John W Crabb

Case Western Reserve University, Cleveland, Ohio, United States

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Publications (174)705.48 Total impact

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    ABSTRACT: Gene activation requires cooperative assembly of multiprotein transcription factor/coregulator complexes. Disruption to cooperative assemblage could underlie repression of tumor suppressor genes in leukemia cells. Mechanisms of cooperation and its disruption were therefore examined for PU.1 and RUNX1, transcription factors that cooperate to activate hematopoietic differentiation genes. PU.1 is highly expressed in leukemia cells, whereas RUNX1 is frequently inactivated by mutation or translocation. Thus, coregulator interactions of Pu.1 were examined by immunoprecipitation coupled with tandem mass spectrometry/Western blot in wild-type and Runx1-deficient hematopoietic cells. In wild-type cells, the NuAT and Baf families of coactivators coimmunoprecipitated with Pu.1. Runx1-deficiency produced a striking switch to Pu.1 interaction with the Dnmt1, Sin3A, Nurd, CoRest and B-Wich corepressor families. Corepressors of the Polycomb family, that are frequently inactivated by mutation or deletion in myeloid leukemia, did not interact with Pu.1. The most significant gene ontology association of Runx1-Pu.1 co-bound genes was with macrophages, therefore, functional consequences of altered corepressor/coactivator exchange were examined at Mcsfr, a key macrophage differentiation gene. In chromatin-immunoprecipitation analyses, high level Pu.1 binding to the Mcsfr promoter was not decreased by Runx1-deficiency. However, the Pu.1-driven shift from histone repression to activation marks at this locus, and terminal macrophage differentiation, were substantially diminished. DNMT1 inhibition, but not Polycomb inhibition, in RUNX1-translocated leukemia cells induced terminal differentiation. Thus, RUNX1 and PU.1 cooperate to exchange corepressors for coactivators, and the specific corepressors recruited to PU.1 as a consequence of RUNX1 deficiency could be rational targets for leukemia differentiation therapy.
    Journal of Biological Chemistry 04/2014; · 4.65 Impact Factor
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    ABSTRACT: The immune response has been implicated in the control of uveal melanoma progression. Epigenetic mechanisms mediated by specific microRNAs (miRs) regulate immune responses. Blood was drawn from six patients with uveal melanoma followed from diagnosis, at which time there was no clinical or radiographic evidence of metastasis, until metastasis manifested. Circulating T cell, natural killer (NK), natural killer T (NKT), and myeloid suppressor cell populations were assessed by flow cytometry. CD3(+), CD15(+), and CD56(+) cells were isolated using immunomagnetic beads. Plasma and cellular levels of immune regulatory miRs were determined by quantitative polymerase chain reaction assays. The development of metastasis was associated with decreases in circulating CD3(-)CD56(dim) NK cells and CD8(+) and double-negative CD3(+)CD56(+) NKT cells. ICOS(+)CD4(+)FoxP3(+) T regulatory cells and CD11b(+)CD14(-)CD15(+) myeloid suppressor cells increased. Plasma levels of miR-20a, 125b, 146a, 155, 181a, and 223 were higher in the study patients at diagnosis compared to controls. Plasma levels of miR-20a, 125b, 146a, 155, and 223 increased, and miR-181a decreased when metastasis manifested. Alterations in immune regulatory miRs were also observed in CD3(+), CD15(+), and CD56(+) cell populations. The development of metastasis in uveal melanoma is associated with changes in immune effector and regulatory cells consistent with lessening tumor immune surveillance. These changes are associated with changes in plasma and cellular levels of immune regulatory miRs. The results may help guide uveal melanoma immunotherapy and biomarker development.
    Molecular Immunology 12/2013; 58(2):182-186. · 2.65 Impact Factor
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    ABSTRACT: Dynamin proteins are involved in vesicle generation, providing mechanical force to excise newly formed vesicles from membranes of cellular compartments. In the brain, dynamin-1, dynamin-2, and dynamin-3 have been well studied; however, their function in the retina remains elusive. A retina-specific splice variant of dynamin-1 interacts with the photoreceptor-specific protein Tubby-like protein 1 (Tulp1), which when mutated causes an early onset form of autosomal recessive retinitis pigmentosa. Here, we investigated the role of the dynamins in the retina, using immunohistochemistry to localize dynamin-1, dynamin-2, and dynamin-3 and immunoprecipitation followed by mass spectrometry to explore dynamin-1 interacting proteins in mouse retina. Dynamin-2 is primarily confined to the inner segment compartment of photoreceptors, suggesting a role in outer segment protein transport. Dynamin-3 is present in the terminals of photoreceptors and dendrites of second-order neurons but is most pronounced in the inner plexiform layer where second-order neurons relay signals from photoreceptors. Dynamin-1 appears to be the dominant isoform in the retina and is present throughout the retina and in multiple compartments of the photoreceptor cell. This suggests that it may function in multiple cellular pathways. Surprisingly, dynamin-1 expression and localization did not appear to be disrupted in tulp1-/- mice. Immunoprecipitation experiments reveal that dynamin-1 associates primarily with proteins involved in cytoskeletal-based membrane dynamics. This finding is confirmed by western blot analysis. Results further implicate dynamin-1 in vesicular protein transport processes relevant to synaptic and post-Golgi pathways and indicate a possible role in photoreceptor stability.
    Visual Neuroscience 06/2013; · 1.48 Impact Factor
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    ABSTRACT: The seventh annual ARVO/Pfizer Ophthalmic Research Institute conference held Friday and Saturday, April 29 and 30, 2011 in Fort Lauderdale, Florida. The conference, funded by The ARVO Foundation for Eye Research through a grant from Pfizer Ophthalmics, provided an opportunity to gather experts from within and outside ophthalmology to determine the state of knowledge pertaining to molecular biomarkers associated with glaucoma, as well as the methods to identify and validate them to predict: (a) those who would be susceptible to development of glaucoma; (b) markers that will enable prediction of glaucoma progression; (c) markers that will predict efficacy of treatment of glaucoma. Identification of such biomarkers will aid in prevention of glaucoma-related vision loss and blindness. The conference focused on an evaluation of glaucoma molecular biomarkers and progress needed for future validation of glaucoma biomarkers.
    Investigative ophthalmology & visual science 01/2013; 54:871-880. · 3.43 Impact Factor
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    ABSTRACT: Carboxyethylpyrrole (CEP) adducts are oxidative modifications derived from docosahexaenoate-containing lipids that are elevated in ocular tissues and plasma in age-related macular degeneration (AMD) and in rodents exposed to intense light. The goal of this study was to determine whether light-induced CEP adducts and autoantibodies are modulated by pretreatment with AL-8309A under conditions that prevent photo-oxidative damage of rat retina. AL-8309A is a serotonin 5-HT1A receptor agonist. Albino rats were dark adapted prior to blue light exposure. Control rats were maintained in normal cyclic light. Rats were injected subcutaneously 3x with 10 mg/kg AL-8309A (2 days, 1 day and 0 hours) before light exposure for 6 h (3.1 mW/cm(2), λ=450 nm). Animals were sacrificed immediately following light exposure and eyes, retinas and plasma were collected. CEP adducts and autoantibodies were quantified by Western analysis or ELISA. ANOVA supported significant differences in mean amounts of CEP adducts and autoantibodies among the light + vehicle, light + drug and dark control groups from both retina and plasma. Light-induced CEP adducts in retina were reduced ~20% following pretreatment with AL-8309A (n = 62 rats, p = 0.006) and retinal CEP immunoreactivity was less intense by immunohistochemistry. Plasma levels of light-induced CEP adducts were reduced at least 30% (n = 15 rats, p = 0.004) by drug pretreatment. Following drug treatment, average CEP autoantibody titer in light exposed rats (n = 22) was unchanged from dark control levels, and ~20% (p = 0.046) lower than in vehicle-treated rats. Light-induced CEP adducts in rat retina and plasma were significantly decreased by pretreatment with AL-8309A. These results are consistent with and extend previous studies showing AL-8309A reduces light-induced retinal lesions in rats and support CEP biomarkers as possible tools for monitoring the efficacy of select therapeutics.
    PLoS ONE 01/2013; 8(10):e76325. · 3.73 Impact Factor
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    ABSTRACT: Age-related changes in the retina are often accompanied by visual impairment but their mechanistic details remain poorly understood. Proteomic studies were pursued toward a better molecular understanding of retinal pigment epithelium (RPE) aging mechanisms. RPE cells were isolated from young adults (3-4 month-old) and old (24-25 month-old) F344BN rats, and separated into subcellular fractions containing apical microvilli (MV) and RPE cell bodies (CB) lacking their apical microvilli. Proteins were extracted in detergent, separated by SDS-PAGE, digested in situ with trypsin and analyzed by LC MS/MS. Select proteins detected in young and old rat RPE were further studied using immunofluorescence and Western blot analysis. A total of 356 proteins were identified in RPE MV from young and 378 in RPE MV from old rats, 48% of which were common to each age group. A total of 897 proteins were identified in RPE CB from young rats and 675 in old CB, 56% of which were common to each age group. Several of the identified proteins, including proteins involved in response to oxidative stress, displayed both quantitative and qualitative changes in overall abundance during RPE aging. Numerous proteins were identified for the first time in the RPE. One such protein, collectrin, was localized to the apical membrane of apical brush border of proximal tubules where it likely regulates several amino acid transporters. Elsewhere, collectrin is involved in pancreatic β cell proliferation and insulin secretion. In the RPE, collectrin expression was significantly modulated during RPE aging. Another age-regulated, newly described protein was DJ-1, a protein extensively studied in brain where oxidative stress-related functions have been described. The data presented here reveals specific changes in the RPE during aging, providing the first protein database of RPE aging, which will facilitate future studies of age-related retinal diseases.
    PLoS ONE 01/2012; 7(6):e38673. · 3.73 Impact Factor
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    ABSTRACT: Glucocorticoids (GCs) are common anti-inflammatory agents that can cause ocular hypertension and secondary glaucoma as a consequence of impaired aqueous humor outflow through the trabecular meshwork (TM). Mechanisms of GC-signaling are complex and poorly understood. To better understand GC-signaling in the eye, we tested the hypothesis that common mechanisms of steroid responsiveness exist in TM cells from normal and glaucomatous donors. Four primary cultures of human TM cells from normal and glaucomatous donors were treated with or without dexamethasone (Dex) for 10 days, then cellular proteins were extracted, identified and quantified by liquid chromatography tandem mass spectrometry (LC MS/MS) iTRAQ (isobaric tags for relative and absolute quantitation) technology. A total of 718 proteins were quantified. Dex-treatment significantly altered the abundance of 40 proteins in ≥3 cell samples, 37 of which have not previously been associated with GC-signaling in TM cells. Most steroid responsive proteins were changed in all four TM cells analyzed, both normal and glaucomatous. GC-induced proteomic changes support remodeling of the extracellular matrix, disorganization of the cytoskeleton/cell-cell interactions, and mitochondrial dysfunction. Such physiologic consequences appear common to those induced in TM cells by transforming growth factor-β(2), another putative contributor to ocular hypertension and glaucoma pathology. The results expand the repertoire of TM proteins involved in GC-signaling, demonstrate common consequences of GC-signaling in normal and glaucomatous TM cells, and reveal similarities in proteomic changes induced by steroids and TGFβ(2) in normal and glaucomatous TM cells. Finally, the data contributes to a TM quantitative proteomic database.
    Molecular vision 01/2012; 18:2001-11. · 1.99 Impact Factor
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    ABSTRACT: Transforming growth factor beta 2 (TGFβ₂) is often elevated in the aqueous humor (AH) and trabecular meshwork (TM) of patients with primary open-angle glaucoma (POAG) and appears to contribute to POAG pathogenesis. To better understand TGFβ₂ signaling in the eye, TGFβ₂-induced proteomic changes were identified in cells cultured from the TM, a tissue involved in intraocular pressure (IOP) elevation in glaucoma. Primary cultures of human TM cells from four donors were treated with or without TGFβ₂ (5 ng/mL) for 48 hours; then cellular protein was analyzed by liquid chromatography-mass spectrometry iTRAQ (isobaric tags for relative and absolute quantitation) technology. A total of 853 proteins were quantified. TGFβ₂ treatment significantly altered the abundance of 47 proteins, 40 of which have not previously been associated with TGFβ₂ signaling in the eye. More than half the 30 elevated proteins support growing evidence that TGFβ₂ induces extracellular matrix remodeling and abnormal cytoskeletal interactions in the TM. The levels of 17 proteins were reduced, including four cytoskeletal and six regulatory proteins. Both elevated and decreased regulatory proteins implicate TGFβ₂-altered processes involving transcription, translation, and the glutamate/glutamine cycle. Altered levels of eight mitochondrial proteins support TGFβ₂-induced mitochondrial dysfunction in the TM that in POAG could contribute to oxidative damage in the AH outflow pathway, TM senescence, and elevated IOP. The results expand the repertoire of proteins known to participate in TGFβ₂ signaling, provide new molecular insight into POAG, and establish a quantitative proteomics database for the TM that includes candidate glaucoma biomarkers for future validation studies.
    Investigative ophthalmology & visual science 09/2011; 52(11):8287-94. · 3.43 Impact Factor
  • Molecular &amp Cellular Proteomics 08/2011; 10(8):A700417MCP200. · 7.25 Impact Factor
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    ABSTRACT: Despite advances in the diagnosis and local tumor control, the overall mortality rate for uveal melanoma remains high because of the development of metastatic disease. The clinical and histopathological systems currently being used to classify patients are not sufficiently accurate to predict metastasis. Tumor genotyping has demonstrated significant promise but obtaining tumor tissue can be problematic. Furthermore, assessment of tumor tissue does not indicate whether tumor cells have actually been shed and cannot indicate whether treatment is reducing metastasis. The detection of circulating tumor cells in blood has been shown to be a prognostic biomarker that can be used to monitor the effectiveness of therapy in patients with metastatic carcinoma. Uveal melanoma disseminates hematogenously, and the detection of circulating melanoma cells may potentially be useful for diagnosis, risk stratification, and the monitoring of disease progression and treatment efficacy. PCR-based and immunomagnetic cell isolation techniques, derived from studies in patients with cutaneous melanoma, have been tested. For various biological and technical reasons, they have not demonstrated the accuracy and reproducibility required for an effective prognostic assay in patients with uveal melanoma. Assessments have been confounded by false positives and negatives and thus, correlations between circulating melanoma cells and survival have not yet been established. Circulating melanoma cell detection is a valuable tool for investigating metastasis in uveal melanoma and also has the potential to become a standard part of uveal melanoma management. However, more research on the biology of uveal melanoma as well as improvements upon the current technologies are needed.
    Future Oncology 01/2011; 7(1):101-9. · 3.20 Impact Factor
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    ABSTRACT: Quantitative proteomic analysis was pursued of retinal ganglion cells (RGCs) from rats with unilateral experimental glaucoma. RGCs were isolated from 22 animals by immunopanning after 8 weeks of sustained elevated intraocular pressure. Proteins were quantified by LC MS/MS iTRAQ technology. Of the 268 proteins quantified, approximately 8% appeared elevated and approximately 13% decreased in glaucomatous RGCs. Voltage-dependent anion channel protein 2, aldose reductase, and ubiquitin were among the significantly elevated proteins while prothymosin was among the significantly decreased. The results demonstrate the feasibility of identifying global proteomic differences in protein expression between purified glaucomatous and control in vivo RGCs.
    Experimental Eye Research 07/2010; 91(1):107-10. · 3.03 Impact Factor
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    ABSTRACT: A quantitative proteomics analysis of the macular Bruch membrane/choroid complex was pursued for insights into the molecular mechanisms of age-related macular degeneration (AMD). Protein in trephine samples from the macular region of 10 early/mid-stage dry AMD, six advanced dry AMD, eight wet AMD, and 25 normal control post-mortem eyes was analyzed by LC MS/MS iTRAQ (isobaric tags for relative and absolute quantitation) technology. A total of 901 proteins was quantified, including 556 proteins from > or =3 AMD samples. Most proteins differed little in amount between AMD and control samples and therefore reflect the proteome of normal macular tissues of average age 81. A total of 56 proteins were found to be elevated and 43 were found to be reduced in AMD tissues relative to controls. Analysis by category of disease progression revealed up to 16 proteins elevated or decreased in each category. About 60% of the elevated proteins are involved in immune response and host defense, including many complement proteins and damage-associated molecular pattern proteins such as alpha-defensins 1-3, protein S100s, crystallins, histones, and galectin-3. Four retinoid processing proteins were elevated only in early/mid-stage AMD, supporting a role for retinoids in AMD initiation. Proteins uniquely decreased in early/mid-stage AMD implicate hematologic malfunctions and weakened extracellular matrix integrity and cellular interactions. Galectin-3, a receptor for advanced glycation end products, was the most significantly elevated protein in advanced dry AMD, supporting a role for advanced glycation end products in dry AMD progression. The results endorse inflammatory processes in both early and advanced AMD pathology, implicate different pathways of progression to advanced dry and wet AMD, and provide a new database for hypothesis-driven and discovery-based studies of AMD.
    Molecular &amp Cellular Proteomics 02/2010; 9(6):1031-46. · 7.25 Impact Factor
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    ABSTRACT: Toward early detection of susceptibility to age-related macular degeneration (AMD), we quantified plasma carboxyethylpyrrole (CEP) oxidative protein modifications and CEP autoantibodies by ELISA in 916 AMD and 488 control donors. Mean CEP adduct and autoantibody levels were elevated in AMD plasma by ∼60 and ∼30%, respectively, and the odds ratio for both CEP markers elevated was ∼3-fold greater in AMD than in control patients. Genotyping was performed for AMD risk polymorphisms associated with age-related maculopathy susceptibility 2 (ARMS2), high-temperature requirement factor A1 (HTRA1), complement factor H (CFH), and complement C3. The AMD risk predicted for those exhibiting elevated CEP markers and risk genotypes was 2- to 3-fold greater than the risk based on genotype alone. AMD donors carrying the ARMS2 and HTRA1 risk alleles were the most likely to exhibit elevated CEP markers. Receiver operating characteristic curves suggest that CEP markers alone can discriminate between AMD and control plasma donors with ∼76% accuracy and in combination with genomic markers, provide up to ∼80% discrimination accuracy. CEP plasma biomarkers, particularly in combination with genomic markers, offer a potential early warning system for predicting susceptibility to this blinding disease.
    Advances in experimental medicine and biology 01/2010; 664:411-7. · 1.83 Impact Factor
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    ABSTRACT: Protein modifications in which the epsilon-amino group of lysyl residues is incorporated into a 2-(omega-carboxyethyl)pyrrole (CEP) are mediators of age-related macular degeneration (AMD). They promote both angiogenesis into the retina ('wet AMD') and geographic retinal atrophy ('dry AMD'). Blood levels of CEPs are biomarkers for clinical prognosis of the disease. To enable mechanistic studies of their role in promoting AMD, for example, through the activation of B- and T-cells, interaction with receptors, or binding with complement proteins, we developed an efficient synthesis of CEP derivatives, that is especially effective for proteins. The structures of tryptic peptides derived from CEP-modified proteins were also determined. A key finding is that 4,7-dioxoheptanoic acid 9-fluorenylmethyl ester reacts with primary amines to provide 9-fluorenylmethyl esters of CEP-modified proteins that can be deprotected in situ with 1,8-diazabicyclo[5.4.0]undec-7-ene without causing protein denaturation. The introduction of multiple CEP-modifications with a wide variety of CEP:protein ratios is readily achieved using this strategy.
    Bioorganic & medicinal chemistry 09/2009; 17(21):7548-61. · 2.82 Impact Factor
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    ABSTRACT: Age-related macular degeneration (AMD) causes severe vision loss in the elderly; early identification of AMD risk could help slow or prevent disease progression. Toward the discovery of AMD biomarkers, we quantified plasma protein N(epsilon)-carboxymethyllysine (CML) and pentosidine from 58 AMD and 32 control donors. CML and pentosidine are advanced glycation end products that are abundant in Bruch membrane, the extracellular matrix separating the retinal pigment epithelium from the blood-bearing choriocapillaris. We measured CML and pentosidine by LC-MS/MS and LC-fluorometry, respectively, and found higher mean levels of CML (approximately 54%) and pentosidine (approximately 64%) in AMD (p < 0.0001) relative to normal controls. Plasma protein fructosyl-lysine, a marker of early glycation, was found by amino acid analysis to be in equal amounts in control and non-diabetic AMD donors, supporting an association between AMD and increased levels of CML and pentosidine independent of other diseases like diabetes. Carboxyethylpyrrole (CEP), an oxidative modification from docosahexaenoate-containing lipids and also abundant in AMD Bruch membrane, was elevated approximately 86% in the AMD cohort, but autoantibody titers to CEP, CML, and pentosidine were not significantly increased. Compellingly higher mean levels of CML and pentosidine were present in AMD plasma protein over a broad age range. Receiver operating curves indicate that CML, CEP adducts, and pentosidine alone discriminated between AMD and control subjects with 78, 79, and 88% accuracy, respectively, whereas CML in combination with pentosidine provided approximately 89% accuracy, and CEP plus pentosidine provided approximately 92% accuracy. Pentosidine levels appeared slightly altered in AMD patients with hypertension and cardiovascular disease, indicating further studies are warranted. Overall this study supports the potential utility of plasma protein CML and pentosidine as biomarkers for assessing AMD risk and susceptibility, particularly in combination with CEP adducts and with concurrent analyses of fructosyl-lysine to detect confounding factors.
    Molecular &amp Cellular Proteomics 05/2009; 8(8):1921-33. · 7.25 Impact Factor
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    ABSTRACT: Hyperglycemia, key factor of the pre-diabetic and diabetic pathology, is associated with cellular oxidative stress that promotes oxidative protein modifications. We report that protein nitration is responsive to changes in glucose concentrations in islets of Langerhans and insulinoma beta cells. Alterations in the extent of tyrosine nitration as well as the cellular nitroproteome profile correlated tightly with changing glucose concentrations. The target proteins we identified function in protein folding, energy metabolism, antioxidant capacity, and membrane permeability. Nitration of heat shock protein 60 in vitro was found to decrease its ATP hydrolysis and interaction with proinsulin, suggesting a mechanism by which protein nitration could diminish insulin secretion. This was supported by our finding of a decrease in stimulated insulin secretion following glycolytic stress in cultured cells. Our results reveal that protein tyrosine nitration may be a previously unrecognized factor in beta-cell dysfunction and the pathogenesis of diabetes.
    Archives of Biochemistry and Biophysics 05/2009; 484(2):221-31. · 3.37 Impact Factor
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    ABSTRACT: Age-related macular degeneration (AMD) is a progressive disease and major cause of severe visual loss. Toward the discovery of tools for early identification of AMD susceptibility, we evaluated the combined predictive capability of proteomic and genomic AMD biomarkers. We quantified plasma carboxyethylpyrrole (CEP) oxidative protein modifications and CEP autoantibodies by ELISA in 916 AMD and 488 control donors. CEP adducts are uniquely generated from oxidation of docosahexaenoate-containing lipids that are abundant in the retina. Mean CEP adduct and autoantibody levels were found to be elevated in AMD plasma by approximately 60 and approximately 30%, respectively. The odds ratio for both CEP markers elevated was 3-fold greater or more in AMD than in control patients. Genotyping was performed for AMD risk polymorphisms associated with age-related maculopathy susceptibility 2 (ARMS2), high temperature requirement factor A1 (HTRA1), complement factor H, and complement C3, and the risk of AMD was predicted based on genotype alone or in combination with the CEP markers. The AMD risk predicted for those exhibiting elevated CEP markers and risk genotypes was 2-3-fold greater than the risk based on genotype alone. AMD donors carrying the ARMS2 and HTRA1 risk alleles were the most likely to exhibit elevated CEP markers. The results compellingly demonstrate higher mean CEP marker levels in AMD plasma over a broad age range. Receiver operating characteristic curves suggest that CEP markers alone can discriminate between AMD and control plasma donors with approximately 76% accuracy and in combination with genomic markers provide up to approximately 80% discrimination accuracy. Plasma CEP marker levels were altered slightly by several demographic and health factors that warrant further study. We conclude that CEP plasma biomarkers, particularly in combination with genomic markers, offer a potential early warning system for assessing susceptibility to this blinding, multifactorial disease.
    Molecular &amp Cellular Proteomics 03/2009; 8(6):1338-49. · 7.25 Impact Factor
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    ABSTRACT: Levuglandins (LGs) and isolevuglandins (isoLGs, also called “isoketals” or “isoKs”) are extraordinarily reactive products of cyclooxygenase- and free radical-induced oxidation of arachidonates. We now report the detection in vivo and quantitative analysis of LG/isoLG adducts that incorporate the amino group of phosphatidylethanolamines (PEs) into LG/isoLG-hydroxylactams. Notably, LC–MS/MS detection of these hydroxylactams is achieved with samples that are an order of magnitude smaller and sample processing is much simpler and less time consuming than required for measuring protein-derived LG/isoLG-lysyl lactams. A key feature of our protocol is treatment of biological phospholipid extracts with phospholipase A2 to generate mainly 1-palmitoyl-2-lysoPE-hydroxylactams from heterogeneous mixtures of phospholipids with a variety of acyl groups on the 2 position. Over 160% higher mean levels of LG/isoLG-PE-hydroxylactam (P < 0.001) were detected in liver from chronic ethanol-fed mice (32.4 ± 6.3 ng/g, n = 6) compared to controls (12.1 ± 1.5 ng/g, n = 4), and mean levels in plasma from patients with age-related macular degeneration (5.2 ± 0.4 ng/ml, n = 15) were elevated ∼ 53% (P < 0.0001) compared to those of healthy volunteers (3.4 ± 0.1 ng/ml, n = 15). Just as LG/isoLG–protein adducts provide a dosimeter of oxidative injury, this study suggests that LG/isoLG-PE-hydroxylactams are potential biomarkers for assessing risk for oxidative stress-stimulated diseases.
    Free Radical Biology & Medicine 01/2009; · 5.27 Impact Factor
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    ABSTRACT: Hyperglycemia, a key factor in insulin resistance and diabetic pathology, is associated with cellular oxidative stress that promotes oxidative protein modifications. We report that protein nitration is responsive to changes in glucose concentrations in 3T3-L1 adipocytes. Alterations in the extent of tyrosine nitration as well as the cellular nitroproteome profile correlated tightly with changing glucose concentrations. The target proteins we identified are involved in fatty acid binding, cell signaling, protein folding, energy metabolism, antioxidant capacity, and membrane permeability. The nitration of adipocyte fatty acid binding protein (FABP4) at Tyr19 decreases, similar to phosphorylation, the binding of palmitic acid to the fatty acid-free protein. This potentially alters intracellular fatty acid transport, nuclear translocation of FABP4, and agonism of PPAR gamma. Our results suggest that protein tyrosine nitration may be a factor in obesity, insulin resistance, and the pathogenesis of diabetes.
    Free Radical Biology & Medicine 01/2009; 46(7):884-892. · 5.27 Impact Factor

Publication Stats

5k Citations
705.48 Total Impact Points

Institutions

  • 2002–2013
    • Case Western Reserve University
      • Department of Chemistry
      Cleveland, Ohio, United States
  • 2001–2013
    • Cleveland Clinic
      Cleveland, Ohio, United States
    • Boston Children's Hospital
      • Plastic and Oral Surgery Research Laboratory
      Boston, MA, United States
  • 2001–2009
    • Lerner Research Institute
      Cleveland, Ohio, United States
  • 1981–2008
    • University of Washington Seattle
      • • Department of Ophthalmology
      • • Division of Medical Genetics
      Seattle, WA, United States
  • 2007
    • Louis Stokes Cleveland VA Medical Center
      Cleveland, Ohio, United States
    • Case Western Reserve University School of Medicine
      Cleveland, Ohio, United States
    • Columbia University
      • Department of Ophthalmology
      New York City, New York, United States
  • 2006
    • Wright State University
      • Department of Biochemistry and Molecular Biology
      Dayton, OH, United States
    • University of Miami
      كورال غيبلز، فلوريدا, Florida, United States
  • 2004
    • University of Everett Washington
      Seattle, Washington, United States
  • 2003
    • Umeå University
      • Department of Medical Biosciences
      Umeå, Vaesterbotten, Sweden
  • 1999
    • Oregon Health and Science University
      Portland, Oregon, United States
    • University of Zurich
      • Biochemisches Institut
      Zürich, ZH, Switzerland
  • 1998
    • Northwestern University
      • Department of Ophthalmology
      Evanston, IL, United States
    • University of California, Los Angeles
      • Department of Neurobiology
      Los Angeles, CA, United States
  • 1994
    • Good Samaritan Hospital
      Suffern, New York, United States
  • 1991–1992
    • Good Samaritan Medical Center
      West Palm Beach, Florida, United States
  • 1984–1989
    • Ruhr-Universität Bochum
      • Institut für Physiologische Chemie
      Bochum, North Rhine-Westphalia, Germany