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641.
Steven E. Schutzer Tao Liu Benjamin H. Natelson Thomas E. Angel Athena A. Schepmoes Samuel O. Purvine Kim K. Hixson Mary S. Lipton David G. Camp II Patricia K. Coyle Richard D. Smith Jonas Bergquist 《PloS one》2010,5(6)
Background
Knowledge of the entire protein content, the proteome, of normal human cerebrospinal fluid (CSF) would enable insights into neurologic and psychiatric disorders. Until now technologic hurdles and access to true normal samples hindered attaining this goal.Methods and Principal Findings
We applied immunoaffinity separation and high sensitivity and resolution liquid chromatography-mass spectrometry to examine CSF from healthy normal individuals. 2630 proteins in CSF from normal subjects were identified, of which 56% were CSF-specific, not found in the much larger set of 3654 proteins we have identified in plasma. We also examined CSF from groups of subjects previously examined by others as surrogates for normals where neurologic symptoms warranted a lumbar puncture but where clinical laboratory were reported as normal. We found statistically significant differences between their CSF proteins and our non-neurological normals. We also examined CSF from 10 volunteer subjects who had lumbar punctures at least 4 weeks apart and found that there was little variability in CSF proteins in an individual as compared to subject to subject.Conclusions
Our results represent the most comprehensive characterization of true normal CSF to date. This normal CSF proteome establishes a comparative standard and basis for investigations into a variety of diseases with neurological and psychiatric features. 相似文献642.
643.
Hydrazine synthase, a unique phylomarker with which to study the presence and biodiversity of anammox bacteria 总被引:4,自引:0,他引:4
Harhangi HR Le Roy M van Alen T Hu BL Groen J Kartal B Tringe SG Quan ZX Jetten MS Op den Camp HJ 《Applied and environmental microbiology》2012,78(3):752-758
Anaerobic ammonium-oxidizing (anammox) bacteria play an important role in the biogeochemical cycling of nitrogen. They derive their energy for growth from the conversion of ammonium and nitrite into dinitrogen gas in the complete absence of oxygen. Several methods have been used to detect the presence and activity of anammox bacteria in the environment, including 16S rRNA gene-based approaches. The use of the 16S rRNA gene to study biodiversity has the disadvantage that it is not directly related to the physiology of the target organism and that current primers do not completely capture the anammox diversity. Here we report the development of PCR primer sets targeting a subunit of the hydrazine synthase (hzsA), which represents a unique phylogenetic marker for anammox bacteria. The tested primers were able to retrieve hzsA gene sequences from anammox enrichment cultures, full-scale anammox wastewater treatment systems, and a variety of freshwater and marine environmental samples, covering all known anammox genera. 相似文献
644.
Vauclare P Kopriva S Fell D Suter M Sticher L von Ballmoos P Krähenbühl U den Camp RO Brunold C 《The Plant journal : for cell and molecular biology》2002,31(6):729-740
The effect of externally applied L-cysteine and glutathione (GSH) on ATP sulphurylase and adenosine 5'-phosphosulphate reductase (APR), two key enzymes of assimilatory sulphate reduction, was examined in Arabidopsis thaliana root cultures. Addition of increasing L-cysteine to the nutrient solution increased internal cysteine, gamma-glutamylcysteine and GSH concentrations, and decreased APR mRNA, protein and extractable activity. An effect on APR could already be detected at 0.2 mm L-cysteine, whereas ATP sulphurylase was significantly affected only at 2 mm L-cysteine. APR mRNA, protein and activity were also decreased by GSH at 0.2 mm and higher concentrations. In the presence of L-buthionine-S, R-sulphoximine (BSO), an inhibitor of GSH synthesis, 0.2 mm L-cysteine had no effect on APR activity, indicating that GSH formed from cysteine was the regulating substance. Simultaneous addition of BSO and 0.5 mm GSH to the culture medium decreased APR mRNA, enzyme protein and activity. ATP sulphurylase activity was not affected by this treatment. Tracer experiments using (35)SO(4)(2-) in the presence of 0.5 mm L-cysteine or GSH showed that both thiols decreased sulphate uptake, APR activity and the flux of label into cysteine, GSH and protein, but had no effect on the activity of all other enzymes of assimilatory sulphate reduction and serine acetyltransferase. These results are consistent with the hypothesis that thiols regulate the flux through sulphate assimilation at the uptake and the APR step. Analysis of radioactive labelling indicates that the flux control coefficient of APR is more than 0.5 for the intracellular pathway of sulphate assimilation. This analysis also shows that the uptake of external sulphate is inhibited by GSH to a greater extent than the flux through the pathway, and that the flux control coefficient of APR for the pathway, including the transport step, is proportionately less, with a significant share of the control exerted by the transport step. 相似文献
645.
Qian WJ Liu T Monroe ME Strittmatter EF Jacobs JM Kangas LJ Petritis K Camp DG Smith RD 《Journal of proteome research》2005,4(1):53-62
Large-scale protein identifications from highly complex protein mixtures have recently been achieved using multidimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) and subsequent database searching with algorithms such as SEQUEST. Here, we describe a probability-based evaluation of false positive rates associated with peptide identifications from three different human proteome samples. Peptides from human plasma, human mammary epithelial cell (HMEC) lysate, and human hepatocyte (Huh)-7.5 cell lysate were separated by strong cation exchange (SCX) chromatography coupled offline with reversed-phase capillary LC-MS/MS analyses. The MS/MS spectra were first analyzed by SEQUEST, searching independently against both normal and sequence-reversed human protein databases, and the false positive rates of peptide identifications for the three proteome samples were then analyzed and compared. The observed false positive rates of peptide identifications for human plasma were significantly higher than those for the human cell lines when identical filtering criteria were used, suggesting that the false positive rates are significantly dependent on sample characteristics, particularly the number of proteins found within the detectable dynamic range. Two new sets of filtering criteria are proposed for human plasma and human cell lines, respectively, to provide an overall confidence of >95% for peptide identifications. The new criteria were compared, using a normalized elution time (NET) criterion (Petritis et al. Anal. Chem. 2003, 75, 1039-1048), with previously published criteria (Washburn et al. Nat. Biotechnol. 2001, 19, 242-247). The results demonstrate that the present criteria provide significantly higher levels of confidence for peptide identifications from mammalian proteomes without greatly decreasing the number of identifications. 相似文献
646.
Jacobs JM Adkins JN Qian WJ Liu T Shen Y Camp DG Smith RD 《Journal of proteome research》2005,4(4):1073-1085
Candidate proteomic biomarker discovery from human plasma holds both incredible clinical potential as well as significant challenges. The dynamic range of proteins within plasma is known to exceed 10(10), and many potential biomarkers are likely present at lower protein abundances. At present, proteomic based MS analyses provide a dynamic range typically not exceeding approximately 10(3) in a single spectrum, and approximately 10(4)-10(6) when combined with on-line separations (e.g., reversed-phase gradient liquid chromatography), and thus are generally insufficient for low level biomarker detection directly from human plasma. This limitation is providing an impetus for the development of experimental methodologies and strategies to increase the possible number of detections within this biofluid. Discussed is the diversity of available approaches currently used by our laboratory and others to utilize human plasma as a viable medium for biomarker discovery. Various separation, depletion, enrichment, and quantitative efforts as well as recent improvements in MS capabilities have resulted in measurable improvements in the detection and identification of lower abundance proteins (by approximately 10-10(2)). Despite these improvements, further advances are needed to provide a basis for discovery of candidate biomarkers at very low levels. Continued development of depletion and enrichment techniques, coupled with improved pre-MS separations (both at the protein and peptide level) holds promise in extending the dynamic range of proteomic analysis. 相似文献
647.
Proteome analysis of liver cells expressing a full-length hepatitis C virus (HCV) replicon and biopsy specimens of posttransplantation liver from HCV-infected patients 下载免费PDF全文
Jacobs JM Diamond DL Chan EY Gritsenko MA Qian W Stastna M Baas T Camp DG Carithers RL Smith RD Katze MG 《Journal of virology》2005,79(12):7558-7569
The development of a reproducible model system for the study of hepatitis C virus (HCV) infection has the potential to significantly enhance the study of virus-host interactions and provide future direction for modeling the pathogenesis of HCV. While there are studies describing global gene expression changes associated with HCV infection, changes in the proteome have not been characterized. We report the first large-scale proteome analysis of the highly permissive Huh-7.5 cell line containing a full-length HCV replicon. We detected >4,200 proteins in this cell line, including HCV replicon proteins, using multidimensional liquid chromatographic (LC) separations coupled to mass spectrometry. Consistent with the literature, a comparison of HCV replicon-positive and -negative Huh-7.5 cells identified expression changes of proteins involved in lipid metabolism. We extended these analyses to liver biopsy material from HCV-infected patients where a total of >1,500 proteins were detected from only 2 mug of liver biopsy protein digest using the Huh-7.5 protein database and the accurate mass and time tag strategy. These findings demonstrate the utility of multidimensional proteome analysis of the HCV replicon model system for assisting in the determination of proteins/pathways affected by HCV infection. Our ability to extend these analyses to the highly complex proteome of small liver biopsies with limiting protein yields offers the unique opportunity to begin evaluating the clinical significance of protein expression changes associated with HCV infection. 相似文献
648.
A colorimetric test for the detection of microcystins based on immobilised protein phosphatase (PP) has been developed. A PP2A produced by molecular engineering has been used and its performance has been compared to those of commercial PP2A and PP1. Covalent immobilisation of the enzyme using glutaraldehyde, encapsulation by sol-gel and entrapment with photocrosslinkable poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ) have been compared, the latter method providing the highest immobilisation yields. Screen-printed carbon electrodes (SPEs), Maxisorp microtiter wells and Ultrabind modified polyethersulfone affinity membranes have been used as immobilisation supports. Whilst the highest immobilisation yields were obtained with microtiter wells, the highest operational and storage stabilities were achieved with carbon SPEs and membranes, respectively. The immobilisation of PP by PVA-SbQ provided a means to preserve the enzymatic activity, which decreased at fast rates when the enzyme was kept in solution. The colorimetric test using p-nitrophenyl phosphate has demonstrated that the immobilised enzyme is able to recognise both microcystin variants (MC-LR and MC-RR), although optimisation work should be performed to achieve appropriate limits of detection. With the purpose to develop an electrochemical biosensor, several phosphorylated substrates have been used. Promising results have been achieved with the commercial enzymes and alpha-naphtyl phosphate, p-aminophenol phosphate and catechol monophosphate as enzyme substrates, guaranteeing the viability of the electrochemical approach. 相似文献
649.
Leverentz B Conway WS Camp MJ Janisiewicz WJ Abuladze T Yang M Saftner R Sulakvelidze A 《Applied and environmental microbiology》2003,69(8):4519-4526
The fresh-cut produce industry has been the fastest-growing portion of the food retail market during the past 10 years, providing consumers with convenient and nutritious food. However, fresh-cut fruits and vegetables raise food safety concerns, because exposed tissue may be colonized more easily by pathogenic bacteria than intact produce. This is due to the higher availability of nutrients on cut surfaces and the greater potential for contamination because of the increased amount of handling. We found that applied Listeria monocytogenes populations survived and increased only slightly on fresh-cut Red Delicious apples stored at 10 degrees C but increased significantly on fresh-cut honeydew melons stored at 10 degrees C over 7 days. In addition, we examined the effect of lytic, L. monocytogenes-specific phages via two phage application methods, spraying and pipetting, on L. monocytogenes populations in artificially contaminated fresh-cut melons and apples. The phage mixture reduced L. monocytogenes populations by 2.0 to 4.6 log units over the control on honeydew melons. On apples, the reduction was below 0.4 log units. In combination with nisin (a bacteriocin), the phage mixture reduced L. monocytogenes populations by up to 5.7 log units on honeydew melon slices and by up to 2.3 log units on apple slices compared to the control. Nisin alone reduced L. monocytogenes populations by up to 3.2 log units on honeydew melon slices and by up to 2.0 log units on apple slices compared to the control. The phage titer was stable on melon slices, but declined rapidly on apple slices. The spray application of the phage and phage plus nisin reduced the bacterial numbers at least as much as the pipette application. The effectiveness of the phage treatment also depended on the initial concentration of L. monocytogenes. 相似文献
650.
Mutations in the WFS1 gene that cause low-frequency sensorineural hearing loss are small non-inactivating mutations 总被引:3,自引:0,他引:3
Cryns K Pfister M Pennings RJ Bom SJ Flothmann K Caethoven G Kremer H Schatteman I Köln KA Tóth T Kupka S Blin N Nürnberg P Thiele H van de Heyning PH Reardon W Stephens D Cremers CW Smith RJ Van Camp G 《Human genetics》2002,110(5):389-394
Hereditary hearing impairment is an extremely heterogeneous trait, with more than 70 identified loci. Only two of these loci are associated with an auditory phenotype that predominantly affects the low frequencies (DFNA1 and DFNA6/14). In this study, we have completed mutation screening of the WFS1 gene in eight autosomal dominant families and twelve sporadic cases in which affected persons have low-frequency sensorineural hearing impairment (LFSNHI). Mutations in this gene are known to be responsible for Wolfram syndrome or DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy, and deafness), which is an autosomal recessive trait. We have identified seven missense mutations and a single amino acid deletion affecting conserved amino acids in six families and one sporadic case, indicating that mutations in WFS1 are a major cause of inherited but not sporadic low-frequency hearing impairment. Among the ten WFS1 mutations reported in LFSNHI, none is expected to lead to premature protein truncation, and nine cluster in the C-terminal protein domain. In contrast, 64% of the Wolfram syndrome mutations are inactivating. Our results indicate that only non-inactivating mutations in WFS1 are responsible for non-syndromic low-frequency hearing impairment. 相似文献