Functionally active targeting domain of the beta-adrenergic receptor kinase: an inhibitor of G beta gamma-mediated stimulation of type II adenylyl cyclase.

The beta-adrenergic receptor kinase (beta ARK) phosphorylates its membrane-associated receptor substrates, such as the beta-adrenergic receptor, triggering events leading to receptor desensitization. beta ARK activity is markedly stimulated by the isoprenylated beta gamma subunit complex of heterotrimeric guanine nucleotide-binding proteins (G beta gamma), which translocates the kinase to the plasma membrane and thereby targets it to its receptor substrate. The amino-terminal two-thirds of beta ARK1 composes the receptor recognition and catalytic domains, while the carboxyl third contains the G beta gamma binding sequences, the targeting domain. We prepared this domain as a recombinant His6 fusion protein from Escherichia coli and found that it had both independent secondary structure and functional activity. We demonstrated the inhibitory properties of this domain against G beta gamma activation of type II adenylyl cyclase both in a reconstituted system utilizing Sf9 insect cell membranes and in a permeabilized 293 human embryonic kidney cell system. Gi alpha-mediated inhibition of adenylyl cyclase was not affected. These data suggest that this His6 fusion protein derived from the carboxyl terminus of beta ARK1 provides a specific probe for defining G beta gamma-mediated processes and for studying the structural features of a G beta gamma-binding domain.

A network of substrates of the E3 ubiquitin ligases MDM2 and HUWE1 control apoptosis independently of p53.

In the intrinsic pathway of apoptosis, cell-damaging signals promote the release of cytochrome c from mitochondria, triggering activation of the Apaf-1 and caspase-9 apoptosome. The ubiquitin E3 ligase MDM2 decreases the stability of the proapoptotic factor p53. We show that it also coordinated apoptotic events in a p53-independent manner by ubiquitylating the apoptosome activator CAS and the ubiquitin E3 ligase HUWE1. HUWE1 ubiquitylates the antiapoptotic factor Mcl-1, and we found that HUWE1 also ubiquitylated PP5 (protein phosphatase 5), which indirectly inhibited apoptosome activation. Breast cancers that are positive for the tyrosine receptor kinase HER2 (human epidermal growth factor receptor 2) tend to be highly aggressive. In HER2-positive breast cancer cells treated with the HER2 tyrosine kinase inhibitor lapatinib, MDM2 was degraded and HUWE1 was stabilized. In contrast, in breast cancer cells that acquired resistance to lapatinib, the abundance of MDM2 was not decreased and HUWE1 was degraded, which inhibited apoptosis, regardless of p53 status. MDM2 inhibition overcame lapatinib resistance in cells with either wild-type or mutant p53 and in xenograft models. These findings demonstrate broader, p53-independent roles for MDM2 and HUWE1 in apoptosis and specifically suggest the potential for therapy directed against MDM2 to overcome lapatinib resistance.

Narrative centrality and negative affectivity: independent and interactive contributors to stress reactions.

Reactions to stressful negative events have long been studied using approaches based on either the narrative interpretation of the event or the traits of the individual. Here, we integrate these 2 approaches by using individual-differences measures of both the narrative interpretation of the stressful event as central to one's life and the personality characteristic of negative affectivity. We show that they each have independent contributions to stress reactions and that high levels on both produce greater than additive effects. The effects on posttraumatic stress symptoms are substantial for both undergraduates (Study 1, n = 2,296; Study 3, n = 488) and veterans (Study 2, n = 104), with mean levels for participants low on both measures near floor on posttraumatic stress symptoms and those high on both measures scoring at or above diagnostic thresholds. Study 3 included 3 measures of narrative centrality and 3 of negative affectivity to demonstrate that the effects were not limited to a single measure. In Study 4 (n = 987), measures associated with symptoms of posttraumatic stress correlated substantially with either measures of narrative centrality or measures of negative affectivity. The concepts of narrative centrality and negative affectivity and the results are consistent with findings from clinical populations using similar measures and with current approaches to therapy. In broad nonclinical populations, such as those used here, the results suggest that we might be able to substantially increase our ability to account for the severity of stress response by including both concepts.

Phytochrome diversity in green plants and the origin of canonical plant phytochromes.

Phytochromes are red/far-red photoreceptors that play essential roles in diverse plant morphogenetic and physiological responses to light. Despite their functional significance, phytochrome diversity and evolution across photosynthetic eukaryotes remain poorly understood. Using newly available transcriptomic and genomic data we show that canonical plant phytochromes originated in a common ancestor of streptophytes (charophyte algae and land plants). Phytochromes in charophyte algae are structurally diverse, including canonical and non-canonical forms, whereas in land plants, phytochrome structure is highly conserved. Liverworts, hornworts and Selaginella apparently possess a single phytochrome, whereas independent gene duplications occurred within mosses, lycopods, ferns and seed plants, leading to diverse phytochrome families in these clades. Surprisingly, the phytochrome portions of algal and land plant neochromes, a chimera of phytochrome and phototropin, appear to share a common origin. Our results reveal novel phytochrome clades and establish the basis for understanding phytochrome functional evolution in land plants and their algal relatives.

Comparative analyses of seven algorithms for copy number variant identification from single nucleotide polymorphism arrays.

Determination of copy number variants (CNVs) inferred in genome wide single nucleotide polymorphism arrays has shown increasing utility in genetic variant disease associations. Several CNV detection methods are available, but differences in CNV call thresholds and characteristics exist. We evaluated the relative performance of seven methods: circular binary segmentation, CNVFinder, cnvPartition, gain and loss of DNA, Nexus algorithms, PennCNV and QuantiSNP. Tested data included real and simulated Illumina HumHap 550 data from the Singapore cohort study of the risk factors for Myopia (SCORM) and simulated data from Affymetrix 6.0 and platform-independent distributions. The normalized singleton ratio (NSR) is proposed as a metric for parameter optimization before enacting full analysis. We used 10 SCORM samples for optimizing parameter settings for each method and then evaluated method performance at optimal parameters using 100 SCORM samples. The statistical power, false positive rates, and receiver operating characteristic (ROC) curve residuals were evaluated by simulation studies. Optimal parameters, as determined by NSR and ROC curve residuals, were consistent across datasets. QuantiSNP outperformed other methods based on ROC curve residuals over most datasets. Nexus Rank and SNPRank have low specificity and high power. Nexus Rank calls oversized CNVs. PennCNV detects one of the fewest numbers of CNVs.

Interactions of chromatin context, binding site sequence content, and sequence evolution in stress-induced p53 occupancy and transactivation.

Cellular stresses activate the tumor suppressor p53 protein leading to selective binding to DNA response elements (REs) and gene transactivation from a large pool of potential p53 REs (p53REs). To elucidate how p53RE sequences and local chromatin context interact to affect p53 binding and gene transactivation, we mapped genome-wide binding localizations of p53 and H3K4me3 in untreated and doxorubicin (DXR)-treated human lymphoblastoid cells. We examined the relationships among p53 occupancy, gene expression, H3K4me3, chromatin accessibility (DNase 1 hypersensitivity, DHS), ENCODE chromatin states, p53RE sequence, and evolutionary conservation. We observed that the inducible expression of p53-regulated genes was associated with the steady-state chromatin status of the cell. Most highly inducible p53-regulated genes were suppressed at baseline and marked by repressive histone modifications or displayed CTCF binding. Comparison of p53RE sequences residing in different chromatin contexts demonstrated that weaker p53REs resided in open promoters, while stronger p53REs were located within enhancers and repressed chromatin. p53 occupancy was strongly correlated with similarity of the target DNA sequences to the p53RE consensus, but surprisingly, inversely correlated with pre-existing nucleosome accessibility (DHS) and evolutionary conservation at the p53RE. Occupancy by p53 of REs that overlapped transposable element (TE) repeats was significantly higher (p<10-7) and correlated with stronger p53RE sequences (p<10-110) relative to nonTE-associated p53REs, particularly for MLT1H, LTR10B, and Mer61 TEs. However, binding at these elements was generally not associated with transactivation of adjacent genes. Occupied p53REs located in L2-like TEs were unique in displaying highly negative PhyloP scores (predicted fast-evolving) and being associated with altered H3K4me3 and DHS levels. These results underscore the systematic interaction between chromatin status and p53RE context in the induced transactivation response. This p53 regulated response appears to have been tuned via evolutionary processes that may have led to repression and/or utilization of p53REs originating from primate-specific transposon elements.

Explicit DNase sequence bias modeling enables high-resolution transcription factor footprint detection.

DNaseI footprinting is an established assay for identifying transcription factor (TF)-DNA interactions with single base pair resolution. High-throughput DNase-seq assays have recently been used to detect in vivo DNase footprints across the genome. Multiple computational approaches have been developed to identify DNase-seq footprints as predictors of TF binding. However, recent studies have pointed to a substantial cleavage bias of DNase and its negative impact on predictive performance of footprinting. To assess the potential for using DNase-seq to identify individual binding sites, we performed DNase-seq on deproteinized genomic DNA and determined sequence cleavage bias. This allowed us to build bias corrected and TF-specific footprint models. The predictive performance of these models demonstrated that predicted footprints corresponded to high-confidence TF-DNA interactions. DNase-seq footprints were absent under a fraction of ChIP-seq peaks, which we show to be indicative of weaker binding, indirect TF-DNA interactions or possible ChIP artifacts. The modeling approach was also able to detect variation in the consensus motifs that TFs bind to. Finally, cell type specific footprints were detected within DNase hypersensitive sites that are present in multiple cell types, further supporting that footprints can identify changes in TF binding that are not detectable using other strategies.

Quantitative genetics of CTCF binding reveal local sequence effects and different modes of X-chromosome association.

Associating genetic variation with quantitative measures of gene regulation offers a way to bridge the gap between genotype and complex phenotypes. In order to identify quantitative trait loci (QTLs) that influence the binding of a transcription factor in humans, we measured binding of the multifunctional transcription and chromatin factor CTCF in 51 HapMap cell lines. We identified thousands of QTLs in which genotype differences were associated with differences in CTCF binding strength, hundreds of them confirmed by directly observable allele-specific binding bias. The majority of QTLs were either within 1 kb of the CTCF binding motif, or in linkage disequilibrium with a variant within 1 kb of the motif. On the X chromosome we observed three classes of binding sites: a minority class bound only to the active copy of the X chromosome, the majority class bound to both the active and inactive X, and a small set of female-specific CTCF sites associated with two non-coding RNA genes. In sum, our data reveal extensive genetic effects on CTCF binding, both direct and indirect, and identify a diversity of patterns of CTCF binding on the X chromosome.

Identification of dopamine receptors across the extant avian family tree and analysis with other clades uncovers a polyploid expansion among vertebrates.

Dopamine is an important central nervous system transmitter that functions through two classes of receptors (D1 and D2) to influence a diverse range of biological processes in vertebrates. With roles in regulating neural activity, behavior, and gene expression, there has been great interest in understanding the function and evolution dopamine and its receptors. In this study, we use a combination of sequence analyses, microsynteny analyses, and phylogenetic relationships to identify and characterize both the D1 (DRD1A, DRD1B, DRD1C, and DRD1E) and D2 (DRD2, DRD3, and DRD4) dopamine receptor gene families in 43 recently sequenced bird genomes representing the major ordinal lineages across the avian family tree. We show that the common ancestor of all birds possessed at least seven D1 and D2 receptors, followed by subsequent independent losses in some lineages of modern birds. Through comparisons with other vertebrate and invertebrate species we show that two of the D1 receptors, DRD1A and DRD1B, and two of the D2 receptors, DRD2 and DRD3, originated from a whole genome duplication event early in the vertebrate lineage, providing the first conclusive evidence of the origin of these highly conserved receptors. Our findings provide insight into the evolutionary development of an important modulatory component of the central nervous system in vertebrates, and will help further unravel the complex evolutionary and functional relationships among dopamine receptors.

Whole genome sequencing identifies circulating Beijing-lineage Mycobacterium tuberculosis strains in Guatemala and an associated urban outbreak.

Limited data are available regarding the molecular epidemiology of Mycobacterium tuberculosis (Mtb) strains circulating in Guatemala. Beijing-lineage Mtb strains have gained prevalence worldwide and are associated with increased virulence and drug resistance, but there have been only a few cases reported in Central America. Here we report the first whole genome sequencing of Central American Beijing-lineage strains of Mtb. We find that multiple Beijing-lineage strains, derived from independent founding events, are currently circulating in Guatemala, but overall still represent a relatively small proportion of disease burden. Finally, we identify a specific Beijing-lineage outbreak centered on a poor neighborhood in Guatemala City.

Distinct routes to metastasis: plasticity-dependent and plasticity-independent pathways.

The cascade that culminates in macrometastases is thought to be mediated by phenotypic plasticity, including epithelial-mesenchymal and mesenchymal-epithelial transitions (EMT and MET). Although there is substantial support for the role of EMT in driving cancer cell invasion and dissemination, much less is known about the importance of MET in the later steps of metastatic colonization. We created novel reporters, which integrate transcriptional and post-transcriptional regulation, to test whether MET is required for metastasis in multiple in vivo cancer models. In a model of carcinosarcoma, metastasis occurred via an MET-dependent pathway; however, in two prostate carcinoma models, metastatic colonization was MET independent. Our results provide evidence for both MET-dependent and MET-independent metastatic pathways.

Cell-to-cell contact-independent regulatory T cells in human tuberculosis

info:eu-repo/semantics/nonPublished

Pathogenicity of Salmonella: SopE-mediated membrane ruffling is independent of inositol phosphate signals

Studies [Zhou, D. Chen, L.-M. Hernandez, L. Shears, S.B. and Galán, J.E. (2001) A Salmonella inositol polyphosphatase acts in conjunction with other bacterial effectors to promote host-cell actin cytoskeleton rearrangements and bacterial internalization. Mol. Microbiol. 39, 248-259] with engineered Salmonella mutants showed that deletion of SopE attenuated the pathogen's ability to deplete host-cell InsP5 and remodel the cytoskeleton. We pursued these observations: In SopE-transfected host-cells, membrane ruffling was induced, but SopE did not dephosphorylate InsP5, nor did it recruit PTEN (a cytosolic InsP5 phosphatase) for this task. However, PTEN strengthened SopE-mediated membrane ruffling. We conclude SopE promotes host-cell InsP5 hydrolysis only with the assistance of other Salmonella proteins. Our demonstration that Salmonella-mediated cytoskeletal modifications are independent of inositolphosphates will focus future studies on elucidating alternate pathogenic consequences of InsP5 metabolism, including ion channel conductance and apoptosis.

Approximation algorithms for two-machine flow shop scheduling with batch setup times

In many practical situations, batching of similar jobs to avoid setups is performed while constructing a schedule. This paper addresses the problem of non-preemptively scheduling independent jobs in a two-machine flow shop with the objective of minimizing the makespan. Jobs are grouped into batches. A sequence independent batch setup time on each machine is required before the first job is processed, and when a machine switches from processing a job in some batch to a job of another batch. Besides its practical interest, this problem is a direct generalization of the classical two-machine flow shop problem with no grouping of jobs, which can be solved optimally by Johnson's well-known algorithm. The problem under investigation is known to be NP-hard. We propose two O(n logn) time heuristic algorithms. The first heuristic, which creates a schedule with minimum total setup time by forcing all jobs in the same batch to be sequenced in adjacent positions, has a worst-case performance ratio of 3/2. By allowing each batch to be split into at most two sub-batches, a second heuristic is developed which has an improved worst-case performance ratio of 4/3. © 1998 The Mathematical Programming Society, Inc. Published by Elsevier Science B.V.

The open shop scheduling problem with a given sequence of jobs on one machine

The paper considers the open shop scheduling problem to minimize the make-span, provided that one of the machines has to process the jobs according to a given sequence. We show that in the preemptive case the problem is polynomially solvable for an arbitrary number of machines. If preemption is not allowed, the problem is NP-hard in the strong sense if the number of machines is variable, and is NP-hard in the ordinary sense in the case of two machines. For the latter case we give a heuristic algorithm that runs in linear time and produces a schedule with the makespan that is at most 5/4 times the optimal value. We also show that the two-machine problem in the nonpreemptive case is solvable in pseudopolynomial time by a dynamic programming algorithm, and that the algorithm can be converted into a fully polynomial approximation scheme. © 1998 John Wiley & Sons, Inc. Naval Research Logistics 45: 705–731, 1998