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11.
Lisa C Willcocks Paul A Lyons Andrew J Rees Kenneth GC Smith 《Arthritis research & therapy》2010,12(1):202
The aetiology of anti-neutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis has not been well defined. Here
we review two factors which may play a role in the pathogenesis of the disease: genetics and infection. In particular, we
discuss the role of autoantibodies to LAMP-2, which may arise following infection with Gram-negative bacteria, and may contribute
to the development of ANCA-associated systemic vasculitis in genetically susceptible individuals. 相似文献
12.
Background
Current techniques used to obtain lung samples have significant limitations and do not provide reproducible biomarkers of inflammation. We have developed a novel technique that allows multiple sampling methods from the same area (or multiple areas) of the lung under direct bronchoscopic vision. It allows collection of mucosal lining fluid and bronchial brushing from the same site; biopsy samples may also be taken. The novel technique takes the same time as standard procedures and can be conducted safely.Methods
Eight healthy smokers aged 40–65 years were included in this study. An absorptive filter paper was applied to the bronchial mucosa under direct vision using standard bronchoscopic techniques. Further samples were obtained from the same site using bronchial brushings. Bronchoalveolar lavage (BAL) was obtained using standard techniques. Chemokine (C-C Motif) Ligand 20 (CCL20), CCL4, CCL5, Chemokine (C-X-C Motif) Ligand 1 (CXCL1), CXCL8, CXCL9, CXCL10, CXCL11, Interleukin 1 beta (IL-1β), IL-6, Vascular endothelial growth factor (VEGF), Matrix metalloproteinase 8 (MMP-8) and MMP-9 were measured in exudate and BAL. mRNA was collected from the bronchial brushings for gene expression analysis.Results
A greater than 10 fold concentration of all the biomarkers was detected in lung exudate in comparison to BAL. High yield of good quality RNA with RNA integrity numbers (RIN) between 7.6 and 9.3 were extracted from the bronchial brushings. The subset of genes measured were reproducible across the samples and corresponded to the inflammatory markers measured in exudate and BAL.Conclusions
The bronchoabsorption technique as described offers the ability to sample lung fluid direct from the site of interest without the dilution effects caused by BAL. Using this method we were able to successfully measure the concentrations of biomarkers present in the lungs as well as collect high yield mRNA samples for gene expression analysis from the same site. This technique demonstrates superior sensitivity to standard BAL for the measurement of biomarkers of inflammation. It could replace BAL as the method of choice for these measurements. This method provides a systems biology approach to studying the inflammatory markers of respiratory disease progression.Trial registration
NHS Health Research Authority (13/LO/0256). 相似文献13.
The field of proteomics is advancing rapidly as a result of powerful new technologies and proteomics experiments yield a vast
and increasing amount of information. Data regarding protein occurrence, abundance, identity, sequence, structure, properties,
and interactions need to be stored. Currently, a common standard has not yet been established and open access to results is
needed for further development of robust analysis algorithms. Databases for proteomics will evolve from pure storage into
knowledge resources, providing a repository for information (meta-data) which is mainly not stored in simple flat files. This
review will shed light on recent steps towards the generation of a common standard in proteomics data storage and integration,
but is not meant to be a comprehensive overview of all available databases and tools in the proteomics community. 相似文献
14.
Phosphoproteome Analysis 总被引:2,自引:0,他引:2
Protein phosphorylation is directly or indirectly involved in all important cellular events. The understanding of its regulatory
role requires the discovery of the proteins involved in these processes and how, where and when protein phosphorylation takes
place. Investigation of the phosphoproteome of a cell is becoming feasible today although it still represents a very difficult
task especially if quantitative comparisons have to be made. Several different experimental strategies can be employed to
explore phosphoproteomes and this review will cover the most important ones such as incorporation of radiolabeled phosphate
into proteins, application of specific antibodies against phosphorylated residues and direct staining of phosphorylated proteins
in polyacrylamide gels. Moreover, methods to enrich phosphorylated proteins such as affinity chromatography (IMAC) and immunoprecipitation
as well as mass spectrometry for identification of phosphorylated peptides and phosphorylation sites are also described. 相似文献
15.
FISH probes are generally made out of BAC clones with genomic DNA containing a variable amount of repetitive DNA that will need to be removed or blocked for FISH analysis. To generate repeat free (RF) Probes without loss in genomic coverage, a random library is made from BAC clones by whole-genome amplification (WGA). Libraries are denatured in the presence of excess C(0)t-1 DNA and allowed to re-anneal followed by digestion of all double-stranded elements by duplex-specific nuclease (DSN). Selective amplification of all elements not containing repetitive sequences is realized by a sequential amplification. The final RF products can be re-amplified and used as a stock for future probe production. The RF probes have a lower background, the signal intensity build up is faster and there is no need for blocking DNA. The signal to background ratio of the RF was higher as compared to repeat containing probes. 相似文献
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17.
Interdomain salt‐bridges in the Ebola virus protein VP40 and their role in domain association and plasma membrane localization 下载免费PDF全文
Jeevan B. GC Kristen A. Johnson Monica L. Husby Cary T. Frick Bernard S. Gerstman Robert V. Stahelin Prem P. Chapagain 《Protein science : a publication of the Protein Society》2016,25(9):1648-1658
The Ebola virus protein VP40 is a transformer protein that possesses an extraordinary ability to accomplish multiple functions by transforming into various oligomeric conformations. The disengagement of the C‐terminal domain (CTD) from the N‐terminal domain (NTD) is a crucial step in the conformational transformations of VP40 from the dimeric form to the hexameric form or octameric ring structure. Here, we use various molecular dynamics (MD) simulations to investigate the dynamics of the VP40 protein and the roles of interdomain interactions that are important for the domain–domain association and dissociation, and report on experimental results of the behavior of mutant variants of VP40. The MD studies find that various salt‐bridge interactions modulate the VP40 domain dynamics by providing conformational specificity through interdomain interactions. The MD simulations reveal a novel salt‐bridge between D45‐K326 when the CTD participates in a latch‐like interaction with the NTD. The D45‐K326 salt‐bridge interaction is proposed to help domain–domain association, whereas the E76‐K291 interaction is important for stabilizing the closed‐form structure. The effects of the removal of important VP40 salt‐bridges on plasma membrane (PM) localization, VP40 oligomerization, and virus like particle (VLP) budding assays were investigated experimentally by live cell imaging using an EGFP‐tagged VP40 system. It is found that the mutations K291E and D45K show enhanced PM localization but D45K significantly reduced VLP formation. 相似文献
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19.
A Nagler R Condiotti R Rabinowitz M Schlesinger M Nguyen LWMM Terstappen 《Cancer immunology, immunotherapy : CII》1999,16(3):177-187
Multi-parameter flow cytometry (MPFC) was used to detect minimal residual disease (MRD) following bone marrow transplantation
(BMT) in 21 patients. Bone marrow (BM) was analyzed pre-transplant and 3–4 months post-BMT while the patients were in clinical
and morphological remission. MRD was detected by identifying cells with aberrant antigen expression and/or leukemia-associated
phenotype (LAP) using MPFC. Prior to BMT, 8 out of 21 patients exhibited normal antigen expression based on normal BM samples
while 13 BM aspirates had abnormal MPFC. Pre-BMT MPFC was abnormal in all 10 patients who were not in complete remission (CR)
(>5% blasts in BM) as well as 3 patients acute lymphoblastic leukemia (ALL) who were in CR. In BM from ALL patients, an abnormal
uniform B cell population was observed however antigen expression patterns varied greatly between patients. BM from acute
myeloblastic leukemia (AML) patients showed an abnormal distribution of CD34+ cells. In addition, a correlation was observed
between pre-BMT cytogenetics and MPFC. Only 2 out of 8 (25%) patients with normal MPFC pre-autologous bone marrow transplantation
(ABMT) relapsed (AML), while 6 out of 13 (46%) patients with abnormal pre-BMT MPFC relapsed including 2 out of 3 patients
who were transplanted in clinical CR. Pre-BMT MPFC may thus be an effective tool for detection of MRD by detection of a pre-transplant
MPFC abnormality. 相似文献
20.
Polarization measurement of orthogonal light scattering is introduced as a new optical parameter in flow cytometry. In the experimental setup, the electrical field of the incident laser beam is polarized in the direction of the sample flow. The intensity of the orthogonal light scattering polarized along the direction of the incoming laser beam is called depolarized orthogonal light scattering. Theoretical analysis shows that for small values of the detection aperture, the measured depolarization is caused by anisotropic cell structures and multiple scattering processes inside the cell. Measurements of the orthogonal depolarized light scattering in combination with the normal orthogonal light scattering of human leucocytes revealed two populations of granulocytes. By means of cell sorting it was shown that the granulocytes with a relatively high depolarization are eosinophilic granulocytes. Similar experiments with human lymphocytes revealed a minor subpopulation of yet-unidentified lymphocytes with a relative large orthogonal light-scattering depolarization. The results were obtained with an argon ion laser tuned at different wavelengths as well as with a 630-nm helium neon laser. These results show that measurement of depolarized orthogonal light scattering is a useful new parameter for flow-cytometric cell differentiation. 相似文献