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Multiple Proteins (multiple + protein)
Selected AbstractsCornelia de Lange syndrome, cohesin, and beyondCLINICAL GENETICS, Issue 4 2009J Liu Cornelia de Lange syndrome (CdLS) (OMIM #122470, #300590 and #610759) is a dominant genetic disorder with multiple organ system abnormalities which is classically characterized by typical facial features, growth and mental retardation, upper limb defects, hirsutism, gastrointestinal and other visceral system involvement. Mutations in three cohesin proteins, a key regulator of cohesin, NIPBL, and two structural components of the cohesin ring SMC1A and SMC3, etiologically account for about 65% of individuals with CdLS. Cohesin controls faithful chromosome segregation during the mitotic and meiotic cell cycles. Multiple proteins in the cohesin pathway are also involved in additional fundamental biological events such as double-strand DNA break repair and long-range regulation of transcription. Moreover, chromosome instability was recently associated with defective sister chromatid cohesion in several cancer studies, and an increasing number of human developmental disorders is being reported to result from disruption of this pathway. Here, we will discuss the human disorders caused by alterations of cohesin function (termed ,cohesinopathies'), with an emphasis on the clinical manifestations of CdLS and mechanistic studies of the CdLS-related proteins. [source] Transcriptional activity of interferon regulatory factor (IRF)-3 depends on multiple protein,protein interactionsFEBS JOURNAL, Issue 24 2002Hongmei Yang Virus infection results in the activation of a set of cellular genes involved in host antiviral defense. IRF-3 has been identified as a critical transcription factor in this process. The activation mechanism of IRF-3 is not fully elucidated, yet it involves a conformational change triggered by the virus-dependent phosphorylation of its C-terminus. This conformational change leads to nuclear accumulation, DNA binding and transcriptional transactivation. Here we show that two distinct sets of Ser/Thr residues of IRF-3, on phosphorylation, synergize functionally to achieve maximal activation. Remarkably, we find that activated IRF-3 lacks transcriptional activity, but activates transcription entirely through the recruitment of the p300/CBP coactivators. Moreover, we show that two separate domains of IRF-3 interact with several distinct regions of p300/CBP. Interference with any of these interactions leads to a complete loss of transcriptional activity, suggesting that a bivalent interaction is essential for coactivator recruitment by IRF-3. [source] Insight into initiator,DNA interactions: a lesson from the archaeal ORCBIOESSAYS, Issue 3 2008Shusuke Tada Although initiation of DNA replication is considered to be highly coordinated through multiple protein,DNA and protein,protein interactions, it is poorly understood how particular locations within the eukaryotic chromosome are selected as origins of DNA replication. Here, we discuss recent reports that present structural information on the interaction characteristics of the archaeal orthologues of the eukaryotic origin recognition complex with their cognate binding sequences.1,2 Since the archaeal replication system is postulated as a simplified version of the one in eukaryotes, by analogy, these works provide insights into the functions of the eukaryotic initiator proteins. BioEssays 30:208,211, 2008. © 2008 Wiley Periodicals, Inc. [source] Guidelines for improving the reproducibility of quantitative multiparameter immunofluorescence measurements by laser scanning cytometry on fixed cell suspensions from human solid tumorsCYTOMETRY, Issue 1 2006Stanley Shackney Abstract Background: Laser scanning Cytometry (LSC) is a versatile technology that makes it possible to perform multiple measurements on individual cells and correlate them cell by cell with other cellular features. It would be highly desirable to be able to perform reproducible, quantitative, correlated cell-based immunofluorescence studies on individual cells from human solid tumors. However, such studies can be challenging because of the presence of large numbers of cell aggregates and other confounding factors. Techniques have been developed to deal with cell aggregates in data sets collected by LSC. Experience has also been gained in addressing other key technical and methodological issues that can affect the reproducibility of such cell-based immunofluorescence measurements. Methods and results: We describe practical aspects of cell sample collection, cell fixation and staining, protocols for performing multiparameter immunofluorescence measurements by LSC, use of controls and reference samples, and approaches to data analysis that we have found useful in improving the accuracy and reproducibility of LSC data obtained in human tumor samples. We provide examples of the potential advantages of LSC in examining quantitative aspects of cell-based analysis. Improvements in the quality of cell-based multiparameter immunofluorescence measurements make it possible to extract useful information from relatively small numbers of cells. This, in turn, permits the performance of multiple multicolor panels on each tumor sample. With links among the different panels that are provided by overlapping measurements, it is possible to develop increasingly more extensive profiles of intracellular expression of multiple proteins in clinical samples of human solid tumors. Examples of such linked panels of measurements are provided. Conclusions: Advances in methodology can improve cell-based multiparameter immunofluorescence measurements on cell suspensions from human solid tumors by LSC for use in prognostic and predictive clinical applications. © 2005 Wiley-Liss, Inc. [source] Protein Immobilization: Capturing Complex Protein Gradients on Biomimetic Hydrogels for Cell-Based Assays (Adv. Funct.ADVANCED FUNCTIONAL MATERIALS, Issue 21 2009Mater. A versatile microfluidic strategy to rapidly and selectively immobilize gradients of virtually any desired protein on soft poly(ethylene glycol) (PEG) hydrogel surfaces is developed by S. Cosson et al. on page 3411. The selectivity and orthogonality of the chosen protein immobilization schemes allows for forming parallel and orthogonal overlapping gradients of multiple proteins. This platform can be exploited to perform a wealth of cell-based assays on biomimetic surfaces. [source] Capturing Complex Protein Gradients on Biomimetic Hydrogels for Cell-Based AssaysADVANCED FUNCTIONAL MATERIALS, Issue 21 2009Steffen Cosson Abstract A versatile strategy to rapidly immobilize complex gradients of virtually any desired protein on soft poly(ethylene glycol) (PEG) hydrogel surfaces that are reminiscent of natural extracellular matrices (ECM) is reported. A microfluidic chip is used to generate steady-state gradients of biotinylated or Fc-tagged fusion proteins that are captured and bound to the surface in less than 5,min by NeutrAvidin or ProteinA, displayed on the surface. The selectivity and orthogonality of the binding schemes enables the formation of parallel and orthogonal overlapping gradients of multiple proteins, which is not possible on conventional cell culture substrates. After patterning, the hydrogels are released from the microfluidic chip and used for cell culture. This novel platform is validated by conducting single-cell migration experiments using time-lapse microscopy. The orientation of cell migration, as well as the migration rate of primary human fibroblasts, depends on the concentration of an immobilized fibronectin fragment. This technique can be readily applied to other proteins to address a wealth of biological questions with different cell types. [source] OmpA is an adhesion factor of Aeromonas veronii, an optimistic pathogen that habituates in carp intestinal tractJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2008A. Namba Abstract Aims:, In the present study, we focused on one of the Aeromonas veronii isolates that exhibited marked adhesion onto carp intestine and studied its membrane-associated proteins for their possible involvement in mucosal adhesion. Methods and Results:, We isolated a strain of Aer. veronii (CWP11) that exhibited a high degree of temperature-dependent adhesion activity onto carp intestinal tract and studied its adhesion factor. A proteomic analysis of the membrane-associated fraction showed the presence of multiple proteins that were specifically expressed in CWP11 cells cultured at 25°C. Of these, a 30 kDa protein was identified to be OmpA by a mass fingerprint analysis. Cloning and nucleotide sequencing of the ompA region of CWP11 revealed the presence of two tandem ompA homologues (ompAI - ompAII). Escherichia coli that expressed either OmpAI or OmpAII exhibited marked adhesion onto carp intestinal surface. Disruption of ompAI by a homologous recombination technique resulted in marked reduction of the adhesion activity in CWP11. Conclusion:, The OmpA homologue plays an important role in the adhesion of the Aer. veronii strain onto the surface of intestinal tract. Significance and Impact of the Study:, We successfully identified an OmpA homologue to be an adhesion factor of Aer. veronii, an optimistic pathogen that habituates in carp intestinal tract. [source] Modulation of white adipose tissue proteome by aging and calorie restrictionAGING CELL, Issue 5 2010Adamo Valle Summary Aging is associated with an accrual of body fat, progressive development of insulin resistance and other obesity comorbidities that contribute to decrease life span. Caloric restriction (CR), which primarily affects energy stores in adipose tissue, is known to extend life span and retard the aging process in animal models. In this study, a proteomic approach combining 2-DE and MS was used to identify proteins modulated by aging and CR in rat white adipose tissue proteome. Proteomic analysis revealed 133 differentially expressed spots, 57 of which were unambiguously identified by MS. Although CR opposed part of the age-associated protein expression patterns, many effects of CR were on proteins unaltered by age, suggesting that the effects of CR on adipose tissue are only weakly related to those of aging. Particularly, CR and aging altered glucose, intermediate and lipid metabolism, with CR enhancing the expression of enzymes involved in oxalacetate and NADPH production, lipid biosynthesis and lipolysis. Consistently, insulin-, and ,3-adrenergic receptors were also increased by CR, which denotes improved sensitivity to lipogenic/lipolytic stimuli. Other beneficial outcomes of CR were an improvement in oxidative stress, preventing the age-associated decrease in several antioxidant enzymes. Proteins involved in cytoskeleton, iron storage, energy metabolism and several proteins with novel or unknown functions in adipose tissue were also modulated by age and/or CR. Such orchestrated changes in expression of multiple proteins provide insights into the mechanism underlying CR effects, ultimately allowing the discovery of new markers of aging and targets for the development of CR-mimetics. [source] Macrophage exposure to particulate titanium induces phosphorylation of the protein tyrosine kinase lyn and the phospholipases C,-1 and C,-2JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 3 2002Phillip L. Palmbos A frequent long-term complication of total joint arthroplasty is aseptic loosening, the end result of wear debris production, synovial macrophage activation, inflammatory mediator release, and osteolysis about the implant,bone or cement,bone interface. To elucidate the mechanisms of particle-induced macrophage activation and mediator production, we studied early signal transduction events using J774A.1 macrophages and 3 ,m titanium particles. Treating macrophages with herbimycin A or genistein, two inhibitors of protein tyrosine kinases (PTKs), inhibited titanium phagocytosis as well as secretion of tumor necrosis factor-, (TNF-,) and prostaglandin-E2 (PGE2) in a dose-dependent manner. Both processes therefore depend on a PTK signaling cascade. Specifically, macrophage exposure to titanium-induced phosphorylation of multiple proteins including the Src kinase Lyn and phospholipase C,-1 and C,-2. Phosphorylation peaked within 2 min and returned to baseline within 45 min. Similar but not identical phosphorylation patterns were obtained when cells were stimulated with titanium preincubated with serum or albumin, suggesting distinct signal transduction pathways dependent on particle coating. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] Chronic Ethanol Consumption Induces Global Hepatic Protein HyperacetylationALCOHOLISM, Issue 2 2010Blythe D. Shepard Background:, Although the clinical manifestations of alcoholic liver disease are well described, little is known about the molecular basis for liver injury. Recent studies have indicated that chronic alcohol consumption leads to the lysine-hyperacetylation of several hepatic proteins, and this list is growing quickly. Methods:, To identify other hyperacetylated proteins in ethanol-fed livers, we chose a proteomics approach. Cytosolic and membrane proteins (excluding nuclei) were separated on 2D gels, transferred to PVDF and immunoblotted with antibodies specific for acetylated lysine residues. Hyperacetylated proteins were selected for trypsin digestion and mass spectrometric analysis. Results:, In all, 40 proteins were identified, 11 of which are known acetylated proteins. Remarkably, the vast majority of hyperacetylated membrane proteins were mitochondrial residents. Hyperacetylated cytosolic proteins ranged in function from metabolism to cytoskeletal support. Notably, 3 key anti-oxidant proteins were identified whose activities are impaired in ethanol-treated cells. We confirmed that the anti-oxidant enzyme, glutathione peroxidase 1, actin and cortactin are hyperacetylated in ethanol-treated livers. Conclusions:, Alcohol-induced hyperacetylation of multiple proteins may contribute to the development of liver injury. The abundance of acetylated mitochondrial proteins further suggests that this modification is important in regulating liver metabolism and when perturbed, may contribute to the progression of a variety of metabolic diseases. [source] Surface plasmon resonance imaging protein arrays for analysis of triple protein interactions of HPV, E6, E6AP, and p53PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 7 2006Hyeon-Su Ro Abstract We have developed a surface plasmon resonance (SPR)-based protein microarray to study protein,protein interactions in a high-throughput mode. As a model system, triple protein interactions have been explored with human papillomaviral E6 protein, tumor suppressor p53, and ubiquitin ligase E6AP. Human papillomavirus (HPV) is known to be a causative agent of cervical cancer. Upon infection, the viral E6 protein forms a heterotrimeric protein complex with p53 and E6AP. The formation of the complex eventually results in the degradation of p53. In the present study, a GST-fused E6AP protein was layered onto a glutathione (GSH)-modified gold chip surface. The specific binding of GST-E6AP protein onto the gold chip surface was facilitated through the affinity of GST to its specific ligand GSH. The interacting proteins (E6 and/or p53) were then spotted. Detection of the interaction was performed using a SPR imaging (SPRI) technique. The resulting SPRI intensity data showed that the protein,protein interactions of E6AP, E6, and p53 were detected in a concentration-dependent manner, suggesting that the SPRI-based microarray system can be an effective tool to study protein,protein interactions where multiple proteins are involved. [source] Stromal cells of fibrodysplasia ossificans progressiva lesions express smooth muscle lineage markers and the osteogenic transcription factor Runx2/Cbfa-1: clues to a vascular origin of heterotopic ossification?THE JOURNAL OF PATHOLOGY, Issue 1 2003Laszlo Hegyi Abstract Fibrodysplasia ossificans progressiva (FOP) is a rare heritable genetic disorder, which is characterized pathologically by sporadic episodes of explosive growth of mesenchymal cells in skeletal muscle followed by cellular differentiation to heterotopic bone through an endochondral process. This study examined the histological origin and differentiation state of stromal cells in early FOP lesions and investigated the association between the phenotype of these FOP cells and bone formation. Interestingly, FOP lesional stromal cells were found to display characteristics of the smooth muscle (SM) cell lineage and are therefore potentially of vascular origin. These cells co-express multiple SM lineage markers along with multiple proteins associated with bone formation including the obligate osteogenic transcription factor Runx2/Cbfa-1. It is hypothesized that the stromal cells of early FOP lesions may be locally recruited vascular cells or cells of the bone marrow stroma and that these cells maintain the potential (given the correct environmental stimuli) to differentiate along an endochondral ossification pathway. Copyright © 2003 John Wiley & Sons, Ltd. [source] Mutation in BAG3 causes severe dominant childhood muscular dystrophy,ANNALS OF NEUROLOGY, Issue 1 2009Duygu Selcen MD Objective Myofibrillar myopathies (MFMs) are morphologically distinct but genetically heterogeneous muscular dystrophies in which disintegration of Z disks and then of myofibrils is followed by ectopic accumulation of multiple proteins. Cardiomyopathy, neuropathy, and dominant inheritance are frequent associated features. Mutations in ,B-crystallin, desmin, myotilin, Zasp, or filamin-C can cause MFMs and were detected in 32 of 85 patients of the Mayo MFM cohort. Bag3, another Z-disk,associated protein, has antiapoptotic properties, and its targeted deletion in mice causes fulminant myopathy with early lethality. We therefore searched for mutations in BAG3 in 53 unrelated MFM patients. Methods We searched for mutations in BAG3 by direct sequencing. We analyzed structural changes in muscle by histochemistry, immunocytochemistry, and electron microscopy, examined mobility of the mutant Bag3 by nondenaturing electrophoresis, and searched for abnormal aggregation of the mutant protein in COS-7 (SV-40 transformed monkey kidney fibroblast-7) cells. Results We identified a heterozygous p.Pro209Leu mutation in three patients. All presented in childhood, had progressive limb and axial muscle weakness, and experienced development of cardiomyopathy and severe respiratory insufficiency in their teens; two had rigid spines, and one a peripheral neuropathy. Electron microscopy showed disintegration of Z disks, extensive accumulation of granular debris and larger inclusions, and apoptosis of 8% of the nuclei. On nondenaturing electrophoresis of muscle extracts, the Bag3 complex migrated faster in patient than control extracts, and expression of FLAG-labeled mutant and wild-type Bag3 in COS cells showed abnormal aggregation of the mutant protein. Interpretation We conclude mutation in Bag3 defines a novel severe autosomal dominant childhood muscular dystrophy. Ann Neurol 2008 [source] Engineering multigene expression in vitro and in vivo with small terminators for T7 RNA polymeraseBIOTECHNOLOGY & BIOENGINEERING, Issue 6 2009Liping Du Abstract Engineering protein expression in vitro or in vivo is usually straightforward for single genes, but remains challenging for multiple genes because of the requirement of coordinated control. RNA and protein overexpression strategies often exploit T7 RNA polymerase and its natural T, Class I terminator. However, this terminator's inefficiency and large size (100,bp) are problematic for multigene construction and expression. Here, we measure the effects of tandem copies of a small (18,bp) Class II T7 terminator from vesicular stomatitis virus on transcription in vitro and on translation in vitro and in vivo. We first test monomeric and dimeric gene constructs, then attempt extension to pentameric gene constructs. "BioBrick" versions of a pET vector and translation factor genes were constructed to facilitate cloning, and His-tags were incorporated to allow copurification of all protein products for relatively unbiased analysis and easy purification. Several results were surprising, including imbalanced expression of the pentameric constructs in vivo, illustrating the value of synthetic biology for investigating gene expression. However, these problems were solved rationally by changing the orders of the genes and by adding extra promoters to the upstream gene or by moving to a more predictable in vitro translation system. These successes were significant, given our initial unexpected results and that we are unaware of another example of coordinated overexpression of five proteins. Our modular, flexible, rational method should further empower synthetic biologists wishing to overexpress multiple proteins simultaneously. Biotechnol. Bioeng. 2009; 104: 1189,1196. © 2009 Wiley Periodicals, Inc. 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