstream of the Salmonella typhimurium trp promoter, both in colE1 and incP plasmid-based vectors. Use of the incP trp promoter plasmid allowed overexpression of a nodABC gene fragment in R. meliloti. We found that nodA encodes a protein of 21 kilodaltons (kDa), and nodB encodes one of 28 kDa; the nodC product appears as two polypeptide bands at 44 and 45 kDa. Expression of the divergently read nodD yields a single polypeptide of 33 kDa. Whether these represent true Rhizobium gene products must be demonstrated by correlating these proteins with genetically defined Rhizobium loci. We purified the 21-kDa putative nodA protein product by gel electrophoresis, selective precipitation, and ion-exchange chromatography and generated antiserum to the purified gene product. This permitted the immunological demonstration that the 21-kDa protein is present in wild-type cells and in nodB- or nodC-defective strains, but is absent from nodA::Tn5 mutants, which confirms that the product expressed in E. coli is identical to that produced by R. meliloti nodA. Using antisera detection, we found that the level of nodA protein is increased by exposure of R. meliloti cells to plant exudate, indicating regulation of the bacterial nod genes by the plant host.     

Mammalian Target of Rapamycin Complex 2 (mTORC2) Is a Critical Determinant of Bladder Cancer Invasion

Bladder cancer is the fourth most common cause of cancer in males in the United States. Invasive behavior is a major determinant of prognosis. In this study, we identified mammalian target of rapamycin complex 2 (mTORC2) as a central regulator of bladder cancer cell migration and invasion. mTORC2 activity was assessed by the extent of phosphorylation of Ser473 in AKT and determined to be approximately 5-fold higher in specimens of invasive human bladder cancer as opposed to non-invasive human bladder cancer. The immortalized malignant bladder cell lines, UMUC-3, J82 and T24 demonstrated higher baseline mTORC2 activity relative to the benign bladder papilloma-derived cell line RT4 and the normal urothelial cell line HU1. The malignant bladder cancer cells also demonstrated increased migration in transwell and denudation assays, increased invasion of matrigel, and increased capacity to invade human bladder specimens. Gene silencing of rictor, a critical component of mTORC2, substantially inhibited bladder cancer cell migration and invasion. This was accompanied by a significant decrease in Rac1 activation and paxillin phosphorylation. These studies identify mTORC2 as a major target for neutralizing bladder cancer invasion.     

Keratin 5-Cre-driven excision of nonmuscle myosin IIA in early embryo trophectoderm leads to placenta defects and embryonic lethality

In studies initially focused on roles of nonmuscle myosin IIA (NMIIA) in the developing mouse epidermis, we have discovered that a previously described cytokeratin 5 (K5)-Cre gene construct is expressed in early embryo development. Mice carrying floxed alleles of the nonmuscle myosin II heavy chain gene (NMHC IIA^flox/flox) were crossed with the K5-Cre line. The progeny of newborn pups did not show a Mendelian genotype distribution, suggesting embryonic lethality. Analysis of post-implantation conceptuses from embryonic day (E)9.5 to E13.5 revealed poorly developed embryos and defective placentas, with significantly reduced labyrinth surface area and blood vessel vascularization. These results suggested the novel possibility that the bovine K5 promoter-driven Cre-recombinase was active early in trophoblast-lineage cells that give rise to the placenta. To test this possibility, K5-Cre transgenic mice were crossed with the mT/mG reporter mouse in which activation of GFP expression indicates Cre transgene expression. We observed activation of K5-Cre-driven GFP expression in the ectoplacental cone, in the extraembryonic ectoderm, and in trophoblast giant cells in the E6.5 embryo. In addition, we observed GFP expression at E11.5 to E13.5 in both the labyrinth of the placenta and the yolk sac. NMIIA expression was detected in these same cell types in normal embryos, as well as in E13.5 yolk sac and labyrinth. These findings taken together suggest that NMHC IIA may play critical roles in the early trophoblast-derived ectoplacental cone and extraembryonic ectoderm, as well as in the yolk sac and labyrinth tissues that form later. Our findings are consistent with phenotypes of constitutive NMIIA knockout mice made earlier, that displayed labyrinth and yolk sac-specific defects, but our findings extend those observations by suggesting possible NMIIA roles in trophoblast lineages as well. These results furthermore demonstrate that K5-Cre gene constructs, previously reported to be activated starting at approximately E12.5 in the forming epidermis, may be widely useful as drivers for activation of cre/lox based gene excision in early embryo extraembronic trophoblast tissues as well.     

Model studies of the flow in abdominal aortic aneurysms during resting and exercise conditions

Pulsatile flow in abdominal aortic aneurysm (AAA) models has been examined in order to understand the hemodynamics that may contribute to growth of an AAA. The model studies were conducted by experiments (flow visualization and laser Doppler velocimetry) and by numerical simulation using physiologically realistic resting and exercise flow conditions. We characterize the flow for two AAA model shapes and sizes emulating early AAA development through moderate AAA growth (mean and peak Reynolds numbers of 362<Re_mean<1053 and 3308<Re_peak<5696 with Womersley parameter 16.4<<21.2). The results of our investigation indicate that AAA flow can be divided into three flow regimes: (i) Attached flow over the entire cycle in small AAAs at resting conditions, (ii) vortex formation and translation in moderate size AAAs at resting conditions, and (iii) vortex formation, translation and turbulence in moderate size AAAs under exercise conditions. The second two regimes are classified in the medical literature as disturbed flow conditions that have been correlated with atherogenesis as well as thrombogenesis. Thus, AAA disturbed hemodynamics may be a contributing factor to AAA growth by accelerating the degeneration of the arterial wall. Our investigation also concluded that vortex development is considerably weaker in an asymmetric AAA. Furthermore, turbulence was not observed in the asymmetric model. Finally, our investigation suggests a new mode of transition to turbulence: vortex ring instability and bursting to turbulence. The transition process depends on a combination of the pulsatile flow conditions and the tube cross-sectional area change.     

Multiple myosin II heavy chain kinases: roles in filament assembly control and proper cytokinesis in Dictyostelium

Myosin II filament assembly in Dictyostelium discoideum is regulated via phosphorylation of residues located in the carboxyl-terminal portion of the myosin II heavy chain (MHC) tail. A series of novel protein kinases in this system are capable of phosphorylating these residues in vitro, driving filament disassembly. Previous studies have demonstrated that at least three of these kinases (MHCK A, MHCK B, and MHCK C) display differential localization patterns in living cells. We have created a collection of single, double, and triple gene knockout cell lines for this family of kinases. Analysis of these lines reveals that three MHC kinases appear to represent the majority of cellular activity capable of driving myosin II filament disassembly, and reveals that cytokinesis defects increase with the number of kinases disrupted. Using biochemical fractionation of cytoskeletons and in vivo measurements via fluorescence recovery after photobleaching (FRAP), we find that myosin II overassembly increases incrementally in the mutants, with the MHCK A(-)/B(-)/C(-) triple mutant showing severe myosin II overassembly. These studies suggest that the full complement of MHC kinases that significantly contribute to growth phase and cytokinesis myosin II disassembly in this organism has now been identified.     

Actin activation of myosin heavy chain kinase A in Dictyostelium: a biochemical mechanism for the spatial regulation of myosin II filament disassembly

Studies in Dictyostelium discoideum have established that the cycle of myosin II bipolar filament assembly and disassembly controls the temporal and spatial localization of myosin II during critical cellular processes, such as cytokinesis and cell locomotion. Myosin heavy chain kinase A (MHCK A) is a key enzyme regulating myosin II filament disassembly through myosin heavy chain phosphorylation in Dictyostelium. Under various cellular conditions, MHCK A is recruited to actin-rich cortical sites and is preferentially enriched at sites of pseudopod formation, and thus MHCK A is proposed to play a role in regulating localized disassembly of myosin II filaments in the cell. MHCK A possesses an aminoterminal coiled-coil domain that participates in the oligomerization, cellular localization, and actin binding activities of the kinase. In the current study, we show that the interaction between the coiled-coil domain of MHCK A and filamentous actin leads to an approximately 40-fold increase in the initial rate of kinase catalytic activity. Actin-mediated activation of MHCK A involves increased rates of kinase autophosphorylation and requires the presence of the coiled-coil domain. Structure-function analyses revealed that the coiled-coil domain alone binds to actin filaments (apparent K(D) = 0.9 microm) and thus mediates the direct interaction with F-actin required for MHCK A activation. Collectively, these results indicate that MHCK A recruitment to actin-rich sites could lead to localized activation of the kinase via direct interaction with actin filaments, and thus this mode of kinase regulation may represent an important mechanism by which the cell achieves localized disassembly of myosin II filaments required for specific changes in cell shape.     

Temporal Variation in Seed Predation by Insects in a Population of Syagrus romanzoffiana (Arecaceae) in Santa Catarina Island, SC, Brazil

Insect seed predation may vary depending on seed production. The present study considers the hypothesis that the rates of seed predation tend to be smaller in years of higher fruit production. Thus, we monitored the production of fruits and predation of seeds of the palm Syagrus romanzoffiana over 2?years in the Atlantic Forest (Parque Municipal da Lagoa do Peri, Florianópolis, SC, Brazil), between July 2006 and June 2008. Plots of 0.25?m² were fitted under 20 mother plants and fruits were monthly collected for assessment of abundance and seed predation. There was variation in fruit production between the 2?years and among reproductive plants. Predation rates were high and occurred in the predispersal phase by the Curculionidae Revena rubiginosa Boheman, Anchylorhynchus aegrotus Fahraeus, and Anchylorhynchus variabilis Gyllenhal. Seed predation by these species of Anchylorhynchus is first registered in the present study. In average, about 60% of the seeds monthly produced in the population tend to escape insect predation in year of high or low production, becoming available for recruitment. The predation rate was not related to the amount of fruits produced per reproductive plant. Also, different than expected, there was a positive relation between the rates of seed predation and the total of fruits produced monthly on the plots. Thus, no evidence for the satiation of insect seed predators was found in this study with S. romanzoffiana.     

Differential localization in cells of myosin II heavy chain kinases during cytokinesis and polarized migration

Background  

Cortical myosin-II filaments in Dictyostelium discoideum display enrichment in the posterior of the cell during cell migration and in the cleavage furrow during cytokinesis. Filament assembly in turn is regulated by phosphorylation in the tail region of the myosin heavy chain (MHC). Early studies have revealed one enzyme, MHCK-A, which participates in filament assembly control, and two other structurally related enzymes, MHCK-B and -C. In this report we evaluate the biochemical properties of MHCK-C, and using fluorescence microscopy in living cells we examine the localization of GFP-labeled MHCK-A, -B, and -C in relation to GFP-myosin-II localization.     

Spatial and temporal control of nonmuscle myosin localization: identification of a domain that is necessary for myosin filament disassembly in vivo

Myosin null mutants of Dictyostelium are defective for cytokinesis, multicellular development, and capping of surface proteins. We have used these cells as transformation recipients for an altered myosin heavy chain gene that encodes a protein bearing a carboxy-terminal 34-kD truncation. This truncation eliminates threonine phosphorylation sites previously shown to control filament assembly in vitro. Despite restoration of growth in suspension, development, and ability to cap cell surface proteins, these delta C34-truncated myosin transformants display severe cytoskeletal abnormalities, including excessive localization of the truncated myosin to the cortical cytoskeleton, impaired cell shaped dynamics, and a temporal defect in myosin dissociation from beneath capped surface proteins. These data demonstrate that the carboxy-terminal domain of myosin plays a critical role in regulating the disassembly of the protein from contractile structures in vivo.     

Is Bagging Effective in the Classification of Small-Sample Genomic and Proteomic Data?

There has been considerable interest recently in the application of bagging in the classification of both gene-expression data and protein-abundance mass spectrometry data. The approach is often justified by the improvement it produces on the performance of unstable, overfitting classification rules under small-sample situations. However, the question of real practical interest is whether the ensemble scheme will improve performance of those classifiers sufficiently to beat the performance of single stable, nonoverfitting classifiers, in the case of small-sample genomic and proteomic data sets. To investigate that question, we conducted a detailed empirical study, using publicly-available data sets from published genomic and proteomic studies. We observed that, under t-test and RELIEF filter-based feature selection, bagging generally does a good job of improving the performance of unstable, overfitting classifiers, such as CART decision trees and neural networks, but that improvement was not sufficient to beat the performance of single stable, nonoverfitting classifiers, such as diagonal and plain linear discriminant analysis, or 3-nearest neighbors. Furthermore, as expected, the ensemble method did not improve the performance of these classifiers significantly. Representative experimental results are presented and discussed in this work.