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ABSTRACT: Mice with deletion of genes for small heat shock proteins αA- and αB-crystallin (αA/αB(-/-)) develop cataracts. We used proteomic analysis to identify lens proteins that change in abundance after deletion of these α-crystallin genes. Wild-type (WT) and αA/αB(-/-) knockout (DKO) mice were compared using two-dimensional difference gel electrophoresis and mass spectrometric analysis, and protein identifications were validated by Mascot proteomic software. The abundance of histones H2A, H4, and H2B fragment, and a low molecular weight β1-catenin increased 2-3-fold in postnatal day 2 lenses of DKO lenses compared with WT lenses. Additional major increases were observed in abundance of βB2-crystallin and vimentin in 30-day-old lenses of DKO animals compared with WT animals. Lenses of DKO mice were comprised of nine protein spots containing βB2-crystallin at 10-40-fold higher abundance and three protein spots containing vimentin at ≥2-fold higher abundance than in WT lenses. Gel permeation chromatography identified a unique 328 kDa protein in DKO lenses, containing β-crystallin, demonstrating aggregation of β-crystallin in the absence of α-crystallins. Together, these changes provide biochemical evidence for possible functions of specific cell adhesion proteins, cytoskeletal proteins, and crystallins in lens opacities caused by the absence of the major chaperones, αA- and αB-crystallins.
Biochemistry 04/2013; · 3.42 Impact Factor
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Jeffery M Klco,
David H Spencer,
Tamara L Lamprecht,
Shawn M Sarkaria,
Todd Wylie,
Vincent Magrini,
Jasreet Hundal,
Jason Walker,
Nobish Varghese,
Petra Erdmann-Gilmore,
Cheryl F Lichti,
Matthew R Meyer, R Reid Townsend,
Richard K Wilson,
Elaine R Mardis,
Timothy J Ley
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ABSTRACT: Acute myeloid leukemia (AML) is characterized by dysregulated gene expression and abnormal patterns of DNA methylation; the relationship between these events is unclear. Many AML patients are now being treated with hypomethylating agents like decitabine (DAC), although the mechanisms by which it induces remissions remain unknown. The goal of this study was to use a novel stromal co-culture assay that can expand primary AML cells to identify the immediate changes induced by DAC with a dose (100 nM) that decreases total 5-methylcytosine content and reactivates imprinted genes (without causing myeloid differentiation, which would confound downstream genomic analyses). Using array-based technologies, we found that DAC treatment caused global hypomethylation in all samples (with a preference for regions with higher levels of baseline methylation), yet there was limited correlation between changes in methylation and gene expression. Moreover, the patterns of methylation and gene expression across the samples were primarily determined by the intrinsic properties of the primary cells, rather than DAC treatment. Although DAC induces hypomethylation, we could not identify canonical target genes that are altered by DAC in primary AML cells, suggesting that the mechanism of action of DAC is more complex than previously recognized.
Blood 01/2013; · 9.90 Impact Factor
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Andrew R Lee,
Rachel R Lamb,
Julietta H Chang,
Petra Erdmann-Gilmore,
Cheryl F Lichti,
Henry W Rohrs,
James P Malone,
Yogesh P Wairkar,
Aaron Diantonio, R Reid Townsend,
Susan M Culican
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ABSTRACT: Retinal ganglion cells (RGCs) transmit visual information topographically from the eye to the brain, creating a map of visual space in retino-recipient nuclei (retinotopy). This process is affected by retinal activity and by activity-independent molecular cues. Phr1, which encodes a presumed E3 ubiquitin ligase (PHR1), is required presynaptically for proper placement of RGC axons in the lateral geniculate nucleus and the superior colliculus, suggesting that increased levels of PHR1 target proteins may be instructive for retinotopic mapping of retinofugal projections. To identify potential target proteins, we conducted a proteomic analysis of optic nerve to identify differentially abundant proteins in the presence or absence of Phr1 in RGCs. 1D gel electrophoresis identified a specific band in controls that was absent in mutants. Targeted proteomic analysis of this band demonstrated the presence of PHR1. Additionally, we conducted an unbiased proteomic analysis that identified 30 proteins as being significantly different between the two genotypes. One of these, heterogeneous nuclear ribonucleoprotein M (hnRNP-M), regulates antero-posterior patterning in invertebrates and can function as a cell surface adhesion receptor in vertebrates. Thus, we have demonstrated that network analysis of quantitative proteomic data is a useful approach for hypothesis generation and for identifying biologically relevant targets in genetically altered biological models.
Journal of Proteome Research 09/2012; · 5.11 Impact Factor
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ABSTRACT: Neurological outcomes of preterm infants with posthemorrhagic hydrocephalus are among the worst in newborn medicine. There remains no consensus regarding the diagnosis or treatment of posthemorrhagic hydrocephalus, and the pathological pathways leading to the adverse neurological sequelae are poorly understood. In the current study, we developed an innovative approach to simultaneously identify potential diagnostic markers of posthemorrhagic hydrocephalus and investigate novel pathways of posthemorrhagic hydrocephalus-related neurological disability. Tandem multi-affinity fractionation for specific removal of plasma proteins from the hemorrhagic cerebrospinal fluid samples was combined with high resolution label-free quantitative proteomics. Analysis of cerebrospinal fluid obtained from infants with posthemorrhagic hydrocephalus demonstrated marked differences in the levels of 438 proteins when compared with cerebrospinal fluid from age-matched control infants. Amyloid precursor protein, neural cell adhesion molecule-L1, neural cell adhesion molecule-1, brevican and other proteins with important roles in neurodevelopment showed profound elevations in posthemorrhagic hydrocephalus cerebrospinal fluid compared with control. Initiation of neurosurgical treatment of posthemorrhagic hydrocephalus resulted in resolution of these elevations. The results from this foundational study demonstrate the significant promise of tandem multi-affinity fractionation-proteomics in the identification and quantitation of protein mediators of neurodevelopment and neurological injury. More specifically, our results suggest that cerebrospinal fluid levels of proteins such as amyloid precursor protein or neural cell adhesion molecule-L1 should be investigated as potential diagnostic markers of posthemorrhagic hydrocephalus. Notably, dysregulation of the levels these and other proteins may directly affect ongoing neurodevelopmental processes in these preterm infants, providing an entirely new hypothesis for the developmental disability associated with posthemorrhagic hydrocephalus.
Molecular & Cellular Proteomics 12/2011; 11(6):M111.011973. · 7.40 Impact Factor
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Mingwei Bao,
Evelyn M Kanter,
Richard Y-C Huang,
Stephan Maxeiner,
Marina Frank,
Yan Zhang,
Richard B Schuessler,
Timothy W Smith, R Reid Townsend,
Henry W Rohrs,
Viviana M Berthoud,
Klaus Willecke,
James G Laing,
Kathryn A Yamada
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ABSTRACT: Gap junction channels in ventricular myocardium are required for electrical and metabolic coupling between cardiac myocytes and for normal cardiac pump function. Although much is known about expression patterns and remodeling of cardiac connexin(Cx)43, little is known about the less abundant Cx45, which is required for embryonic development and viability, is downregulated in adult hearts, and is pathophysiologically upregulated in human end-stage heart failure. We applied quantitative immunoblotting and immunoprecipitation to native myocardial extracts, immunogold electron microscopy to cardiac tissue and membrane sections, electrophysiological recordings to whole hearts, and high-resolution tandem mass spectrometry to Cx45 fusion protein, and developed two new tools, anti-Cx45 antisera and Cre(+);Cx45 floxed mice, to facilitate characterization of Cx45 in adult mammalian hearts. We found that Cx45 represents 0.3% of total Cx protein (predominantly 200 fmol Cx43 protein/μg ventricular protein) and colocalizes with Cx43 in native ventricular gap junctions, particularly in the apex and septum. Cre(+);Cx45 floxed mice express 85% less Cx45, but do not exhibit overt electrophysiologic abnormalities. Although the basal phosphorylation status of native Cx45 remains unknown, CaMKII phosphorylates 8 Ser/Thr residues in Cx45 in vitro. Thus, although downregulation of Cx45 does not produce notable deficits in electrical conduction in adult, disease-free hearts, Cx45 is a target of the multifunctional kinase CaMKII, and the phosphorylation status of Cx45 and the role of Cx43/Cx45 heteromeric gap junction channels in both normal and diseased hearts merits further investigation.
Channels (Austin, Tex.) 11/2011; 5(6):489-99. · 1.91 Impact Factor
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ABSTRACT: To determine whether class 1 UV-blocking contact lenses protect against UVB radiation-induced damage in a human lens epithelial cell line (HLE B-3) and postmortem human lenses using a proteomics approach.
HLE B-3 cells were exposed to 6.4 mW/cm(2) UVB radiation at 302 nm for 2 minutes (768 mJ/cm(2)) with or without covering by senofilcon A class 1 UV-blocking contact lenses or lotrafilcon A non-UV-blocking (lotrafilcon A has some UV-blocking ability, albeit minimal) contact lenses. Control cells were not exposed to UVB radiation. Four hours after treatment, cells were analyzed by two-dimensional difference gel electrophoresis and tandem mass spectrometry, and changes in protein abundance were quantified. F-actin and microtubule cytoskeletons were examined by fluorescence staining. In addition, human donor lenses were exposed to UVB radiation at 302 nm for 4 minutes (1536 mJ/cm(2)). Cortical and epithelial cell proteins were scraped from lens surfaces and subjected to the same protein analyses.
Senofilcon A lenses were beneficial for protecting HLE B-3 cells against UVB radiation-induced changes in caldesmon 1 isoform, lamin A/C transcript variant 1, DEAD (Asp-Glu-Ala-Asp) box polypeptide, β-actin, glyceraldehyde 3-phosphate dehydrogenase (G3PDH), annexin A2, triose phosphate isomerase, and ubiquitin B precursor. These contact lenses also prevented actin and microtubule cytoskeleton changes typically induced by UVB radiation. Conversely, non-UV-blocking contact lenses were not protective. UVB-irradiated human lenses showed marked reductions in αA-crystallin, αB-crystallin, aldehyde dehydrogenase 1, βS-crystallin, βB2-crystallin, and G3PDH, and UV-absorbing contact lenses significantly prevented these alterations.
Senofilcon A class 1 UV-blocking contact lenses largely prevented UVB-induced changes in protein abundance in lens epithelial cells and in human lenses.
Investigative ophthalmology & visual science 08/2011; 52(11):8330-41. · 3.43 Impact Factor
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ABSTRACT: Connexin43 (Cx43) is a major cardiac gap junction channel protein required for normal electrical and contractile activity. Gap junction channel assembly, function, and turnover are regulated by phosphorylation under both normal and disease conditions. The carboxyl terminus (CT) of Cx43 contains numerous amino acid residues that are phosphorylated by protein kinases. However, our knowledge of the specific residues and kinases involved is incomplete. The objective of this study was to identify amino acid residues in the Cx43-CT that are targets of the multifunctional protein kinase, Ca(2+)/calmodulin protein kinase II (CaMKII), an enzyme known to play critical roles in Ca(2+) homeostasis, transcription, apoptosis, and ischemic heart disease. We subjected fusion protein containing the Cx43-CT to phosphorylation by CaMKII in vitro, digestion with Lys-C and trypsin followed by enrichment for phosphorylated peptides using TiO(2), and analysis in an LTQ XL Orbitrap with collision-induced dissociation and electron transfer dissociation. We deduced the sites of modification by interpreting tandem spectra from these "orthogonal" methods of gas phase peptide fragmentation. We have identified 15 serine residues, including one novel site, in the Cx43-CT that are phosphorylated by CaMKII, the activity of which may be important in regulating Cx43 in normal and diseased hearts.
Journal of Proteome Research 02/2011; 10(3):1098-109. · 5.11 Impact Factor
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Richard J Perrin,
Rebecca Craig-Schapiro,
James P Malone,
Aarti R Shah,
Petra Gilmore,
Alan E Davis,
Catherine M Roe,
Elaine R Peskind,
Ge Li,
Douglas R Galasko,
Christopher M Clark,
Joseph F Quinn,
Jeffrey A Kaye,
John C Morris,
David M Holtzman, R Reid Townsend,
Anne M Fagan
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ABSTRACT: Ideally, disease modifying therapies for Alzheimer disease (AD) will be applied during the 'preclinical' stage (pathology present with cognition intact) before severe neuronal damage occurs, or upon recognizing very mild cognitive impairment. Developing and judiciously administering such therapies will require biomarker panels to identify early AD pathology, classify disease stage, monitor pathological progression, and predict cognitive decline. To discover such biomarkers, we measured AD-associated changes in the cerebrospinal fluid (CSF) proteome.
CSF samples from individuals with mild AD (Clinical Dementia Rating [CDR] 1) (n = 24) and cognitively normal controls (CDR 0) (n = 24) were subjected to two-dimensional difference-in-gel electrophoresis. Within 119 differentially-abundant gel features, mass spectrometry (LC-MS/MS) identified 47 proteins. For validation, eleven proteins were re-evaluated by enzyme-linked immunosorbent assays (ELISA). Six of these assays (NrCAM, YKL-40, chromogranin A, carnosinase I, transthyretin, cystatin C) distinguished CDR 1 and CDR 0 groups and were subsequently applied (with tau, p-tau181 and Aβ42 ELISAs) to a larger independent cohort (n = 292) that included individuals with very mild dementia (CDR 0.5). Receiver-operating characteristic curve analyses using stepwise logistic regression yielded optimal biomarker combinations to distinguish CDR 0 from CDR>0 (tau, YKL-40, NrCAM) and CDR 1 from CDR<1 (tau, chromogranin A, carnosinase I) with areas under the curve of 0.90 (0.85-0.94 95% confidence interval [CI]) and 0.88 (0.81-0.94 CI), respectively.
Four novel CSF biomarkers for AD (NrCAM, YKL-40, chromogranin A, carnosinase I) can improve the diagnostic accuracy of Aβ42 and tau. Together, these six markers describe six clinicopathological stages from cognitive normalcy to mild dementia, including stages defined by increased risk of cognitive decline. Such a panel might improve clinical trial efficiency by guiding subject enrollment and monitoring disease progression. Further studies will be required to validate this panel and evaluate its potential for distinguishing AD from other dementing conditions.
PLoS ONE 01/2011; 6(1):e16032. · 4.09 Impact Factor
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Sarah J Fentress,
Michael S Behnke,
Ildiko R Dunay,
Mona Mashayekhi,
Leah M Rommereim,
Barbara A Fox,
David J Bzik,
Gregory A Taylor,
Benjamin E Turk,
Cheryl F Lichti, R Reid Townsend,
Wei Qiu,
Raymond Hui,
Wandy L Beatty,
L David Sibley
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ABSTRACT: Macrophages are specialized to detect and destroy intracellular microbes and yet a number of pathogens have evolved to exploit this hostile niche. Here we demonstrate that the obligate intracellular parasite Toxoplasma gondii disarms macrophage innate clearance mechanisms by secreting a serine threonine kinase called ROP18, which binds to and phosphorylates immunity-related GTPases (IRGs). Substrate profiling of ROP18 revealed a preference for a conserved motif within switch region I of the GTPase domain, a modification predicted to disrupt IRG function. Consistent with this, expression of ROP18 was both necessary and sufficient to block recruitment of Irgb6, which was in turn required for parasite destruction. ROP18 phosphorylation of IRGs prevented clearance within inflammatory monocytes and IFN-γ-activated macrophages, conferring parasite survival in vivo and promoting virulence. IRGs are implicated in clearance of a variety of intracellular pathogens, suggesting that other virulence factors may similarly thwart this innate cellular defense mechanism.
Cell host & microbe 12/2010; 8(6):484-95. · 13.02 Impact Factor
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Rebecca Craig-Schapiro,
Richard J Perrin,
Catherine M Roe,
Chengjie Xiong,
Deborah Carter,
Nigel J Cairns,
Mark A Mintun,
Elaine R Peskind,
Ge Li,
Douglas R Galasko,
Christopher M Clark,
Joseph F Quinn,
Gina D'Angelo,
James P Malone, R Reid Townsend,
John C Morris,
Anne M Fagan,
David M Holtzman
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ABSTRACT: Disease-modifying therapies for Alzheimer's disease (AD) would be most effective during the preclinical stage (pathology present, cognition intact) before significant neuronal loss occurs. Therefore, biomarkers that detect AD pathology in its early stages and predict dementia onset and progression will be invaluable for patient care and efficient clinical trial design.
AD-associated changes in cerebrospinal fluid (CSF) were measured using two-dimensional difference gel electrophoresis and liquid chromatography tandem mass spectrometry. Subsequently, CSF YKL-40 was measured by enzyme-linked immunosorbent assay in the discovery cohort (n = 47), validation cohort (n = 292) with paired plasma samples (n = 237), frontotemporal lobar degeneration (n=9) [corrected], and progressive supranuclear palsy (PSP; n = 6). Immunohistochemistry was performed to identify source(s) of YKL-40 in human AD brain.
Discovery and validation cohorts, showed higher mean CSF YKL-40 in very mild and mild AD-type dementia (Clinical Dementia Rating [CDR] 0.5 and 1) versus control subjects (CDR 0) and PSP subjects. Importantly, CSF YKL-40/Aβ42 ratio predicted risk of developing cognitive impairment (CDR 0 to CDR > 0 conversion), as well as the best CSF biomarkers identified to date, tau/Aβ42 and p-tau 181/Aβ42. Mean plasma YKL-40 was higher in CDR 0.5 and 1 versus CDR 0, and correlated with CSF levels. YKL-40 immunoreactivity labeled astrocytes near a subset of amyloid plaques, implicating YKL-40 in the neuroinflammatory response to Aβ deposition.
These data demonstrate that YKL-40, a putative indicator of neuroinflammation, is elevated in AD and, together with Aβ42, has potential prognostic utility as a biomarker for preclinical AD.
Biological psychiatry 11/2010; 68(10):903-12. · 8.93 Impact Factor
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Timothy J Ley,
Li Ding,
Matthew J Walter,
Michael D McLellan,
Tamara Lamprecht,
David E Larson,
Cyriac Kandoth,
Jacqueline E Payton,
Jack Baty,
John Welch, [......],
William D Shannon,
Nobish Varghese,
Rakesh Nagarajan,
Peter Westervelt,
Michael H Tomasson,
Daniel C Link,
Timothy A Graubert,
John F DiPersio,
Elaine R Mardis,
Richard K Wilson
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ABSTRACT: The genetic alterations responsible for an adverse outcome in most patients with acute myeloid leukemia (AML) are unknown.
Using massively parallel DNA sequencing, we identified a somatic mutation in DNMT3A, encoding a DNA methyltransferase, in the genome of cells from a patient with AML with a normal karyotype. We sequenced the exons of DNMT3A in 280 additional patients with de novo AML to define recurring mutations.
A total of 62 of 281 patients (22.1%) had mutations in DNMT3A that were predicted to affect translation. We identified 18 different missense mutations, the most common of which was predicted to affect amino acid R882 (in 37 patients). We also identified six frameshift, six nonsense, and three splice-site mutations and a 1.5-Mbp deletion encompassing DNMT3A. These mutations were highly enriched in the group of patients with an intermediate-risk cytogenetic profile (56 of 166 patients, or 33.7%) but were absent in all 79 patients with a favorable-risk cytogenetic profile (P<0.001 for both comparisons). The median overall survival among patients with DNMT3A mutations was significantly shorter than that among patients without such mutations (12.3 months vs. 41.1 months, P<0.001). DNMT3A mutations were associated with adverse outcomes among patients with an intermediate-risk cytogenetic profile or FLT3 mutations, regardless of age, and were independently associated with a poor outcome in Cox proportional-hazards analysis.
DNMT3A mutations are highly recurrent in patients with de novo AML with an intermediate-risk cytogenetic profile and are independently associated with a poor outcome. (Funded by the National Institutes of Health and others.).
New England Journal of Medicine 11/2010; 363(25):2424-33. · 53.30 Impact Factor
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Nadzeya V Marozkina,
Sean Yemen,
Molly Borowitz,
Lei Liu,
Melissa Plapp,
Fei Sun,
Rafique Islam,
Petra Erdmann-Gilmore, R Reid Townsend,
Cheryl F Lichti,
Sneha Mantri,
Phillip W Clapp,
Scott H Randell,
Benjamin Gaston,
Khalequz Zaman
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ABSTRACT: The endogenous signaling molecule S-nitrosoglutathione (GSNO) and other S-nitrosylating agents can cause full maturation of the abnormal gene product DeltaF508 cystic fibrosis (CF) transmembrane conductance regulator (CFTR). However, the molecular mechanism of action is not known. Here we show that Hsp70/Hsp90 organizing protein (Hop) is a critical target of GSNO, and its S-nitrosylation results in DeltaF508 CFTR maturation and cell surface expression. S-nitrosylation by GSNO inhibited the association of Hop with CFTR in the endoplasmic reticulum. This effect was necessary and sufficient to mediate GSNO-induced cell-surface expression of DeltaF508 CFTR. Hop knockdown using siRNA recapitulated the effect of GSNO on DeltaF508 CFTR maturation and expression. Moreover, GSNO acted additively with decreased temperature, which promoted mutant CFTR maturation through a Hop-independent mechanism. We conclude that GSNO corrects DeltaF508 CFTR trafficking by inhibiting Hop expression, and that combination therapies--using differing mechanisms of action--may have additive benefits in treating CF.
Proceedings of the National Academy of Sciences 06/2010; 107(25):11393-8. · 9.68 Impact Factor
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ABSTRACT: Telomeres are DNA-protein structures that protect chromosome ends from the actions of the DNA repair machinery. When telomeric integrity is compromised, genomic instability ensues. Considerable effort has focused on identification of telomere-binding proteins and elucidation of their functions. To date, protein identification has relied on classical immunoprecipitation and mass spectrometric approaches, primarily under conditions that favor isolation of proteins with strong or long lived interactions that are present at sufficient quantities to visualize by SDS-PAGE. To facilitate identification of low abundance and transiently associated telomere-binding proteins, we developed a novel approach that combines in vivo protein-protein cross-linking, tandem affinity purification, and stringent sequential endoprotease digestion. Peptides were identified by label-free comparative nano-LC-FTICR-MS. Here, we expressed an epitope-tagged telomere-binding protein and utilized a modified chromatin immunoprecipitation approach to cross-link associated proteins. The resulting immunoprecipitant contained telomeric DNA, establishing that this approach captures bona fide telomere binding complexes. To identify proteins present in the immunocaptured complexes, samples were reduced, alkylated, and digested with sequential endoprotease treatment. The resulting peptides were purified using a microscale porous graphite stationary phase and analyzed using nano-LC-FTICR-MS. Proteins enriched in cells expressing HA-FLAG-TIN2 were identified by label-free quantitative analysis of the FTICR mass spectra from different samples and ion trap tandem mass spectrometry followed by database searching. We identified all of the proteins that constitute the telomeric shelterin complex, thus validating the robustness of this approach. We also identified 62 novel telomere-binding proteins. These results demonstrate that DNA-bound protein complexes, including those present at low molar ratios, can be identified by this approach. The success of this approach will allow us to create a more complete understanding of telomere maintenance and have broad applicability.
Molecular & Cellular Proteomics 06/2010; 9(6):1144-56. · 7.40 Impact Factor
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ABSTRACT: The Haemophilus influenzae HMW1 adhesin is a high-molecular weight protein that is secreted by the bacterial two-partner secretion pathway and mediates adherence to respiratory epithelium, an essential early step in the pathogenesis of H. influenzae disease. In recent work, we discovered that HMW1 is a glycoprotein and undergoes N-linked glycosylation at multiple asparagine residues with simple hexose units rather than N-acetylated hexose units, revealing an unusual N-glycosidic linkage and suggesting a new glycosyltransferase activity. Glycosylation protects HMW1 against premature degradation during the process of secretion and facilitates HMW1 tethering to the bacterial surface, a prerequisite for HMW1-mediated adherence. In the current study, we establish that the enzyme responsible for glycosylation of HMW1 is a protein called HMW1C, which is encoded by the hmw1 gene cluster and shares homology with a group of bacterial proteins that are generally associated with two-partner secretion systems. In addition, we demonstrate that HMW1C is capable of transferring glucose and galactose to HMW1 and is also able to generate hexose-hexose bonds. Our results define a new family of bacterial glycosyltransferases.
PLoS Pathogens 05/2010; 6(5):e1000919. · 9.13 Impact Factor
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ABSTRACT: Biliary atresia (BA) is the most serious liver disease in infants. Diagnosis currently depends on surgical exploration of the biliary tree. Noninvasive tests that distinguish BA from other types of neonatal liver disease are not available.
To identify potential serum biomarkers that classify children with neonatal cholestasis, we performed 2-dimensional difference gel electrophoresis, statistical analysis, and tandem mass spectrometry using serum samples from 19 infants with BA and 19 infants with non-BA neonatal cholestasis.
Eleven potential serum biomarkers were found that could in combination classify children with neonatal cholestasis.
Although no single biomarker or imaging test adequately distinguishes BA from other types of neonatal cholestasis, combinations of biomarkers, imaging tests, and noninvasive clinical criteria should be further explored as potential tests for rapid and accurate diagnosis of BA.
Journal of pediatric gastroenterology and nutrition 02/2010; 50(4):411-6. · 2.18 Impact Factor
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ABSTRACT: Baseline level of the cysteinyl leukotriene (CysLT), leukotriene E4 (LTE4), is associated with an increased pain rate in children and adults with sickle cell disease (SCD). To provide additional evidence for a role of CysLTs in the pathogenesis of vaso-occlusion, we tested the hypothesis that LTE4 levels will increase within an individual during painful episodes compared to baseline. In a cohort of 19 children and adults with SCD, median LTE4 levels increased from 82.36 pg/mg creatinine at baseline to 162.81 pg/mg creatinine during a painful episode (P < 0.001). These data further support a contribution of CysLTs to the process of vaso-occlusion.
American Journal of Hematology 04/2009; 84(4):231-3. · 4.67 Impact Factor
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ABSTRACT: While some species and tissue types are injured by oxygen deprivation, anoxia tolerant organisms display a protective response that has not been fully elucidated and is well-suited to genomic and proteomic analysis. However, such methodologies have focused on transcriptional responses, prolonged anoxia, or have used cultured cells or isolated tissues. In this study of intact zebrafish embryos, a species capable of >24 h survival in anoxia, we have utilized 2D difference in gel electrophoresis to identify changes in the proteomic profile caused by near-lethal anoxic durations as well as acute anoxia (1 h), a timeframe relevant to ischemic events in human disease when response mechanisms are largely limited to post-transcriptional and post-translational processes. We observed a general stabilization of the proteome in anoxia. Proteins involved in oxidative phosphorylation, antioxidant defense, transcription, and translation changed over this time period. Among the largest proteomic alterations was that of muscle cofilin 2, implicating the regulation of the cytoskeleton and actin assembly in the adaptation to acute anoxia. These studies in an intact embryo highlight proteomic components of an adaptive response to anoxia in a model organism amenable to genetic analysis to permit further mechanistic insight into the phenomenon of anoxia tolerance.
Comparative Biochemistry and Physiology Part D Genomics and Proteomics 03/2009; 4(1):21-31. · 1.72 Impact Factor
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ABSTRACT: The protozoan intestinal parasite Entamoeba histolytica infects millions of people worldwide and is capable of causing amebic dysentery and amebic liver abscess. The closely related species Entamoeba dispar colonizes many more individuals, but this organism does not induce disease. To identify molecular differences between these two organisms that may account for their differential ability to cause disease in humans, we used two-dimensional gel-based (DIGE) proteomic analysis to compare whole cell lysates of E. histolytica and E. dispar. We observed 141 spots expressed at a substantially (>5-fold) higher level in E. histolytica HM-1:IMSS than E. dispar and 189 spots showing the opposite pattern. Strikingly, 3 of 4 proteins consistently identified as different at a greater than 5-fold level between E. histolytica HM-1:IMSS and E. dispar were identical to proteins recently identified as differentially expressed between E. histolytica HM-1:IMSS and the reduced virulence strain E. histolytica Rahman. One of these was E. histolytica alcohol dehydrogenase 3 (EhADH3). We found that E. histolytica possesses a higher level of NADP-dependent alcohol dehydrogenase activity than E. dispar and that some EhADH3 can be localized to the surface of E. histolytica. Episomal overexpression of EhADH3 in E. histolytica trophozoites resulted in only subtle phenotypic differences in E. histolytica virulence in animal models of amebic colitis and amebic liver abscess, making it difficult to directly link EhADH3 levels to virulence differences between E. histolytica and less-pathogenic Entamoeba.
PLoS Neglected Tropical Diseases 01/2009; 3(4):e415. · 4.69 Impact Factor
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Janet S. Rader Professor,
James P. Malone,
Julia Gross,
Petra Gilmore,
Rebecca A. Brooks,
Loan Nguyen,
Dan L. Crimmins,
Sheng Feng,
Jason D. Wright,
Nicholas Taylor,
Israel Zighelboim,
Margo C. Funk,
Phyllis C. Huettner,
Jack H. Ladenson,
David Gius, R. Reid Townsend
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ABSTRACT: Cervical cancer screening is ideally suited for the development of biomarkers due to the ease of tissue acquisition and the well-established histological transitions. Furthermore, cell and biologic fluid obtained from cervix samples undergo specific molecular changes that can be profiled. However, the ideal manner and techniques for preparing cervical samples remains to be determined. To address this critical issue a patient screening protein and nucleic acid collection protocol was established. RNAlater was used to collect the samples followed by proteomic methods to identify proteins that were differentially expressed in normal cervical epithelial versus cervical cancer cells. Three hundred ninety spots were identified via 2-D DIGE that were expressed at either higher or lower levels (>three-fold) in cervical cancer samples. These proteomic results were compared to genes in a cDNA microarray analysis of microdissected neoplastic cervical specimens to identify overlapping patterns of expression. The most frequent pathways represented by the combined dataset were: cell cycle: G2/M DNA damage checkpoint regulation; aryl hydrocarbon receptor signaling; p53 signaling; cell cycle: G1/S checkpoint regulation; and the ER stress pathway. HNRPA2B1 was identified as a biomarker candidate with increased expression in cancer compared to normal cervix and validated by Western blot.
PROTEOMICS - CLINICAL APPLICATIONS 10/2008; 2(12):1658 - 1669. · 1.81 Impact Factor
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ABSTRACT: The Haemophilus influenzae HMW1 adhesin mediates adherence to respiratory epithelial cells, a critical early step in the pathogenesis of H. influenzae disease. In recent work, we demonstrated that HMW1 undergoes glycosylation. In addition, we observed that glycosylation of HMW1 is essential for HMW1 tethering to the bacterial surface, a prerequisite for HMW1-mediated adherence to host epithelium. In this study, we examined HMW1 proteolytic fragments by mass spectrometry, achieved 89% amino acid sequence coverage, and identified 31 novel modification sites. All of the modified sites were asparagine residues, in all but one case in the conventional consensus sequence of N-linked glycans, viz. NX(S/T). Liquid chromatography-tandem mass spectrometry analysis using a hybrid linear quadrupole ion trap Fourier transform ion cyclotron mass spectrometer, accurate mass measurements, and deuterium exchange studies established that the modifying glycan structures were mono- or dihexoses rather than the N-acetylated chitobiosyl core that is characteristic of N-glycosylation. This unusual carbohydrate modification suggests that HMW1 glycosylation requires a glycosyltransferase with a novel activity.
Journal of Biological Chemistry 08/2008; 283(38):26010-5. · 4.77 Impact Factor
