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Audit Benjamin - - 2013
In higher eukaryotes, the absence of specific sequence motifs, marking the origins of replication has been a serious hindrance to the understanding of (i) the mechanisms that regulate the spatio-temporal replication program, and (ii) the links between origins activation, chromatin structure and transcription. In this chapter, we review the partitioning ...
Berrington William R WR Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington School of Medicine, Seattle, WA, - - 2013
Legionella pneumophila is a facultative intracellular pathogen that is an important cause of pneumonia. Although host factors that may predispose to acquisition of Legionnaire's Disease (LD) include comorbid illnesses (e.g., diabetes, chronic lung disease), age, male sex, and smoking, many individuals have no identifiable risk factors. Some studies suggest that ...
Kozak Natalia A - - 2013
Legionella is ubiquitous in freshwater systems worldwide and can also be found in soil. Legionellosis may be caused by inhalation of aerosolized water or soil particles containing Legionella. Isolation of Legionella from the environment is an essential step in outbreak investigation and may also be performed within the context of ...
Seitz Roswitha - - 2013
The animal model of N-methyl-D-aspartate (NMDA)-induced excitotoxic damage of retinal ganglion cells (RGC) is widely used to study the molecular mechanisms of RGC apoptosis and/or its prevention by neuroprotective agents. This chapter provides protocols for applying NMDA-induced excitotoxic damage to RGC of mouse eyes and for subsequent measuring of the ...
Jäger Jens - - 2013
The production of outer membrane vesicles (OMVs) is a widespread phenomenon employed by bacteria to secrete cell envelope components into the environment. A contribution of Legionella pneumophila OMVs to the pathogenesis of Legionnaires' disease is likely due to the high number of virulence-related proteins in the vesicles. OMVs are isolated ...
Bozzaro Salvatore - - 2013
The professional phagocyte Dictyostelium discoideum is a simple eukaryotic microorganism, whose natural habitat is deciduous forest soil and decaying leaves, where the amoebae feed on bacteria and grow as separate, independent, single cells. In the last decade, the organism has been successfully used as a host for several human pathogens, ...
Goeman Frauke - - 2013
Chromatin immunoprecipitation (ChIP) followed by microarray hybridization (on-chip) is a technique well suited for a comprehensive analysis of transcription factor binding sites, histone modification patterns, and nucleosome occupancy. It can be restricted to a subset of genes or regions but also expanded up to a genome-wide range yielding insight into ...
Day Shandra R - - 2013
Legionella pneumophila is an intracellular pathogen of free-living protozoa that can also infect alveolar macrophages, L929 fibroblast cells, and HeLa cells. Infection of nonphagocytic cells by L. pneumophila can be used to study invasion mechanisms, compare infectivity of different strains and identify factors important for virulence. Virulent strains of L. ...
Ratcliff Rodney M - - 2013
Legionella strains are considered biologically inert with respect to traditional identification schemes. Various phenotypic alternatives have been attempted but all have lacked resolution as additional species have been added to what is proving to be a large genus. Only sequence-based schemes have the required resolution to confidently speciate or recognize ...
Dill Holger - - 2013
Disturbances in the general mRNA metabolism have been recognized as a major defect in a growing number of hereditary human diseases. One prominent example of this disease group is Retinitis pigmentosa (RP), characterized by selective loss of photoreceptor cells. RP can be caused by dominant mutations in key factors of ...
Shah Mansi - - 2013
Stem cells are unique cells that can self-renew and differentiate into many cell types. Plasticity is a fundamental characteristic of stem cells and it is regulated by reversible epigenetic modifications. Although gene-restriction programs are established during embryonic development when cell lineages are formed, stem cells retain a degree of flexibility ...
Sahr Tobias - - 2013
The adaptation of Legionella pneumophila to the different conditions it encounters in the environment and in the host is governed by a complex regulatory system. Current knowledge of these regulatory networks and the transcriptome responses of L. pneumophila is mainly based on microarray analysis and limited to transcriptional products of ...
Joseph Carol - - 2013
Outbreaks of Legionnaires' disease create high levels of public anxiety and media interest and inevitably consume a great deal of public health resources. Investigations should begin as early as possible in order to rapidly identify suspected sources of infection, control the outbreak and prevent further cases occurring. The investigations should ...
Villamar Mauricio F MF Laboratory of Neuromodulation, Spaulding Rehabilitation Hospital, Harvard Medical School, 02114 Boston, MA, - - 2013
To conduct a systematic review evaluating the reporting of blinding in randomized controlled trials published in the field of Physical Medicine and Rehabilitation over two time periods. We searched MEDLINE via PubMed for all randomized controlled trials published in American Journal of Physical Medicine and Rehabilitation, Archives of Physical Medicine ...
Sethna Saumil S Department of Biological Sciences, Fordham University, Bronx, NY, - - 2013
Counting rhodopsin-positive phagosomes residing in the retinal pigment epithelium (RPE) in the eye at different times of day allows a quantitative assessment of engulfment and digestion phases of diurnal RPE phagocytosis, which efficiently clears shed photoreceptor outer segment fragments (POS) from the neural retina. Comparing such activities among age- and ...
Liao Jun-Ming - - 2013
Chromatin immunoprecipitation assay (ChIP) has been frequently used to determine whether a transcriptional regulator can bind to a specific DNA element in the chromatin content of cells. Here, we describe a detailed protocol for this assay with hands-on tips based on our own experience in working on the transcriptional regulator ...
Tiaden André N - - 2013
Legionella pneumophila infects and replicates in environmental protozoa and metazoan macrophages within a specific vacuole. The infection of phagocytes by L. pneumophila can be assessed by an agar plating assay or by fluorescence microscopy. Here, we describe the analysis of Legionella infection by automated flow cytometry using wild-type and mutant ...
Bryan Andrew - - 2013
The ability to construct recombinant alleles efficiently in strains of interest, particularly unmarked deletions that reduce the potential for polar effects, is essential to studies of both pathogenesis and basic bacterial physiology. Here we describe a three-phase approach for generating unmarked deletions in Legionella pneumophila by constructing a mutant allele ...
Frum Rebecca A - - 2013
DNA replication involves a coordinated progression through S phase, and disruption of these regulated steps may cause gene abnormalities, which may lead to cancer. Different stages of DNA replication can be detected immunofluorescently that would indicate how replication is progressing in a cell population or under specific conditions. We describe ...
Vaughan Catherine - - 2013
In order to study the functions of a cell's endogenous mutant p53, the p53 protein levels must be knocked-down. Transient transfection of small interfering RNAs is one way to accomplish this. Another is the stable expression of short hairpin RNAs. This chapter presents a method by which a short hairpin ...
Tanimoto Naoyuki - - 2013
In many situations it is important to be able to assess the degree of retinal function, e.g., for the characterization of mouse models with unknown retinal involvement, when studying degenerative processes, for the analysis of visual signal processing, and during the follow-up of therapeutic interventions. Full-field electroretinography (ERG), yielding a ...
Vaseva Angelina V - - 2013
p53 is a master regulator of cell death pathways and has transcription-dependent and transcription-independent modes of action. Mitochondria are major signal transducers in apoptosis and are critical for p53-dependent cell death. Our lab and others have discovered that a fraction of stress-induced wild-type p53 protein rapidly translocates to mitochondria upon ...
Strauß Olaf - - 2013
Ca(2+) is an important regulator of many cell functions including proliferation, apoptosis, movements, secretion, contraction, excitation, and differentiation. The regulation of these different cell functions is encoded by the specific temporal and spatial distribution of Ca(2+) signals. In degenerative diseases mutations can lead to changes in cell functions in the ...
Marques Sueli - - 2013
Neurodegenerative disorders, such as Parkinson's and Alzheimer's disease, are highly complex, due to their multifactorial origin, not only depending on genetic but also on environmental factors. Several genetic risk factors have already been associated with both the diseases, however, the precise way through which the environment contributes to neurodegeneration is ...
Senturk Emir - - 2013
The p53 tumor suppressor protein plays a central role in mediating the cellular response to a variety of stresses. Activation of p53 signaling will trigger cell cycle arrest or apoptosis in normal cells, depending on such factors as cell type and genetic context. The ability of a cell to circumvent ...
Goehe Rachel W - - 2013
This chapter presents methods for interrogating the involvement of p53 in signaling to apoptosis, autophagy, and senescence. The well-known association of p53 with the stress response to chemotherapy and radiation is the basis for presenting these approaches. The development of quantitative and efficient in vitro assays has enabled researchers to ...
Lück Christian - - 2013
The lipopolysaccharide(LPS) of Legionella spp. is an immuno-dominant antigen and the basis for Legionella pneumophila serogroup classification. The LPS shows a peculiar structure composed of a very hydrophobic lipid A acylated by long chain fatty acids and an O-antigen-specific chain consisting of homopolymeric legionaminic acid. In this chapter we describe ...
Campbell Matthew - - 2013
The inner blood-retina barrier (iBRB) is essential in restricting the movement of systemic components such as enzymes, anaphylatoxins, or pathogens that could otherwise enter the neural retina and cause extensive damage. The barrier has evolved to confer protection to the delicate microenvironment of the retina, and the tight junctions located ...
Pascali Chiara - - 2013
RNA polymerase III (Pol III) transcription is regulated by modifications of the chromatin. DNA methylation and post-translational modifications of histones, such as acetylation, phosphorylation and methylation have been linked to Pol III transcriptional activity. In addition to being regulated by modifications of DNA and histones, Pol III genes and its ...
Gadepalli Venkat S - - 2013
Cancer stem cells possess the ability to self-renew and differentiate into specific cells found in tumor types, a characteristic feature of normal multipotent stem cells. These cells harbor within the bulk of tumors and if the tumor suppressor p53 is mutated in these cells, can be more likely to cause ...
Doonan Francesca - - 2013
Terminal dUTP nick end labeling (TUNEL) is an invaluable technique used in the study of late-stage apoptosis. The technique is based upon detection of fragmented DNA, a well-recognized characteristic of apoptosis, usually with fluorescent markers. Here, we describe the TUNEL technique (1) employing two different detection techniques, fluorescence microscopy and ...
Chang Bo B The Jackson Laboratory, Bar Harbor, ME, USA, - - 2013
Mouse models, with their well-developed genetics and similarity to human physiology and anatomy, serve as powerful tools with which to investigate the etiology of human retinal degeneration. Mutant mice also provide reproducible, experimental systems for elucidating pathways of normal development and function. Here, I describe the tools used in the ...
Barrero Maria J - - 2013
RNA polymerase II (Pol II) is the main engine that drives transcription of protein-encoding genes in eukaryotes. Despite its intrinsic subunit complexity, Pol II is subject to a host of factors that regulate the multistep transcription process. Indeed, the hallmark of the transcription cycle is the dynamic association of Pol ...
Weber Stephen - - 2013
By means of the Icm/Dot type IV secretion system Legionella pneumophila translocates several effector proteins into host cells, where they anchor to the cytoplasmic face of the LCV membrane by binding to phosphoinositide (PI) lipids. Thus, phosphatidylinositol-4-phosphate anchors the effector proteins SidC and SidM, which promote the interaction of LCVs ...
Lee Susanne - - 2013
To avoid further cases arising from an infectious source it is essential to ensure the early identification of all potential source(s) within an identified area, or buildings, to determine if they are being managed safely; to take appropriate samples and ensure appropriate remedial actions are taken to remove the risk ...
Montana Cynthia L - - 2013
Transcription factors control gene expression by binding to noncoding regions of DNA known as -cis-regulatory elements (CREs; i.e., enhancer/promoters). Traditionally, cis-regulatory analysis has been carried out via mouse transgenesis which is time-consuming and nonquantitative. Electroporation of DNA reporter constructs into living mouse tissue is a rapid and effective alternative to ...
Zhang Qi - - 2013
Fluorescence polarization (FP) has become a powerful technique to quantitatively analyze the binding of a small soluble fluorescence-labeled probe to a larger soluble protein and its displacement by other molecules. Here, we describe a detailed protocol to identify small molecules capable of targeting p53-MDM2/MDMX interactions using a fluorescence polarization assay ...
Pécastaings Sophie - - 2013
In aquatic environments such as water distribution systems, Legionella pneumophila persistence is -correlated to the presence of a biofilm. The method described here permits the formation of a monospecies L. pneumophila biofilm in microplates, enabling the screening of multiple parameters. The culture medium used has a low nutrient concentration compared ...
Mühlfriedel Regine - - 2013
Subretinal injections in mice become increasingly important. Currently, the most prominent application is in gene therapy of inherited eye diseases by means of viral vector delivery to photoreceptors or the retinal pigment epithelium (RPE). Since there are no large animal models for most of these diseases, genetically modified mouse models ...
Gokul Gopinathan - - 2013
Cancer has been considered a genetic disease with a wide array of well-characterized gene mutations and chromosomal abnormalities. Of late, aberrant epigenetic modifications have been elucidated in cancer, and together with genetic alterations, they have been helpful in understanding the complex traits observed in neoplasia. "Cancer Epigenetics" therefore has contributed ...
Kumar Ritesh - - 2013
In prokaryotes, alteration in gene expression was observed with the modification of DNA, especially DNA methylation. Such changes are inherited from generation to generation with no alterations in the DNA sequence and represent the epigenetic signal in prokaryotes. DNA methyltransferases are enzymes involved in DNA modification and thus in epigenetic ...
Wei Zhiyun Z Department of Medicine, Institute for Genomic Medicine, University of California at San Diego, VA San Diego Healthcare System, La Jolla, California 92093-0838, - - 2013
The neuropeptide Y(2) G-protein-coupled receptor (NPY2R) relays signals from PYY or neuropeptide Y toward satiety and control of body mass. Targeted ablation of the NPY2R locus in mice yields obesity, and studies of NPY2R promoter genetic variation in more than 10,000 human participants indicate its involvement in control of obesity ...
Portier Emilie - - 2013
Biofilm formation could be studied in various conditions. Most of the studies with Legionella pneumophila used monospecies biofilm in culture media. In some cases, it is important to study bacteria in conditions more close to environmental conditions. In this paper, we describe protocols to produce natural biofilms from river water ...
Dhamija Neeru - - 2013
Despite the intense effort put by researchers globally to understand Human Immunodeficiency Virus (HIV-1) pathogenesis since its discovery 30 years ago, the acquired knowledge till date is not good enough to eradicate HIV-1 from an infected individual. HIV-1 infects cells of the human immune system and integrates into the host ...
Yeudall W Andrew - - 2013
Pro-oncogenic properties of mutant p53 were investigated with the aid of migration assays, adhesion assays, and soft agar growth assays using cells stably expressing gain-of-function p53 mutants. To determine cell migration, "wound-healing" (scratch) assays and haptotactic (chamber) assays were used. H1299 cells expressing mutant p53 were found to migrate more ...
Huber-Reggi Sabina P - - 2013
Large-field movements in the visual surround trigger spontaneous, compensatory eye movements known as optokinetic response (OKR) in all vertebrates. In zebrafish (Danio rerio) the OKR is well developed at 5 days post fertilization and can be used in the laboratory for screening of visual performance following genetic manipulations or pharmaceutical ...
Goswami Shyamal K - - 2013
Since the Central dogma of Molecular Biology was proposed about 40 years ago; our understanding of the intricacies of gene regulation has undergone tectonic shifts almost every decade. It is now widely accepted that the complexity of an organism is not directed by the sheer number of genes it carries ...
Buchrieser Carmen - - 2013
Many Gram-positive and Gram-negative bacteria possess natural competence mechanisms for DNA -capture and internalization that play an important role in diversifying adaptation of bacteria through horizontal gene transfer. Natural transformation and other mechanisms of horizontal gene transfer are dependent on DNA recombination. Natural competence can be exploited both for studying ...
Fischer M Dominik - - 2013
Optical coherence tomography (OCT) is an invaluable technique to perform noninvasive retinal imaging in small animal models such as mice. It provides virtual cross sections that correlate well with histomorphometric data with the advantage that multiple iterative measurements can be acquired in time line analyses to detect dynamic changes and ...
Love Ian M - - 2013
p53 levels and activity are controlled in large part through regulated ubiquitination and subsequent destruction by the 26S proteasome. Monoubiquitination of p53 is mediated primarily by the RING-finger E3 ubiquitin ligase MDM2 and impacts p53 activity through modulation of p53 localization and transcription activities. Recently, several E4 ubiquitin ligases (E4s) ...
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