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Faucher Sebastien P - - 2013
The study of transcriptome responses can provide insight into the regulatory pathways and genetic factors that contribute to a specific phenotype. For bacterial pathogens, it can identify putative new virulence systems and shed light on the mechanisms underlying the regulation of virulence factors. Microarrays have been previously used to study ...
du Preez Louis L - - 2013
The core histone N-terminal tails dissociate from their binding positions in nucleosomes at moderate salt concentrations, and appear unstructured in the crystal. This suggested that the tails contributed minimally to chromatin structure. However, in vitro studies have shown that the tails were involved in a range of intra- and inter-nucleosomal ...
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 ...
Marcel Virginie - - 2013
The human p53 protein isoforms are expressed in several cell lines and modulate p53 tumor suppressor -activity, mainly through modulation of gene expression (1-4). Thus, identifying the pattern of p53 isoforms expression in cell lines is a key step for future studies of the p53 network (5). At the moment, ...
Hori Juliana I - - 2013
Legionella pneumophila is an intracellular bacterium that was evolutionarily selected to survive in freshwater environments by infecting free-living unicellular protozoa. Once humans inhale contaminated water droplets, the bacteria reach the pulmonary alveoli where they are phagocytized by resident alveolar macrophages. Depending on host immunity and bacterial virulence genes, the infection ...
Enoki Ryosuke - - 2013
Various types of retinal neurons, including amacrine, ganglion, and horizontal cells, expand neurites (dendrites or axons) in horizontal direction and make synaptic or electrical contacts with other cells to integrate the visual information. Many types of ion-channels and receptors are located along these neurites, and these horizontal connections critically contribute ...
Neveling Kornelia - - 2013
Inherited retinal diseases display a very high degree of clinical and genetic heterogeneity, which poses challenges in identifying the underlying defects in known genes and in identifying novel retinal disease genes. Here, we outline the state-of-the-art techniques to find the causative DNA variants, with special attention for next-generation sequencing which ...
Shanmugam Muthu K - - 2013
There is considerable evidence suggesting that epigenetic mechanisms may mediate development of chronic inflammation by modulating the expression of pro-inflammatory cytokine TNF-α, interleukins, tumor suppressor genes, oncogenes and autocrine and paracrine activation of the transcription factor NF-κB. These molecules are constitutively produced by a variety of cells under chronic inflammatory ...
Bharathy Narendra - - 2013
Skeletal muscle cells have served as a paradigm for understanding mechanisms leading to cellular differentiation. Formation of skeletal muscle involves a series of steps in which cells are commited towards the myogenic lineage, undergo expansion to give rise to myoblasts that differentiate into multinucleated myotubes, and mature to form adult ...
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 ...
Sahr Tobias - - 2013
Transcriptional and posttranscriptional regulators play a critical role in allowing a bacterium to adapt to the diverse environments and conditions it encounters. In order to characterize the role of these regulators the identification of their specific interaction partners is of utmost importance. Co-immunoprecipitation (IP) is based on antigen/antibody complex formation ...
Chatfield Christa H - - 2013
This chapter describes methods for culturing Legionella pneumophila in both complex and defined media. The first protocol describes the use of buffered charcoal yeast extract (BCYE) agar, the solid medium that is most commonly used for culturing L. pneumophila. The next procedure details the cultivation of L. pneumophila in buffered ...
Deshmukh Abhijit S - - 2013
Plasmodium, one of the most important members of Apicomplexan protozoans imposes a significant economic and health impact on human populations around the world. Plasmodium parasite has complex life cycle with morphologically distinct asexual and sexual developmental stages in the human host and Anopheline mosquito vector. The parasites undergo rapid transition ...
Goodfellow Sarah J - - 2013
RNA Polymerase (Pol) I produces ribosomal (r)RNA, an essential component of the cellular protein synthetic machinery that drives cell growth, underlying many fundamental cellular processes. Extensive research into the mechanisms governing transcription by Pol I has revealed an intricate set of control mechanisms impinging upon rRNA production. Pol I-specific transcription ...
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 ...
Nagai Hiroki - - 2013
Bacterial virulence proteins often mimic host eukaryotic proteins to modify or disturb host cellular -pathways. Increasing lines of evidence show that many bacterial effector proteins have E3 ubiquitin ligase activity. The effector protein LubX is one such bacterial E3 ubiquitin ligase. We describe here the method to purify soluble LubX ...
Weber Stefan S - - 2013
Upon uptake into a host cell, the intracellular bacterium Legionella pneumophila is not degraded on the lysosomal pathway but efficiently establishes a highly specialized replicative vacuole in which it readily multiplies. As many Icm/Dot type 4 secretion translocated bacterial effectors contribute to the establishment of this subcellular compartment in close ...
Alexandrova Evguenia M - - 2013
Here we describe a method for generating induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). Recombinant retroviruses carrying human transcription factors for Klf4, Oct3/4, Sox2, with or without c-Myc, are used to transduce early passage MEFs several times. Based on morphologic criteria, the resulting iPS colonies are picked ...
Deb Sumitra - - 2013
Protein-protein interaction studies can provide valuable insight into protein function. One of the most practical and high-yielding approaches is immunoprecipitation of a bait protein followed by mass spectrometry to identify co-precipitating proteins. Here we describe an effective and simplified version of this method that can be performed in most laboratories ...
Seipel Kathleen - - 2013
The intracellularly replicating lung pathogen Legionella pneumophila expresses a multitude of different phospholipases which are important virulence tools during host cell infection. To study the lipolytic properties including substrate specificities of potential L. pneumophila phospholipases A (PLA), we used different assays to monitor lipid hydrolysis. Here we describe methods for ...
Peng Guang-Hua - - 2013
Combinatorial binding of transcription factors (TFs) and cofactors to specific regulatory regions of target genes in vivo is an important mechanism of transcriptional regulation. Chromatin immunoprecipitation (ChIP) is a powerful technique to detect protein binding to specific regions of target genes in vivo. However, conventional ChIP analysis for individual factors ...
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 ...
Reddy Marpadga A - - 2013
Diabetes and metabolic disorders are leading causes of micro- and macrovascular complications. Furthermore, efforts to treat these complications are hampered by metabolic memory, a phenomenon in which prior exposure to hyperglycemia predisposes diabetic patients to the continued development of vascular diseases despite subsequent glycemic control. Persistently increased levels of oxidant ...
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 ...
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 ...
Suzuki Hiroshi I - - 2013
The tumor suppressor p53 orchestrates multiple cellular pathways as a central node of anti-oncogenic programs in response to DNA damage, oncogene activation, and several stresses. In addition to the principal role as a transcription factor that transactivates many target genes involved in apoptosis and cell cycle control, p53 has been ...
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 ...
Lück Christian - - 2013
In this chapter we describe the methods currently used for subgrouping Legionella pneumophila and other non-pneumophila species. In the first part we describe monoclonal antibody (mAb) subgrouping, either by indirect immunofluorescence or indirect ELISA methods. These monoclonal antibodies are not commercially available but can be obtained for noncommercial purposes from ...
Igarashi Kazuhiko - - 2013
Histone and DNA methyltransferases utilize S-adenosyl-L-methionine (SAM), a key intermediate of sulfur amino acid metabolism, as a donor of methyl group. SAM is biosynthesized by methionine adenosyltransferase (MAT) using two substrates, methionine and ATP. Three distinct forms of MAT (MATI, MATII and MATIII), encoded by two distinct genes (MAT1A and ...
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 ...
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 ...
Khoury Marie P - - 2013
The TP53 gene expresses at least nine different mRNA variants (p53 isoform mRNAs), including the one encoding the canonical p53 tumor suppressor protein. We have developed scientific tools to specifically detect and quantify p53 isoform expression at mRNA level by nested RT-PCR (reverse transcription-polymerase chain reaction) and quantitative real-time RT-PCR ...
Case Christopher L - - 2013
Caspase-1 is a critical factor in the innate immune response to Legionella pneumophila. The development of methods for analyzing caspase-1 activation pathways and downstream caspase-1-associated activities has helped in understanding the regulation of this protease and the signaling components involved. Here we outline methods for directly detecting active caspase-1, measuring ...
Mishra Monalisa - - 2013
The Drosophila compound eye is a regular structure, in which about 750 units, called ommatidia, are arranged in a highly regular pattern. Eye development proceeds in a stereotypical fashion, where epithelial cells of the eye imaginal discs are specified, recruited, and differentiated in a sequential order that leads to the ...
Ramamoorthy Mahesh - - 2013
Chemoresistance and increased growth rate are two gain-of-function functions that mutant p53 is thought to possess. Here, we describe two methods for measuring the sensitiveness of cells to chemotherapeutic drugs and the rate of cell growth. Both of which can be used with a wide range of cell types. The ...
Jarrett Stuart G - - 2013
Mitochondrial dysfunction and genomic instability are associated with a number of retinal pathologies including age-related macular degeneration, diabetic retinopathy, and glaucoma. Consequences of mitochondrial dysfunction within cells include elevation of the rate of ROS production due to damage of electron transport chain proteins, mitochondrial DNA (mtDNA) damage, and loss of ...
Dhanasekaran Karthigeyan - - 2013
The Genome of a eukaryotic cell harbors genetic material in the form of DNA which carries the hereditary information encoded in their bases. Nucleotide bases of DNA are transcribed into complimentary RNA bases which are further translated into protein, performing defined set of functions. The central dogma of life ensures ...
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 ...
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