Collectively, the data indicate that mtDNA is preferentially damaged with AMD progression. These results suggest a potential link between mt dysfunction due to increased mtDNA lesions and AMD.

The results are consistent with the hypothesis that mitochondrial dysfunction is associated with AMD and further suggest specific pathophysiological mechanisms involving altered mitochondrial translation, import of nuclear-encoded proteins, and ATP synthase activity.

PURPOSE-To examine changes of select reduction-oxidation (redox) sensitive proteins from human donor retinal pigment epithelium (RPE) at four stages of age-related macular degeneration (AMD). DESIGN-Experimental study.METHODS-Human donor eyes were obtained from the Minnesota Lions Eye Bank and graded using the Minnesota Grading System (MGS) into four stages that correspond to stages defined by the age-related eye disease study (AREDS). Protein content in RPE homogenates was measured using Western immunoblotting with protein-specific antibodies.RESULTS-The content of several antioxidant enzymes and specific proteins that facilitate refolding or degradation of oxidatively damaged proteins increased significantly in MGS stage 3. These proteins are involved in the primary (copper-zinc superoxide dismutase [CuZnSOD], manganese superoxide dismutase [MnSOD], and catalase) and secondary (heat shock protein [HSP] 27, HSP 90, and proteasome) defense against oxidative damage. Additionally, the insulin prosurvival receptor exhibited disease-related upregulation.CONCLUSIONS-The pattern of protein changes identified in human donor tissue graded using the MGS support the role of oxidative mechanisms in the pathogenesis and progression of AMD. The MGS uses nearly identical clinical definitions and grading criteria of AMD that are used in the AREDS, so our results apply to clinical and epidemiologic studies using similar definitions. Results from our protein analysis of human donor tissue helps to explain altered oxidative stress regulation and cell-survival pathways that occur in progressive stages of AMD.Age-related macular degeneration (amd) is the leading cause of blindness in developed countries 1-9 . The number of affected individuals in the United States alone is expected to increase nearly twofold, to approximately three million by the year 2020. 10 Fortunately, therapeutic options are improving. The use of antioxidant vitamins has been shown to delay disease progression at an intermediate stage, 11 and rapid innovation in the use of antiangiogenic therapies has resulted in new clinical methods to treat the exudative phase of AMD. 12-18 However, developing new treatment and prevention strategies targeting earlier stages of the disease requires a better understanding of the underlying disease mechanisms.Findings from the Age-Related Eye Disease Study (AREDS) clearly support the hypothesis that oxidative mechanisms play a significant role in the progression of AMD. Although several studies have shown that the intake of antioxidant-rich foods lowers the risk of AMD, 19-24 others have not supported this conclusion. 25-27 Cigarette smoking, a pro-oxidant, NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript significantly increases the risk of AMD, and this association is well supported in numerous, well-designed studies. 28-35The retinal pigment epithelium (RPE) is subject to a particularly high level of oxidative stress because of locally elevated oxygen tension, high polyunsaturated lipid con...

These results provide the first direct evidence of AMD stage-specific changes in human RPE protein expression and provide a basis for functional investigation of AMD that may ultimately suggest new therapeutic strategies.

Aims/hypothesis Diabetic retinopathy is the most common complication of diabetes and a leading cause of blindness among working-age adults. Anatomical and functional changes occur in the retina and retinal pigment epithelium (RPE) prior to clinical symptoms of the disease. However, the molecular mechanisms responsible for these early changes, particularly in the RPE, remain unclear. To begin defining the molecular changes associated with pre-retinopathic diabetes, we conducted a comparative proteomics study of human donor RPE. Methods The RPE was dissected from diabetic human donor eyes with no clinically apparent diabetic retinopathy (n=6) and from eyes of age-matched control donors (n= 17). Soluble proteins were separated based upon their mass and charge using two-dimensional (2-D) gel electrophoresis. Protein spots were visualised with a fluorescent dye and spot densities were compared between diabetic and control gels. Proteins from spots with significant disease-related changes in density were identified using mass spectrometry.Results Analysis of 325 spots on 2-D gels identified 31 spots that were either up-or downregulated relative to those from age-matched control donors. The protein identity of 18 spots was determined by mass spectrometry. A majority of altered proteins belonged to two major functional groups, metabolism and chaperones, while other affected categories included protein degradation, synthesis and transport, oxidoreductases, cytoskeletal structure and retinoid metabolism. Conclusions/interpretation Changes identified in the RPE proteome of pre-retinopathic diabetic donor eyes compared with age-matched controls suggest specific cellular alterations that may contribute to diabetic retinopathy. Defining the pre-retinopathic changes affecting the RPE could provide important insight into the molecular events that lead to this disease.

CTC-binding factor (CTCF) is a DNA-binding protein of verte-brates that plays essential roles in regulating genome activity through its capacity to act as an enhancer blocker. We performed a yeast two-hybrid screen to identify protein partners of CTCF that could regulate its activity. Using full-length CTCF as bait we recovered Kaiso, a POZ-zinc finger transcription factor, as a specific binding partner. The interaction occurs through a C-terminal region of CTCF and the POZ domain of Kaiso. CTCF and Kaiso are co-expressed in many tissues, and CTCF was specifically co-immunoprecipitated by several Kaiso monoclonal antibodies from nuclear lysates. Kaiso is a bimodal transcription factor that recognizes methylated CpG dinucleotides or a conserved unmethylated sequence (TNGCAGGA, the Kaiso binding site). We identified one consensus unmethylated Kaiso binding site in close proximity to the CTCF binding site in the human 5 ␤-globin insulator. We found, in an insulation assay, that the presence of this Kaiso binding site reduced the enhancer-blocking activity of CTCF. These data suggest that the Kaiso-CTCF interaction negatively regulates CTCF insulator activity.

The presence of volatile organic compounds (VOCs) in unprocessed natural gas (NG) is well documented; however, the degree to which VOCs are present in NG at the point of end use is largely uncharacterized. We collected 234 whole NG samples across 69 unique residential locations across the Greater Boston metropolitan area, Massachusetts. NG samples were measured for methane (CH 4 ), ethane (C 2 H 6 ), and nonmethane VOC (NMVOC) content (including tentatively identified compounds) using commercially available USEPA analytical methods. Results revealed 296 unique NMVOC constituents in end use NG, of which 21 (or approximately 7%) were designated as hazardous air pollutants. Benzene (bootstrapped mean = 164 ppbv; SD = 16; 95% CI: 134–196) was detected in 95% of samples along with hexane (98% detection), toluene (94%), heptane (94%), and cyclohexane (89%), contributing to a mean total concentration of NMVOCs in distribution-grade NG of 6.0 ppmv (95% CI: 5.5–6.6). While total VOCs exhibited significant spatial variability, over twice as much temporal variability was observed, with a wintertime NG benzene concentration nearly eight-fold greater than summertime. By using previous NG leakage data, we estimated that 120–356 kg/yr of annual NG benzene emissions throughout Greater Boston are not currently accounted for in emissions inventories, along with an unaccounted-for indoor portion. NG-odorant content ( tert -butyl mercaptan and isopropyl mercaptan) was used to estimate that a mean NG-CH 4 concentration of 21.3 ppmv (95% CI: 16.7–25.9) could persist undetected in ambient air given known odor detection thresholds. This implies that indoor NG leakage may be an underappreciated source of both CH 4 and associated VOCs.

Use of the CDSS was associated with more appropriate AM use. To achieve greater improvements, strategies are needed to improve provider diagnoses of syndromes that are infectious or possibly infectious.