59 research outputs found

    Influence of chondroitin 6-sulfate oligosaccharide unit addition on the immunopathogenicity of human thyroglobulin in cba/j(h-2k) mice. Multiple effects on antigen processing and t cell responses

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    The site of type D (chondroitin 6-sulfate) oligosaccharide unit addition to human thyroglobulin (hTg) was localized. Furthermore, hTg and its fractions endowed with chondroitin 6-sulfate oligosaccaride units (hTg-CS) and devoid of it (hTg-CS-), were compared, with respect to their ability to induce experimental autoimmune thyroiditis (EAT) in CBA/J(H-2k) mice, by subcutaneous administration, in the presence of complete adjuvant. HTg was chromatographically separated into hTg-CS and hTg-CS- molecules, on the base of their uronic acid content. In an ample number of hTg preparations, the fraction of hTg-CS in total hTg ranged from 32.0 to 71.6 percent. By exploiting the electrophoretic mobility shift and metachromasia conferred by chondrotin-6-sulfate upon the products of limited proteolysis of hTg, chondroitin 6-sulfate was first restricted to a carboxy-terminal region, starting at residue 2513. A single chondroitin 6-sulfate-containing nonapeptide was isolated in pure form from the products of digestion of hTg with endoproteinase Glu-C, and its sequence was determined as being LTAGXGLRE (residues 2725-2733, X being Ser2729 linked to the oligosaccharide chain). In an in vitro assay of enzymatic iodination, hTg-CS produced higher yields of 3,5,5’-triiodothyronine (T3) (171%) and 3,5,3’,5’-tetraiodothyronine (T4) (134%), than hTg-CS-. Unfractionated hTg behaved as hTg-CS. Thus, chondroitin 6-sulfate addition to a subset of hTg molecules enhanced the overall level of T4 and, particularly, T3 formation. Furthermore, the chondroitin 6-sulfate oligosaccharide unit of hTg-CS protected peptide bond Gly2713-Lys2714 from proteolysis, during the limited digestion of hTg-CS with trypsin. Although immunization with all forms of hTg was accompanied by thyroid cell damage, as judged from the increase of T4 in blood, a higher degree of mononuclear infiltration of the thyroid was associated with unfractionated hTg, in comparison both with hTg-CS and with hTg-CS-. Thus, it appears that both hTg subfractions contributed to the immunopathogenic potency of unfractionated hTg, as neither one reproduced fully the histological picture associated with the latter. Significant differences were observed also upon restimulation in vitro of splenic lymphocytes obtained from mice immunized in vivo with the different forms of hTg. Restimulation in vitro with hTg-CS of splenocytes from mice immunized with the same antigen was followed by low-level, dose-dependent proliferation and IFN-γ production, whereas cross-stimulation with hTg-CS- of the same cells was followed by proliferative and secretory responses of even lower degree. On the other hand, restimulation in vitro with hTg-CS- of splenocytes primed in vivo with the same antigen was followed by higher-level, dose-dependent increases of IFN-γ production, accompanied by proliferative responses of low degree and inversely related with the antigen dose, while cross-stimulation with hTg-CS of the same cells was followed by dose-dependent increases, both of proliferation and IFN-γ production, of the highest level observed in this study. Similar results were obtained when splenocytes, primed in vivo with hTg-CS-, were restimulated with purified glycopeptide hTg-CSgp, containing the chondroitin 6-sulfate unit, but not with its non-glycosylated, synthetic homologue. These data indicate that hTg-CS- was more effective than hTg-CS in priming autoreactive T lymphocytes, recognizing thyroiditogenic epitopes shared between murine and human Tg, whereas hTg-CS was a stronger inducer of proliferation of antigen-sensitized T cells. Moreover, different molecular signals, including structural determinant(s) associated with the chondroitin 6-sulfate chain, were required, in addition to epitope recognition, for the activation of T cell proliferation, together with IFN-γ production. These findings provide insights into the molecular mechanism of regulation of the hormonogenic efficiency and of the T4/T3 ratio in hTg, and may bear important implications in the processing and presentation of hTg as an autoantigen, and in the mechanisms of activation of Th-1-mediated and cytotoxic lymphocyte responses involved in EAT

    Abstracts for the 72nd Annual Meeting

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    Analysis of the effects of BrdU on DLKP human lung cancer cells by two-dimensional difference gel electrophoresis and mass spectrometry

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    Bromodeoxyuridine (BrdU) is a thymidiie analogue that incorporates into DNA of dividing cells during the S-phase of the cell cycle. Previous work in laboratories reported that treatment with lOyM BrdU in the human lung carcinoma cell line (DLKP) resulted in increased expression of the cytoskeletal proteins Keratin 8 and 18 and the cell adhesion proteins a2 and b1 integrin. This study investigated protein expression changes in differentiating DLKP cells following exposure to 10yM BrdU. DLKP cells were grown in culture flasks and harvested after 7 days exposure to BrdU. Two-dimensional gel electrophoresis was used to investigate BrdU specific changes in the proteome of DLKP BrdU treated and control cells. Cy3-labeled DLKP control were combined with Cy-5 labeled BrdU DLKP treated proteins and separated on the same 2-D gel along with a Cy-2 labelled mixture of both samples as an internal standard. Using DIGE technology, the statistically significant comparisons of each protein abundance was made over three biological replicates. 43 protein spots were identified as differentially regulated. Among the 43 protein spots, 25 were found to be up-regulated and 18 were found to be downregulated

    Unraveling the intricacies of spatial organization of the ErbB receptors and downstream signaling pathways

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    Faced with the complexity of diseases such as cancer which has 1012 mutations, altering gene expression, and disrupting regulatory networks, there has been a paradigm shift in the biological sciences and what has emerged is a much more quantitative field of biology. Mathematical modeling can aid in biological discovery with the development of predictive models that provide future direction for experimentalist. In this work, I have contributed to the development of novel computational approaches which explore mechanisms of receptor aggregation and predict the effects of downstream signaling. The coupled spatial non-spatial simulation algorithm, CSNSA is a tool that I took part in developing, which implements a spatial kinetic Monte Carlo for capturing receptor interactions on the cell membrane with Gillespies stochastic simulation algorithm, SSA, for temporal cytosolic interactions. Using this framework we determine that receptor clustering significantly enhances downstream signaling. In the next study the goal was to understand mechanisms of clustering. Cytoskeletal interactions with mobile proteins are known to hinder diffusion. Using a Monte Carlo approach we simulate these interactions, determining at what cytoskeletal distribution and receptor concentration optimal clustering occurs and when it is inhibited. We investigate oligomerization induced trapping to determine mechanisms of clustering, and our results show that the cytoskeletal interactions lead to receptor clustering. After exploring the mechanisms of clustering we determine how receptor aggregation effects downstream signaling. We further proceed by implementing the adaptively coarse grained Monte Carlo, ACGMC to determine if \u27receptor-sharing\u27 occurs when receptors are clustered. In our proposed \u27receptor-sharing\u27 mechanism a cytosolic species binds with a receptor then disassociates and rebinds a neighboring receptor. We tested our hypothesis using a novel computational approach, the ACGMC, an algorithm which enables the spatial temporal evolution of the system in three dimensions by using a coarse graining approach. In this framework we are modeling EGFR reaction-diffusion events on the plasma membrane while capturing the spatial-temporal dynamics of proteins in the cytosol. From this framework we observe \u27receptor-sharing\u27 which may be an important mechanism in the regulation and overall efficiency of signal transduction. In summary, I have helped to develop predictive computational tools that take systems biology in a new direction.\u2

    Aerospace medicine and biology: A continuing bibliography with indexes (supplement 352)

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    This bibliography lists 147 reports, articles and other documents introduced into the NASA Scientific and Technical Information System during July 1991. Subject coverage includes: aerospace medicine and psychology, life support systems and controlled environments, safety equipment, exobiology and extraterrestrial life, and flight crew behavior and performance

    Identification and functional analysis of thylakoid membrane proteome

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    Membrane proteins play crucial roles in many biological functions. Identities and functions of most membrane proteins remain to be revealed. New technological breakthroughs in proteomics together with the availability of genomic sequence information make it possible to study functions of membrane proteins on a genome-wide scale. We used a multidisciplinary approach combining biochemistry, genetics, proteomics and bioinformatics to study the functions of the thylakoid proteome of Synechocystis sp. PCC6803. The thylakoid membrane proteins were separated into peripheral and integral fractions and resolved into 2-D gels with different pH ranges. The protein spots in the 2-D gels were subjected to peptide mass fingerprinting analysis, and totally 390 out of 558 analyzed spots were identified as protein products of 128 individual genes, of which 38 gene encode hypothetical proteins with unknown function. To study the function of some hypothetical proteins, we inactivated a set of genes, and 10 knockout mutants were obtained. The growth analysis for the mutant cells revealed that only one mutant (H1) which has a deletion in the ORF slr0110, showed conditional growth phenotype. Detailed analysis indicated that the H1 mutant is sensitive to both glucose and light, which is caused by the over-reduction of the PQ pool in the thylakoid membrane. The ID and the structural and functional information of the identified proteins as well as the 2-D reference maps were included in a web-based relational database for thylakoid membrane proteins. The database was constructed with MySQL, and the application programs were developed with SQL, PERL, JAVASCRIPT and HTML. Users can search the information of identified proteins and compare their own identified proteins with the identified proteins in the database. A manager interface is also provided for the routine maintenance of the database

    Imaging and 3D reconstruction of membrane protein complexes by cryo-electron microscopy and single particle analysis

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    Cryo-electron microscopy (cryo-EM) in combination with single particle image processing and volume reconstruction is a powerful technology to obtain medium-resolution structures of large protein complexes, which are extremely difficult to crystallize and not amenable to NMR studies due to size limitation. Depending on the stability and stiffness as well as on the symmetry of the complex, three-dimensional reconstructions at a resolution of 10-30 ˚ can be achieved. In this range of resolution, we may not be able to answer A chemical questions at the level of atomic interactions, but we can gain detailed insight into the macromolecular architecture of large multi-subunit complexes and their mechanisms of action. In this thesis, several prevalently large membrane protein complexes of great physiological importance were examined by various electron microscopy techniques and single particle image analysis. The core part of my work consists in the imaging of a mammalian V-ATPase, frozen-hydrated in amorphous ice and of the completion of the first volume reconstruction of this type of enzyme, derived from cryo-EM images. This ubiquitous rotary motor is essential in every eukaryotic cell and is of high medical importance due to its implication in various diseases such as osteoporosis, skeletal cancer and kidney disorders. My contribution to the second and third paper concerns the volume reconstruction of two bacterial outer membrane pore complexes from cryo-EM images recorded by my colleague Mohamed Chami. PulD from Klebsiella oxytoca constitutes a massive translocating pore capable of transporting a fully folded cell surface protein PulA through the membrane. It is part of the Type II secretion system, which is common for Gram-negative bacteria. The second volume regards ClyA, a pore-forming heamolytic toxin of virulent Escherichia coli and Salmonella enterica strains that kill target cells by inserting pores into their membranes. To the last two papers, I contributed with cryo-negative stain imaging of the cell division protein DivIVA from Bacillus subtilis and with image processing of the micrographs displaying the siderophore receptor FrpB from Neisseria meningitidis

    The proteomic analysis of resistance to anticancer therapy in human breast cancer

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    Resistance to anticancer therapy represents a major barrier in the successful management of human breast cancer. The identification of novel biomarkers that correlate with treatment response would increase our understanding of the resistance mechanisms and may allow therapy to be tailored on an individual patient basis. Those patients unlikely to respond to a particular treatment strategy would be spared the serious life-threatening side effects for no therapeutic gain. The immediate implementation of a more appropriate treatment strategy may also prevent disease progression and such biomarkers would also suggest new drug targets and/or sensitising agents for future therapeutic intervention.The primary aim of this thesis was to establish and optimise two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption/ ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and to exploit this global proteomics approach to identify molecular markers associated with resistance to anticancer therapy in human breast cancer cells. This technique was applied to compare the MCF7 cell line with novel derivatives displaying significant resistance to cisplatin (MCF7CR) and radiotherapy (MCF740Gy). Differentially expressed proteins were identified by MALDI-TOF MS analysis. Several molecular markers have been identified, which may play a role in resistance to anticancer therapy. Western blotting was used to confirm the expression of selected targets and to ensure the accuracy of protein identification. Examples of those targets which were confirmed by western blotting include GST-M3, cytokeratin 17, peroxiredoxin 4 and HSP27 (which were associated with cisplatin resistance) and L-Plastin, GST-M3 and cytokeratin 17 (which were associated with radio-resistance). These provide interesting targets and are worthy of further studies.Although 2DE-MALDI-TOF MS remains the gold standard for the global analysis of protein expression this technique is associated with several drawbacks. The antibody microarray is a powerful new technique designed to overcome many of these drawbacks and provides complementary information. Antibody microarrays were applied to identify further molecular markers associated with resistance to anticancer therapy in human breast cancer cells. The MDA-MB-231 cell line and a novel derivative (MDAMB- 231DR) displaying significant resistance to doxorubicin were analysed using Panorama Cell Signalling antibody microarrays (Sigma-Aldrich) comprised of 224 antibodies. Several proteins were associated with resistance to doxorubicin and some proteins were confirmed by western blotting. These targets included p-ERK, cyclin D2 and cyclin B 1. The technique was subsequently extended using the Labvision Antibody Microarray Kit (Neomarkers) to analyse the MCF7 parental cell line and the derivative (MCF740Gy) displaying significant resistance to radiotherapy. The Labvision antibody microarrays were comprised of 720 antibodies. Results indicated that this kit requires further optimisation and a preliminary analysis of the data revealed that further developmental and statistical work is required for interpretation of the results.This thesis has generated a list of potential candidate proteins, which may play a role in the development of resistance to anticancer therapy. Some of these targets overlap between different treatment modalities and have been confirmed by western blotting. Further work is now required in order to determine the functional relevance of these interesting targets. Ultimately, overcoming resistance to both chemotherapy and radiotherapy would represent a major advance in the effective management of breast cancer today
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