Proteomic changes in the photoreceptor outer segment upon intense light exposure.

Acute light-induced photoreceptor degeneration has been studied in experimental animals as a model for photoreceptor cell loss in human retinal degenerative diseases. Light absorption by rhodopsin in rod photoreceptor outer segments (OS) induces oxidative stress and initiates apoptotic cell death. However, the molecular events that induce oxidative stress and initiate the apoptotic cascade remain poorly understood. To better understand the molecular mechanisms of light-induced photoreceptor cell death, we studied the proteomic changes in OS upon intense light exposure by using a proteolytic (18)O labeling method. Of 171 proteins identified, the relative abundance of 98 proteins in light-exposed and unexposed OS was determined. The quantities of 11 proteins were found to differ by more than 2-fold between light-exposed OS and those remaining in darkness. Among the 11 proteins, 8 were phototransduction proteins and 7 of these were altered such that the efficiency of phototransduction would be reduced or quenched during light exposure. In contrast, the amount of OS rhodopsin kinase was reduced by 2-fold after light exposure, suggesting attenuation in the mechanism of quenching phototransduction. Liquid chromatography multiple reaction monitoring (LC-MRM) was performed to confirm this reduction in the quantity of rhodopsin kinase. As revealed by immunofluorescence microscopy, this reduction of rhodopsin kinase is not a result of protein translocation from the outer to the inner segment. Collectively, our findings suggest that the absolute quantity of rhodopsin kinase in rod photoreceptors is reduced upon light stimulation and that this reduction may be a contributing factor to light-induced photoreceptor cell death. This report provides new insights into the proteomic changes in the OS upon intense light exposure and creates a foundation for understanding the mechanisms of light-induced photoreceptor cell death.

Falsely Undetectable Prostate-Specific Antigen (PSA) Due to Presence of an Inhibitory Serum Factor: A Case Report and Review of Pertinent Literature.

BACKGROUND Few cases of falsely undetectable PSA due to the presence of an inhibitory serum factor have been reported in the world literature. We present a case of falsely low-to-undetectable PSA with data from a serum dilution series, the current literature on biochemical assay interference, and the implications for prostate cancer salvage treatment. CASE REPORT A 63-year-old man was treated with prostatectomy for high-risk prostate cancer and was found to have a rising PSA after approximately 3 years following surgery. He subsequently transferred his care to a different health system and was found to have an undetectable PSA. He was eventually found to have an elevated PSA once again after the particular assay at this institution was changed. He thus received salvage prostate radiotherapy and androgen deprivation therapy. CONCLUSIONS While falsely low PSA results cannot be explained by the presence of serum heterophile antibodies, competitive antibody interference against the immunoassay reagents or anti-PSA antibodies are possible explanations for the results of the dilution experiments performed in this case study. We suggest that unexpected PSA testing results should raise concern for assay interference and warrant further clinical workup.

A comparison of three apolipoprotein B methods and their associations with incident coronary heart disease risk over a 12-year follow-up period: The Multi-Ethnic Study of Atherosclerosis.

BACKGROUND: Apolipoprotein B-100 (ApoB) is a well-researched lipoprotein marker used in assessing the risk of coronary heart disease (CHD) development. Despite its continued use at the bedside, ApoB methodologies have not been thoroughly compared and may differentially discriminate CHD risk, resulting in patient misclassification. OBJECTIVE: This study compared 3 ApoB immunoassays and their associations with incident CHD risk over a 12-year follow-up period in the Multi-Ethnic Study of Atherosclerosis. METHODS: Plasma ApoB concentrations were measured in 4679 participants of Multi-Ethnic Study of Atherosclerosis at baseline, using 3 immunoturbidimetric methods. Roche and Kamiya reagent-based methods were analyzed on a Roche modular P analyzer, and the Diazyme reagent-based method was analyzed on a Siemens Dimension analyzer. Cox proportional analysis estimated ApoB-related risk of incident CHD over a median follow-up period of 12.5 years with adjustments for nonlipid CHD risk factors. ApoB concentrations were examined as continuous variables but were also dichotomized based on clinical designations of borderline (100 mg/dL), high (120 mg/dL), and very high ApoB levels (140 mg/dL). RESULTS: Moderate to strong correlations among ApoB methods were observed (r = 0.79-0.98). ApoB concentrations (per standard deviation) were similarly associated with CHD risk and hazard ratio (95% confidence interval): Roche: 1.16 (1.03-1.30); Kamiya: 1.14 (1.02-1.28); and Diazyme: 1.14 (1.02-1.28). CONCLUSION: Although all 3 ApoB were similarly associated with risk of incident CHD over the study period regardless of the reagent type, the bias between methods suggests that these reagents are not fungible, and assay harmonization may be warranted.

Light-induced changes in protein nitration in photoreceptor rod outer segments.

PURPOSE: Light has been shown to modulate protein nitration in rat retinas. To better understand the role of protein nitration in photoreceptor cell death induced by intense light, we examined retinal protein nitration and identified target proteins in rod outer segments (ROS). METHODS: Cyclic light-reared rats, treated or not with the antioxidant, dimethylthiourea (DMTU), were exposed to intense green light for 8 h. A subset of these rats was kept in the dark for 24 h after 8 h of light exposure. Western analysis of ROS proteins with an anti-nitrotyrosine antibody was performed to examine changes in protein nitration. 2D-immunoblots with anti-nitrotyrosine antibody followed by liquid chromatography tandem mass spectrometry was used to identify nitrated proteins in ROS. The expression levels of three nitric oxide synthase (NOS) isoforms, inducible, neuronal-, and endothelial-NOS were semi-quantified by immunoblot analysis. RESULTS: Western analysis revealed that the level of ROS protein nitration increased during the dark recovery period after 8 h of light treatment in both DMTU treated and untreated rats. However, DMTU effectively reduced protein nitration in ROS during light exposure and during the subsequent dark recovery period. Using 2D-immunoblotting followed by liquid chromatography tandem mass spectrometry analysis, we identified ten ROS proteins as nitration targets. Most of these proteins were glycolytic enzymes. The level of inducible-NOS in the retina was increased by light exposure. CONCLUSIONS: The effect of DMTU in reducing ROS protein nitration during and after light suggests the involvement of protein nitration during light-induced photoreceptor cell death. Nitration of glycolytic enzymes specifically may alter their activities. Increased levels of iNOS during and after intense light exposure suggest that this isoform is responsible for intense light induced protein nitration in ROS during the dark recovery period. The limited nitration seen in ROS during light exposure may reflect a quenching effect by endogenous antioxidants on the generation of reactive oxygen and nitrogen species.

Diagnostic performance of four point of care cardiac troponin I assays to rule in and rule out acute myocardial infarction.

OBJECTIVE: This study evaluated the diagnostic performance of four point-of-care (POC) cardiac troponin I (cTnI) assays compared to a central laboratory cTnI assay for detecting myocardial injury and diagnosing acute myocardial infarction (AMI). DESIGN AND METHODS: Plasma obtained at admission, 3 h, and 6 h post-admission in 169 patients presenting with symptoms suggestive of acute coronary syndrome (ACS) was studied. cTnI concentrations were measured on the Instrumentation Laboratory prototype GEM Immuno, Radiometer AQT90, Mitsubishi PATHFAST, Abbott i-STAT and the Ortho-Clinical Diagnostic Vitros assays. MI was determined based on 99th percentiles according to Universal MI guidelines. RESULTS: For ruling in MI at presentation (0 h), the GEM Immuno (sensitivity 63%, specificity 85%) and PATHFAST (sensitivity 53%, specificity 86%) were comparable to the OCD (sensitivity 68%, specificity 81%), and significantly better (p<0.05) than the AQT90 (sensitivity 26%, specificity 93%) and i-STAT (sensitivity 32%, specificity 92%). cTnI concentrations and serial rising patterns after MI differed by each assay. Negative predictive values were >90% and ROC AUCs were >0.90 after 6h for all assays. Detection of myocardial injury in non-ischemic pathologies accounted for lower than 100% specificity for MI. CONCLUSION: cTnI is a sensitive biomarker for detection of myocardial injury. The analytical variability that exists between POC cTnI assays demonstrates substantial diagnostic differences for ruling in and ruling out MI in patients presenting with symptoms suggestive of ACS.

Superiority of postmortem liver fentanyl concentrations over peripheral blood influenced by postmortem interval for determination of fentanyl toxicity.

OBJECTIVE: The current study was undertaken to determine the relationship between postmortem (PM) peripheral blood (PB) and liver fentanyl concentrations and the role of measuring liver fentanyl concentrations in cause of death investigations in medical examiner cases in which fentanyl was identified. DESIGN AND METHODS: FB and liver tissue were routinely collected at autopsy from 4 Minnesota medical examiners' offices in 2010-2011. Samples were analyzed by gas chromatography-mass spectrometry (GC-MS). RESULTS: PB fentanyl ranged from <2-15µg/L in non-drug related deaths (n=5), <2-22µg/L from mixed drug toxicity (n=26) and 3.7-56µg/L from fentanyl toxicity (n=33). Liver fentanyl ranged from 11 to 104µg/kg, 6 to 235µg/kg, and 18 to 365µg/kg, respectively. PB and liver fentanyl showed a modest correlation (r=0.67). PM interval to the liver/blood ratio showed a decreasing ratio over increasing PM interval in cases from fentanyl and mixed drug toxicity. Liver fentanyl concentrations best define therapeutic use at <23µg/kg and fatal toxicity at >56µg/kg, without substantial overlap as found in blood fentanyl concentrations. CONCLUSION: Discriminatory liver fentanyl concentrations suggestive of therapeutic or toxic drug levels may better assist cause of death determination in cases of suspected fentanyl toxicity than postmortem PB concentrations. Peripheral blood fentanyl concentrations appear to undergo postmortem redistribution, associated with an increasing PM interval.

Mechanism of glyceraldehyde-3-phosphate dehydrogenase inactivation by tyrosine nitration.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifaceted protein that is involved in numerous processes including glycolysis, translational silencing, transcriptional regulation of specific genes, and acting as a nitric oxide sensor. The precise mechanism on how GAPDH is targeted to these different roles is unclear but believed to involve specific posttranslational modification to the protein. Numerous studies have demonstrated that GAPDH is a target for tyrosine nitration. However, the site of modification and the molecular consequence have not been defined. Rabbit GAPDH with a reversibly protected catalytic cysteine was nitrated in vitro with tetranitromethane, resulting in complete loss of GAPDH catalytic activity. Nitration was estimated as 0.32 mol of nitrotyrosine residue per mole of GAPDH. Mass spectrometry analysis of nitrated GAPDH indicated that Tyr311 and Tyr317 were the sole sites of nitration. The X-ray crystal structure revealed that the distances between Tyr311 and Tyr317 and the cofactor nicotinamide adenine dinucleotide (NAD(+)) were less than 7.2 and 3.7 A, respectively, implying that nitration of these two residues may affect NAD(+) binding. This possibility was assessed using an NAD(+) binding assay, which showed that nitrated GAPDH was incapable of binding NAD(+). Thus, these results strongly suggest that Tyr311 and Tyr317 nitration prohibits NAD(+) binding, leading to the loss of catalytic activity.

Peptidyl-Lys metalloendopeptidase-catalyzed 18O labeling for comparative proteomics: application to cytokine/lipolysaccharide-treated human retinal pigment epithelium cell line.

We recently proposed a comparative proteomic method utilizing proteolytic 18O labeling of peptides catalyzed by peptidyl-Lys metalloendopeptidase (Lys-N) (Rao, K. C. S., Carruth, R. T., and Miyagi, M. (2005) Proteolytic 18O labeling by peptidyl-Lys metalloendopeptidase for comparative proteomics. J. Proteome Res. 4, 507-514). Unlike trypsin, which generates a mixture of isotopic isoforms resulting from the incorporation of one or two 18O atoms into each peptide species, Lys-N incorporates only a single 18O atom into the carboxyl terminus of each proteolytically generated peptide in H2(18)O solvent. This study reports the first biological application of the Lys-N-based proteolytic 18O labeling method, characterizing the proteome changes of cytokine/lipopolysaccharide-treated verses untreated human retinal pigment epithelium (ARPE-19) cells. The study resulted not only in the identification of 584 proteins but also the determination of the relative abundances of 562 proteins in the two proteomes. The results demonstrate the usefulness of the Lys-N-based proteolytic 18O labeling method in comparative proteomic studies. The results also provide the most comprehensive description of the retinal pigment epithelium proteome to date.