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Search Results: 1 - 10 of 339859 matches for " J. David Lambeth "
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Molecular evolution of Phox-related regulatory subunits for NADPH oxidase enzymes
Tsukasa Kawahara, J David Lambeth
BMC Evolutionary Biology , 2007, DOI: 10.1186/1471-2148-7-178
Abstract: Ancestral p47phox, p67phox, and p22phox genes are broadly seen in the metazoa, except for the ecdysozoans. The choanoflagellate Monosiga brevicollis, the unicellular organism that is the closest relatives of multicellular animals, encodes early prototypes of p22phox, p47phox as well as the earliest known Nox2-like ancestor of the Nox1-3 subfamily. p67phox- and p47phox-like genes are seen in the sea urchin Strongylocentrotus purpuratus and the limpet Lottia gigantea that also possess Nox2-like co-orthologs of vertebrate Nox1-3. Duplication of primordial p47phox and p67phox genes occurred in vertebrates, with the duplicated branches evolving into NOXO1 and NOXA1. Analysis of characteristic domains of regulatory subunits suggests a novel view of the evolution of Nox: in fish, p40phox participated in regulating both Nox1 and Nox2, but after the appearance of mammals, Nox1 (but not Nox2) became independent of p40phox. In the fish Oryzias latipes, a NOXO1 ortholog retains an autoinhibitory region that is characteristic of mammalian p47phox, and this was subsequently lost from NOXO1 in later vertebrates. Detailed amino acid sequence comparisons identified both putative key residues conserved in characteristic domains and previously unidentified conserved regions. Also, candidate organizer/activator proteins in fungi and amoeba are identified and hypothetical activation models are suggested.This is the first report to provide the comprehensive view of the molecular evolution of regulatory subunits for Nox enzymes. This approach provides clues for understanding the evolution of biochemical and physiological functions for regulatory-subunit-dependent Nox enzymes.Nox enzymes (reactive oxygen-generating NADPH-oxidases) diverged early in evolution into calcium-regulated Noxes (e.g., Nox5 and the Duox enzymes) and Noxes that are activated by binding to regulatory subunits [1,2]. The most extensively studied of the latter is the phagocyte NAPDH-oxidase (Phox), whose role in host d
Molecular evolution of the reactive oxygen-generating NADPH oxidase (Nox/Duox) family of enzymes
Tsukasa Kawahara, Mark T Quinn, J David Lambeth
BMC Evolutionary Biology , 2007, DOI: 10.1186/1471-2148-7-109
Abstract: We assembled and analyzed the deduced amino acid sequences of 101 Nox/Duox orthologs from 25 species, including vertebrates, urochordates, echinoderms, insects, nematodes, fungi, slime mold amoeba, alga and plants. In contrast to ROS defense enzymes, such as superoxide dismutase and catalase that are present in prokaryotes, ROS-generating Nox/Duox orthologs only appeared later in evolution. Molecular taxonomy revealed seven distinct subfamilies of Noxes and Duoxes. The calcium-regulated orthologs representing 4 subfamilies diverged early and are the most widely distributed in biology. Subunit-regulated Noxes represent a second major subdivision, and appeared first in fungi and amoeba. Nox5 was lost in rodents, and Nox3, which functions in the inner ear in gravity perception, emerged the most recently, corresponding to full-time adaptation of vertebrates to land. The sea urchin Strongylocentrotus purpuratus possesses the earliest Nox2 co-ortholog of vertebrate Nox1, 2, and 3, while Nox4 first appeared somewhat later in urochordates. Comparison of evolutionary substitution rates demonstrates that Nox2, the regulatory subunits p47phox and p67phox, and Duox are more stringently conserved in vertebrates than other Noxes and Nox regulatory subunits. Amino acid sequence comparisons identified key catalytic or regulatory regions, as 68 residues were highly conserved among all Nox/Duox orthologs, and 14 of these were identical with those mutated in Nox2 in variants of X-linked chronic granulomatous disease. In addition to canonical motifs, the B-loop, TM6-FAD, VXGPFG-motif, and extreme C-terminal regions were identified as important for Nox activity, as verified by mutational analysis. The presence of these non-canonical, but highly conserved regions suggests that all Nox/Duox may possess a common biological function remained in a long history of Nox/Duox evolution.This report provides the first comprehensive analysis of the evolution and conserved functions of Nox and Duox
Overexpression of Aromatase Alone is Sufficient for Ovarian Development in Genetically Male Chicken Embryos
Luke S. Lambeth, David M. Cummins, Timothy J. Doran, Andrew H. Sinclair, Craig A. Smith
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0068362
Abstract: Estrogens play a key role in sexual differentiation of both the gonads and external traits in birds. The production of estrogen occurs via a well-characterised steroidogenic pathway, which is a multi-step process involving several enzymes, including cytochrome P450 aromatase. In chicken embryos, the aromatase gene (CYP19A1) is expressed female-specifically from the time of gonadal sex differentiation. To further explore the role of aromatase in sex determination, we ectopically delivered this enzyme using the retroviral vector RCASBP in ovo. Aromatase overexpression in male chicken embryos induced gonadal sex-reversal characterised by an enlargement of the left gonad and development of ovarian structures such as a thickened outer cortex and medulla with lacunae. In addition, the expression of key male gonad developmental genes (DMRT1, SOX9 and Anti-Müllerian hormone (AMH)) was suppressed, and the distribution of germ cells in sex-reversed males followed the female pattern. The detection of SCP3 protein in late stage sex-reversed male embryonic gonads indicated that these genetically male germ cells had entered meiosis, a process that normally only occurs in female embryonic germ cells. This work shows for the first time that the addition of aromatase into a developing male embryo is sufficient to direct ovarian development, suggesting that male gonads have the complete capacity to develop as ovaries if provided with aromatase.
Amyloid-β and Proinflammatory Cytokines Utilize a Prion Protein-Dependent Pathway to Activate NADPH Oxidase and Induce Cofilin-Actin Rods in Hippocampal Neurons
Keifer P. Walsh, Laurie S. Minamide, Sarah J. Kane, Alisa E. Shaw, David R. Brown, Bruce Pulford, Mark D. Zabel, J. David Lambeth, Thomas B. Kuhn, James R. Bamburg
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0095995
Abstract: Neurites of neurons under acute or chronic stress form bundles of filaments (rods) containing 1:1 cofilin:actin, which impair transport and synaptic function. Rods contain disulfide cross-linked cofilin and are induced by treatments resulting in oxidative stress. Rods form rapidly (5–30 min) in >80% of cultured hippocampal or cortical neurons treated with excitotoxic levels of glutamate or energy depleted (hypoxia/ischemia or mitochondrial inhibitors). In contrast, slow rod formation (50% of maximum response in ~6 h) occurs in a subpopulation (~20%) of hippocampal neurons upon exposure to soluble human amyloid-β dimer/trimer (Aβd/t) at subnanomolar concentrations. Here we show that proinflammatory cytokines (TNFα, IL-1β, IL-6) also induce rods at the same rate and within the same neuronal population as Aβd/t. Neurons from prion (PrPC)-null mice form rods in response to glutamate or antimycin A, but not in response to proinflammatory cytokines or Aβd/t. Two pathways inducing rod formation were confirmed by demonstrating that NADPH-oxidase (NOX) activity is required for prion-dependent rod formation, but not for rods induced by glutamate or energy depletion. Surprisingly, overexpression of PrPC is by itself sufficient to induce rods in over 40% of hippocampal neurons through the NOX-dependent pathway. Persistence of PrPC-dependent rods requires the continuous activity of NOX. Removing inducers or inhibiting NOX activity in cells containing PrPC-dependent rods causes rod disappearance with a half-life of about 36 min. Cofilin-actin rods provide a mechanism for synapse loss bridging the amyloid and cytokine hypotheses for Alzheimer disease, and may explain how functionally diverse Aβ-binding membrane proteins induce synaptic dysfunction.
Development of Retroviral Vectors for Tissue-Restricted Expression in Chicken Embryonic Gonads
Luke S. Lambeth, Thomas Ohnesorg, David M. Cummins, Andrew H. Sinclair, Craig A. Smith
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0101811
Abstract: The chicken embryo has long been a useful model organism for studying development, including sex determination and gonadal differentiation. However, manipulating gene expression specifically in the embryonic avian gonad has been difficult. The viral vector RCASBP can be readily used for embryo-wide transgene expression; however global mis-expression using this method can cause deleterious off-target effects and embryo-lethality. In an attempt to develop vectors for the over-expression of sequences in chicken embryonic urogenital tissues, the viral vector RCANBP was engineered to contain predicted promoter sequences of gonadal-expressed genes. Several promoters were analysed and it was found that although the SF1 promoter produced a tissue-restricted expression pattern that was highest in the mesonephros and liver, it was also higher in the gonads compared to the rest of the body. The location of EGFP expression from the SF1 promoter overlapped with several key gonad-expressed sex development genes; however expression was generally low-level and was not seen in all gonadal cells. To further validate this sequence the key testis determinant DMRT1 was over-expressed in female embryos, which due to insufficient levels had no effect on gonad development. The female gene aromatase was then over-expressed in male embryos, which disrupted the testis pathway as demonstrated by a reduction in AMH protein. Taken together, although these data showed that the SF1 promoter can be used for functional studies in ovo, a stronger promoter sequence would likely be required for the functional analysis of gonad genes that require high-level expression.
Chimpanzee Autarky
Sarah F. Brosnan, Mark F. Grady, Susan P. Lambeth, Steven J. Schapiro, Michael J. Beran
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0001518
Abstract: Background Economists believe that barter is the ultimate cause of social wealth—and even much of our human culture—yet little is known about the evolution and development of such behavior. It is useful to examine the circumstances under which other species will or will not barter to more fully understand the phenomenon. Chimpanzees (Pan troglodytes) are an interesting test case as they are an intelligent species, closely related to humans, and known to participate in reciprocal interactions and token economies with humans, yet they have not spontaneously developed costly barter. Methodology/Principle Findings Although chimpanzees do engage in noncostly barter, in which otherwise value-less tokens are exchanged for food, this lack of risk is not typical of human barter. Thus, we systematically examined barter in chimpanzees to ascertain under what circumstances chimpanzees will engage in costly barter of commodities, that is, trading food items for other food items with a human experimenter. We found that chimpanzees do barter, relinquishing lower value items to obtain higher value items (and not the reverse). However, they do not trade in all beneficial situations, maintaining possession of less preferred items when the relative gains they stand to make are small. Conclusions/Significance Two potential explanations for this puzzling behavior are that chimpanzees lack ownership norms, and thus have limited opportunity to benefit from the gains of trade, and that chimpanzees' risk of defection is sufficiently high that large gains must be imminent to justify the risk. Understanding the conditions that support barter in chimpanzees may increase understanding of situations in which humans, too, do not maximize their gains.
Characterisation and application of a bovine U6 promoter for expression of short hairpin RNAs
Luke S Lambeth, Robert J Moore, Morley Muralitharan, Brian P Dalrymple, Sean McWilliam, Timothy J Doran
BMC Biotechnology , 2005, DOI: 10.1186/1472-6750-5-13
Abstract: To develop a shRNA expression vector specifically for bovine RNAi applications, we identified and characterised a novel bovine U6 small nuclear RNA (snRNA) promoter from bovine sequence data. This promoter is the putative bovine homologue of the human U6-8 snRNA promoter, and features a number of functional sequence elements that are characteristic of these types of pol. III promoters. A PCR based cloning strategy was used to incorporate this promoter sequence into plasmid vectors along with shRNA sequences for RNAi. The promoter was then used to express shRNAs, which resulted in the efficient knockdown of an exogenous reporter gene and an endogenous bovine gene.We have mined data from the bovine genome sequencing project to identify a functional bovine U6 promoter and used the promoter sequence to construct a shRNA expression vector. The use of this native bovine promoter in shRNA expression is an important component of our future development of RNAi therapeutic and transgenic applications in bovine species.RNA interference (RNAi), a method of sequence specific gene knockdown, has been used to analyse gene function in plants, invertebrates, and more recently mammalian cells [1-3]. The conserved RNAi pathway involves the processing of double stranded RNA (dsRNA) duplexes into 21–23 nucleotide (nt) molecules known as small interfering RNAs (siRNA) to initiate gene knockdown [4-6]. Since the discovery of RNAi in animals [7] the use of long dsRNA in lower eukaryotes, especially in the model organism Caenorhabditis elegans, has been used to determine gene function [8,9]. However, in mammalian systems the cellular uptake of long dsRNA induces an antiviral defence mechanism initiated by interferon (IFN), leading to non-specific translational shutdown and apoptosis [10-12].This non-specific cellular activity can be circumvented by the direct transfection of either chemically synthesised or in vitro transcribed siRNAs of approximately 21 nt in length into mammalian cells [1
A direct comparison of strategies for combinatorial RNA interference
Luke S Lambeth, Nick J Van Hateren, Stuart A Wilson, Venugopal Nair
BMC Molecular Biology , 2010, DOI: 10.1186/1471-2199-11-77
Abstract: Double-shRNA expression vectors were generated for each co-RNAi platform and their ability to suppress both single and double-gene reporter targets were compared. The most reliable and effective gene silencing was achieved from the multiple promoter/shRNA approach, as this method induced additive suppression of single-gene targets and equally effective knockdown of double-gene targets. Although both lhRNA and microRNA-embedded strategies provided efficient gene knockdown, suppression levels were inconsistent and activity varied greatly for different siRNAs tested. Furthermore, it appeared that not only the position of siRNAs within these multi-shRNA constructs impacted upon silencing activity, but also local properties of each individual molecule. In addition, it was also found that the insertion of up to five promoter/shRNA cassettes into a single construct did not negatively affect the efficacy of each individual shRNA.By directly comparing the ability of shRNAs delivered from different co-RNA platforms to initiate knockdown of the same gene targets, we found that multiple U6/shRNA cassettes offered the most reliable and predictable suppression of both single and multiple-gene targets. These results highlight some important strengths and pitfalls of the currently used methods for multiple shRNA delivery, and provide valuable insights for the design and application of reliable co-RNAi.Since the first application of DNA-delivered RNA interference (RNAi), the expression of short hairpin RNAs (shRNAs) for targeted gene silencing has become a benchmark technology. Using plasmid and viral vectoring systems, the transcription of double stranded RNA precursors that are processed by the RNAi pathway has lead to potent gene-specific knockdown. Importantly, such strategies can permit the long-term delivery of shRNAs to overcome the limitation of transient suppression by small interfering RNAs (siRNAs). Building upon the early experimental success of expressed shRNAs, the del
When given the opportunity, chimpanzees maximize personal gain rather than “level the playing field”
Lydia M. Hopper,Susan P. Lambeth,Steven J. Schapiro,Sarah F. Brosnan
PeerJ , 2015, DOI: 10.7717/peerj.165
Abstract: We provided chimpanzees (Pan troglodytes) with the ability to improve the quality of food rewards they received in a dyadic test of inequity. We were interested to see if this provision influenced their responses and, if so, whether it was mediated by a social partner’s outcomes. We tested eight dyads using an exchange paradigm in which, depending on the condition, the chimpanzees were rewarded with either high-value (a grape) or low-value (a piece of celery) food rewards for each completed exchange. We included four conditions. In the first, “Different” condition, the subject received different, less-preferred, rewards than their partner for each exchange made (a test of inequity). In the “Unavailable” condition, high-value rewards were shown, but not given, to both chimpanzees prior to each exchange and the chimpanzees were rewarded equally with low-value rewards (a test of individual contrast). The final two conditions created equity. In these High-value and Low-value “Same” conditions both chimpanzees received the same food rewards for each exchange. Within each condition, the chimpanzees first completed ten trials in the Baseline Phase, in which the experimenter determined the rewards they received, and then ten trials in the Test Phase. In the Test Phase, the chimpanzees could exchange tokens through the aperture of a small wooden picture frame hung on their cage mesh in order to receive the high-value reward. Thus, in the Test Phase, the chimpanzees were provided with an opportunity to improve the quality of the rewards they received, either absolutely or relative to what their partner received. The chimpanzees responded in a targeted manner; in the Test Phase they attempted to maximize their returns in all conditions in which they had received low-value rewards during the Baseline Phase. Thus, the chimpanzees were apparently motivated to increase their reward regardless of their partners’, but they only used the mechanism provided when it afforded the opportunity for them to increase their rewards. We also found evidence that the chimpanzees’ responses were enhanced by social facilitation. Specifically, the chimpanzees were more likely to exchange their tokens through the frame when their test partner also did so, even in circumstances in which their reward value could not be improved. Our paradigm provided the chimpanzees with the possibility to improve the quality of rewards they received in the Test Phase. We found that refusals – to exchange tokens or to eat rewards – decreased significantly in the Test Phase compared to the Baseline Phase,
Israel's Second Lebanon War Reconsidered
Benjamin S. Lambeth
Military and Strategic Affairs , 2012,
Abstract: Operation Change of Direction, the code name given to Israel’s war against Hizbollah in Lebanon in 2006 by the Operations Directorate of the Israel Defense Forces (IDF), was the most inconclusive performance by far in the IDF’s many trials by re since 1948, in that it represented the rst time that a major regional confrontation ended without a clear cut victory on Israel’s part. The campaign’s uneven course and outcome did not emanate from any particular single point failure but rather, in the words of two informed commentators, from “an overall accumulation of circumstances.” More speci cally, it did not re ect any failure of Israel’s well endowed air arm to perform to the fullest extent of its considerable but not unlimited capabilities, as many were quick to complain. Rather, it resulted from a more overarching de ciency in strategy choice, whose most awed elements were inconsistency between avowed goals and the available means and will to pursue them, and the Israeli government’s initial placement of friendly casualty avoidance above mission accomplishment in its ranking of campaign priorities.
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