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The anti-inflammatory drug, PMX205, is an antagonist of the C5a complement receptor and has been shown to be effective in rodent models of amyotrophic lateral sclerosis and Alzheimer's disease. This cyclic hexapeptide (c[Arg-Trp-D-Cha-Pro-Orn]-Hca) has been reported to produce relatively low yields for both the linear peptide assembly and the cyclization reaction in solution and solid phase syntheses. During attempts to reproduce the solid phase methodology, a catastrophic loss of substitution was encountered which could be avoided or reduced by the use of 2-chlorotrityl resin. Likewise, the cyclization reaction could be significantly improved by the use of FDPP (pentafluorophenyl diphenylphosphinate) at high dilution (up to 80% purified yield). Both improvements are accomplished with commercially available products.

Purpose: To evaluate efficacy, safety, and causes of vision loss among 813 patients (1,392 eyes) with moderately severe to very severe nonproliferative diabetic retinopathy from the Protein Kinase C β Inhibitor-Diabetic Retinopathy Study and Protein Kinase C β Inhibitor-Diabetic Retinopathy Study 2 ruboxistaurin (RBX) protein kinase C β inhibitor trials., Methods: Patients in these 3-year, randomized, placebo-controlled, double-masked, Phase 3 trials had best-corrected Early Treatment Diabetic Retinopathy Study visual acuity ≥45 letters (∼20/125 Snellen), Early Treatment Diabetic Retinopathy Study retinopathy level 47A/B-53E, and no previous panretinal photocoagulation in ≥1 eye. Patients received placebo (N = 401) or RBX 32 mg/day (N = 412). Data from the 2 studies were combined and masked evaluation of retinal photographs was performed for cause of visual decline in all patients experiencing sustained moderate visual loss (≥15-letter loss sustained for the last 6 months of study)., Results: In the studies combined, sustained moderate visual loss occurred in 10.2% of placebo-treated patients versus 6.1% of RBX-treated patients (P = 0.011). A ≥15-letter gain occurred in 2.4% of placebo versus 4.7% of RBX eyes (P = 0.021) and a ≥15-letter loss occurred in 11.4% versus 7.4%, respectively (P = 0.012). Diabetic macular edema was the probable primary cause of vision loss. Among eyes without focal/grid photocoagulation at baseline, fewer RBX group eyes (26.7%) required initial focal/grid photocoagulation versus placebo (35.6%; P = 0.008). No safety concerns were identified., Conclusion: Analysis of data combined from two similar studies adds further statistical significance to RBX's beneficial effects on visual loss, need for focal laser, and vision gain, most likely through effects on macular edema.

Purpose: To examine the grading (interrater) reliability of the Age-Related Eye Disease Study (AREDS) Clinical Lens Grading System (ARLNS)., Design: Evaluation of diagnostic test or technology., Participants: One hundred fifty volunteers (284 eyes)., Methods: Participants with lens opacities of varying severity were independently graded at the slit lamp for cataract severity by 2 examiners (retinal or anterior segment specialists) using the ARLNS, which employs 3 standard photographs of increasing severity for classifying each of the 3 major types of opacity. Lens photographs were taken and graded at a reading center using the more detailed AREDS System for Classifying Cataracts from photographs., Main Outcome Measures: The Pearson correlation, weighted-kappa, and limits-of-agreement statistics were used to assess the interrater agreement of the gradings., Results: Examinations were performed on 284 lenses (150 participants). Tests of interrater reliability between pairs of clinicians showed substantial agreement between clinicians for cortical and posterior subcapsular opacities and moderate agreement for nuclear opacities. A similar pattern and strength of agreement was present when comparing scores of retinal versus anterior segment specialists. Interrater agreement between clinical and reading center gradings was not as great as inter-clinician agreement., Conclusions: Interrater agreements were in the moderate to substantial range for the clinical assessment of lens opacities. Inherent differences in cataract classification systems that rely on slit lamp vs photographic assessments of lens opacities may explain some of the disagreement noted between slit lamp and photographic gradings. Given the interrater reliability statistics for clinicians and the simplicity of the grading procedure, ARLNS is presented for use in studies requiring a simple, inexpensive method for detecting the presence and severity of the major types of lens opacities., Financial Disclosure(s): The authors have no proprietary or commercial interest in any of the materials discussed in this article., (Copyright © 2010 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Background: Recent information suggests that the Age-Related Eye Disease Study (AREDS) supplement, enhanced intake of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and reducing dietary glycaemic index (dGI) are protective against advanced age-related macular degeneration (AMD)., Methods: Dietary information was collected at baseline, and fundus photograph grades were obtained during the 8-year trial period from 2924 eligible AREDS AMD trial participants. Using the eye as the unit of analysis and multifailure Cox proportional-hazards regression, the risk of AMD progression was related to dietary intake in the four arms of the trial., Results: Independent of AREDS supplementation, higher intakes of DHA (> or =64.0 vs <26.0 mg/day) (hazard ratio (HR) = 0.73, 95% confidence interval (CI), 0.57 to 0.94), EPA (> or =42.3 vs <12.7 mg/day) (HR = 0.74, 95% CI 0.59 to 0.94), and lower dGI (dGI, <75.2 vs > or =81.5) (HR = 0.76, 95% CI 0.60 to 0.96) were associated with a lower risk for progression to advanced AMD. Participants consuming a lower dGI and higher DHA or EPA had the lowest risk (p value for synergistic interaction <0.001). Only participants in the "placebo" (p value for antagonistic interaction = 0.006) benefited from a higher DHA intake against early AMD progression (HR = 0.58, 95% CI 0.37 to 0.92; P(trend) = 0.01)., Conclusions: The findings show an association of consuming a diet rich in DHA with a lower progression of early AMD. In addition to the AREDS supplement, a lower dGI with higher intakes of DHA and EPA was associated with a reduced progression to advanced AMD., Trial Registration Number: NCT00000145.

Purpose: Because foods provide many nutrients that may interact to modify risk for multifactorial diseases such as age-related macular degeneration (AMD), we sought to develop a composite scoring system to summarize the combined effect of multiple dietary nutrients on AMD risk. This has not been done previously., Design: Cross-sectional study., Participants: From the 4003 participants in the Age-Related Eye Disease Study (AREDS), there were 7,934 eyes included in this study., Methods: Considering dietary intakes of vitamins C and E, zinc, lutein/zeaxanthin, docosahexaenoic acid, eicosapentaenoic acid, and low-dietary glycemic index (dGI) from AREDS baseline information, we assigned each nutrient a percentile rank score then summed them into a compound score for each participant. Using eye as the unit of analysis, we evaluated the association between the compound score and risk of prevalent AMD. Validation, fitness, and performance of the model were evaluated using bootstrapping techniques, adjusted quasi-likelihood under the independence model criterion, and the c-index, respectively., Main Outcome Measures: Stereoscopic fundus photographs of the macula were taken and graded at baseline using the AREDS protocol and AMD Classification System., Results: Our results showed that higher compound scores were associated with lower risk for early AMD, indicated by drusen, and advanced AMD. Validation analyses indicated that these relationships are robust (the average 50-time bootstrapping per quartile odds ratios = 0.727, 0.827, and 0.753, respectively, for drusen, and 0.616, 0.536, and 0.572, respectively, for advanced AMD). Model selection analyses suggested that the compound score should be included, but that measures of dietary beta-carotene should not be included., Conclusions: We found that consuming diets that provide low dGI and higher intakes of these nutrients were associated with the greatest reduction in risk for prevalent drusen and advanced AMD, whereas dietary beta-carotene did not affect these relationships. These findings warrant further prospective studies., Financial Disclosure(s): Proprietary or commercial disclosure may be found after the references.

Purpose: To assess the risk of advanced age-related macular degeneration (AMD) developing after cataract surgery., Design: Cohort study., Participants: Four thousand five hundred seventy-seven participants (8050 eyes) from a multicenter, controlled, randomized clinical trial, the Age-Related Eye Disease Study (AREDS)., Methods: Development of advanced AMD, either neovascular (NV) AMD or geographic atrophy (GA), was evaluated with annual fundus photographs, and history of cataract surgery was assessed every 6 months. Cox proportional hazard models with time-dependent covariates were conducted for NV AMD and GA separately., Main Outcome Measures: Neovascular AMD, GA, and central GA (CGA; involving the center of the macula)., Results: The Cox proportional hazards model of right eyes showed nonsignificant hazard ratios of 1.20 (95% confidence interval [CI], 0.82-1.75) for NV AMD, 0.80 (95% CI, 0.61-1.06) for GA, and 0.87 (95% CI, 0.64-1.18) for CGA. Similar results were obtained for left eyes: 1.07 (95% CI, 0.72-1.58) for NV AMD, 0.94 (95% CI, 0.71-1.25) for GA, and 0.86 (95% CI, 0.63-1.19) for CGA. For participants with advanced AMD in 1 eye (AREDS category 4), the hazard ratios for fellow eyes were 1.08 (95% CI, 0.65-1.72) for NV AMD and 0.98 (95% CI, 0.64-1.49) for CGA., Conclusions: The AREDS results showed no clear effect of cataract surgery on the risk of progression to advanced AMD., Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

The use of multivitamin-mineral supplements has become increasingly common, but whether the use of such supplements improves micronutrient status remains still unclear. The objective of this report is to investigate how a long-term vitamin-mineral supplementation following the US Recommended Daily Intake (RDI) affected the plasma levels of selected nutrients in a subset (No. = 407) of participants in the Italian-American Clinical Trial of Nutritional Supplements and Age-related Cataract (CTNS). The CTNS was a double-blind, single centre, controlled clinical trial of 1020 participants aged 55-75 years randomized to a daily tablet of Centrum(R) or placebo. A representative sample of 40% of the 1020 subjects, whom plasma level of selected vitamins was determined at the baseline, was retested throughout the treatment period that averaged 9.0 +/- 2.4 years. Participants assigned to Centrum(R) showed a significant increase (p < 0.005) in mean/median plasma levels of vitamin E, beta-carotene, folate, and vitamin B12, and an improved riboflavin status when compared with participants assigned to placebo. Differences concerning vitamin C were statistically less relevant and those concerning vitamin A were at a borderline level. In the treated group the effect of supplementation on plasma levels of vitamins A, E, and C, and on the glutathione reductase activation coefficient was significantly higher in participants with lower nutritional status at baseline.

Purpose: To compare relationships between severity and duration of diabetic macular edema (DME) and visual acuity (VA) observed in the PKC-DRS2 with those from the Early Treatment Diabetic Retinopathy Study (ETDRS) and to assess the effect of the orally administered PKC beta inhibitor ruboxistaurin (RBX) on these parameters., Methods: In the PKC-DRS2, patients with moderately severe to very severe nonproliferative diabetic retinopathy (n = 685) were randomly assigned to 32 mg/d RBX or placebo and followed up for 36 months with ETDRS VA measurements and fundus photographs (FP) every 3 to 6 months. Mean VA was calculated across all FP visits for eyes in each level of the ETDRS DME severity scale at those visits. For eyes with baseline VA > or = 20/40, relationships between change in VA from baseline to last visit and duration of severe DME were analyzed with linear regression., Results: Mean VA decreased by approximately 22 letters between the mildest and most severe levels of the DME scale in the PKC-DRS2, compared with 27 letters in the ETDRS. In the placebo group, the rate of decrease in VA over time associated with duration of severe DME was 0.67 letters per month (24 letters over 36 months, compared with 20 letters over 28-36 months in the ETDRS). This rate was 30% less in the RBX group (0.47 letter per month, P = 0.022)., Conclusions: The VA decrease in the PKC-DRS2 associated with long-standing DME agrees well with estimates from the ETDRS. RBX appears to ameliorate this decrease, an effect that could be important clinically. (ClinicalTrials.gov number, NCT00604383.).

Purpose: In PKC-DRS2, the efficacy of the oral PKC-beta inhibitor, ruboxistaurin 32 mg/day, was measured by the primary end point of sustained moderate visual loss (SMVL: a > or = 15 letter decrease from baseline on the ETDRS (Early Treatment Diabetic Retinopathy Study) chart sustained at least for the last 6 months of study participation). We now evaluate whether SMVL is more accurate than moderate visual loss (MVL: a single occurrence of a decrease from baseline of > or = 15 ETDRS letters) for predicting future visual loss., Methods: Study eyes with moderately severe to very-severe non-proliferative diabetic retinopathy, best-corrected visual acuity of at least 45 letters on the ETDRS chart (approximately Snellen 20/125), and no prior pan retinal photocoagulation were evaluated in 506 patients (869 eyes) who completed 36 months of treatment., Results: Sixty-five percentage (26/40) of study eyes with the onset of SMVL within 24 months of enrolment still had SMVL at study completion (36 months). In comparison, only 24% (30/126) with MVL within 24 months had SMVL at study completion. Analyses based on data from 6, 12, and 18 months of treatment were similar., Conclusions: SMVL is a more predictable measure of subsequent visual loss than is a single time point measure of MVL.

Objective: To examine the association of dietary omega-3 long-chain polyunsaturated fatty acid and fish intake with incident neovascular age-related macular degeneration (AMD) and central geographic atrophy (CGA)., Methods: Multicenter clinic-based prospective cohort study from a clinical trial including Age-Related Eye Disease Study (AREDS) participants with bilateral drusen at enrollment. Main outcome measures were incident neovascular AMD and CGA, ascertained from annual stereoscopic color fundus photographs (median follow-up, 6.3 years). We estimated nutrient and food intake from a validated food frequency questionnaire (FFQ) at baseline, with intake of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), combined EPA and DHA, and fish as primary exposures., Results: After controlling for known covariates, we observed a reduced likelihood of progression from bilateral drusen to CGA among people who reported the highest levels of EPA (odds ratio [OR], 0.44; 95% confidence interval [CI], 0.23-0.87) and EPA+DHA (OR, 0.45; 95% CI, 0.23-0.90) consumption. Levels of DHA were associated with CGA in age-, sex-, and calorie-adjusted models (OR, 0.51; 95% CI, 0.26-1.00); however, this statistical relationship did not persist in multivariable models., Conclusions: Dietary lipid intake is a modifiable factor that may influence the likelihood of developing sight-threatening forms of AMD. Our findings suggest that dietary omega-3 long-chain polyunsaturated fatty acid intake is associated with a decreased risk of progression from bilateral drusen to CGA.

Objective: To evaluate the effect of a multivitamin/mineral supplement on development or progression of age-related lens opacities., Design: Randomized, double-masked, single center, placebo-controlled clinical trial., Participants: One thousand twenty participants, 55 to 75 years old and with early or no cataract, were randomly assigned to a daily tablet of a multivitamin/mineral formulation or a placebo., Methods: Baseline and annual lens photographs were graded for severity of lens opacities according to a modification of the Age-Related Eye Disease Study system for classifying cataracts., Main Outcome Measures: The primary outcome was a prespecified increase from baseline in nuclear, cortical, or posterior subcapsular cataract (PSC) opacity grades or cataract surgery. Secondary outcomes included an increase in type-specific opacity grades, cataract surgery, and visual acuity (VA) loss from baseline > or =15 letters., Results: Participants were observed for an average of 9.0+/-2.4 years. There was a decrease in total lens events in participants assigned to the multivitamin/mineral formulation compared with those assigned to the placebo (hazard ratio [HR], 0.82; 95% confidence interval [CI], 0.68-0.98; P = 0.03). Nuclear events were significantly less common (HR, 0.66; 95% CI, 0.50-0.88; P = 0.004) and PSC events significantly more common (HR, 2.00; 95% CI, 1.35-2.98; P<0.001) in participants taking the multivitamin/mineral formulation than in those assigned to the placebo. No statistically significant treatment effects were seen for cortical opacities, moderate VA loss, or cataract surgery., Conclusions: Lens events were less common in participants who took the multivitamin/mineral formulation, but treatment had opposite effects on the development or progression of nuclear and PSC opacities, the 2 most visually important opacity subtypes.

Background: Cross-sectional studies indicate that diets that provide a higher dietary glycemic index (dGI) are associated with a greater risk of age-related macular degeneration (AMD). No prospective studies have addressed this issue., Objective: The objective was to prospectively evaluate the effect of baseline dGI on the progression of AMD., Design: dGI was calculated as the weighted average of GIs from foods and was evaluated as being above or below the sex median (women: 77.9; men: 79.3) for 3977 participants aged 55-80 y (58% women) in the Age-Related Eye Disease Study. The 7232 eligible eyes without advanced AMD were classified into 1 of 3 AMD categories: group 1 (nonextensive small drusen), group 2 (intermediate drusen, extensive small drusen, or pigmentary abnormalities), or group 3 (large drusen or extensive intermediate drusen). With the use of multifailure Cox proportional-hazards regression, we modeled the time to the maximal progression to evaluate the relation between dGI and the risk of AMD., Results: Overall, the multivariate-adjusted risk of progression over 8 y of follow-up (x: 5.4 y) was significantly higher (risk ratio: 1.10; 95% CI: 1.00, 1.20; P = 0.047) in the high-dGI group than in the low-dGI group. The risk of progression for groups 1, 2, and 3 eyes was 5%, 8%, and 17% greater, respectively (P for trend < 0.001). The latter gives an estimate that 7.8% of new advanced AMD cases would be prevented in 5 y if people consumed the low-dGI diet., Conclusion: Persons at risk of AMD progression, especially those at high risk of advanced AMD, may benefit from consuming a smaller amount of refined carbohydrates.

Objective: To evaluate the relationship of dietary carotenoids, vitamin A, alpha-tocopherol, and vitamin C with prevalent age-related macular degeneration (AMD) in the Age-Related Eye Disease Study (AREDS)., Methods: Demographic, lifestyle, and medical characteristics were ascertained on 4519 AREDS participants aged 60 to 80 years at enrollment. Stereoscopic color fundus photographs were used to categorize participants into 4 AMD severity groups and a control group (participants with < 15 small drusen). Nutrient intake was estimated from a self-administered semiquantitative food frequency questionnaire at enrollment. Intake values were energy adjusted and classified by quintiles. The relationship between diet and AMD status was assessed using logistic regression analyses., Results: Dietary lutein/zeaxanthin intake was inversely associated with neovascular AMD (odds ratio [OR], 0.65; 95% confidence interval [CI], 0.45-0.93), geographic atrophy (OR, 0.45; 95% CI, 0.24-0.86), and large or extensive intermediate drusen (OR, 0.73; 95% CI, 0.56-0.96), comparing the highest vs lowest quintiles of intake, after adjustment for total energy intake and nonnutrient-based covariates. Other nutrients were not independently related to AMD., Conclusion: Higher dietary intake of lutein/zeaxanthin was independently associated with decreased likelihood of having neovascular AMD, geographic atrophy, and large or extensive intermediate drusen.

Background: Age-related macular degeneration (AMD) is the major cause of irreversible blindness. AMD appears to share several carbohydrate-related mechanisms and risk factors with diabetes-related diseases, including retinopathy and cardiovascular disease (CVD); however, to date, only one small study has addressed this issue., Objective: The objective was to test the hypothesis that dietary glycemic index (dGI), which has been related to the risk of diabetes and CVD, is associated with the risk and severity of AMD in nondiabetic elderly populations., Design: Dietary information was obtained from 4099 participants aged 55-80 y (56% women) in the Age-Related Eye Disease Study (AREDS). A total of 8125 eligible eyes at baseline were classified into 1 of 5 AMD groups according to the size and extent of drusen, the presence of geographic atrophy, and neovascular changes. We used a generalized estimating approach to evaluate the relations between dGI and risk and severity of AMD with eyes as the unit of analysis., Results: Compared with eyes in the first quintile of dGI, eyes in the fourth and fifth quintiles had a significantly or suggestively higher risk of large drusen, geographic atrophy, and neovascularization. The multivariate-adjusted odds ratios (95% CIs) for the highest quintile were 1.42 (1.09, 1.84), 1.78 (0.81, 3.90), and 1.41 (0.95, 2.08), respectively, of which only the odds ratio for large drusen was significant. A significant positive relation between dGI and severity of AMD was also noted (P for trend < 0.001). There was a 49% increase in the risk of advanced AMD (geographic atrophy plus neovascularization) for persons with a dGI higher than the sex median (women: >or=77.9; men: >or=79.3). This result indicated that 20% of prevalent cases of AMD would have been eliminated if the AREDS participants consumed diets with a dGI below the median., Conclusion: The association between dGI and AMD from the AREDS cross-sectional analysis at baseline suggests that a reduction in the dGI, a modifiable risk factor, may provide a means of diminishing the risk of AMD.

Objective: To evaluate the association of lipid intake with baseline severity of age-related macular degeneration (AMD) in the Age-Related Eye Disease Study (AREDS)., Methods: Age-Related Eye Disease Study participants aged 60 to 80 years at enrollment (N = 4519) provided estimates of habitual nutrient intake through a self-administered semiquantitative food frequency questionnaire. Stereoscopic color fundus photographs were used to categorize participants into 4 AMD severity groups and a control group (participants with <15 small drusen)., Results: Dietary total omega-3 long-chain polyunsaturated fatty acid (LCPUFA) intake was inversely associated with neovascular (NV) AMD (odds ratio [OR], 0.61; 95% confidence interval [CI], 0.41-0.90), as was docosahexaenoic acid, a retinal omega-3 LCPUFA (OR, 0.54; 95% CI, 0.36-0.80), comparing highest vs lowest quintile of intake, after adjustment for total energy intake and covariates. Higher fish consumption, both total and broiled/baked, was also inversely associated with NV AMD (OR, 0.61; 95% CI, 0.37-1.00 and OR, 0.65; 95% CI, 0.45-0.93, respectively). Dietary arachidonic acid was directly associated with NV AMD prevalence (OR, 1.54; 95% CI, 1.04-2.29). No statistically significant relationships existed for the other lipids or AMD groups., Conclusion: Higher intake of omega-3 LCPUFAs and fish was associated with decreased likelihood of having NV AMD.

Here, we provide methods for the creation of protein microarrays in microplates. The microplate consists of 96 wells with each well capable of holding a protein microarray at a spot density of up to 400 (20 x 20) individual elements. Arrays of capture monoclonal antibodies, corresponding to specific interleukins, were printed onto the bottom of the wells which had been surface activated for covalent attachment. A Biomek 2000 laboratory automation workstation (Beckman Coulter, Inc., Fullerton, CA) equipped with a high-density replicating tool was used for printing low density 3 x 3 to 5 x 5 arrays. For higher density arrays, a microarrayer system (Cartesian PS7200, Genomic Solutions, Inc., Ann Arbor, MI) was employed. Multiple antigens were simultaneously analyzed without detectable cross-reactivity associated with capture antibody or secondary antibody interactions. Detection of interleukin antigens, spiked into cell culture media containing 10% fetal calf serum, was specific and sensitive.

The ability to perform microarray-based immunoassays without the need for wells or other fluid barriers were demonstrated. Both contact and noncontact microarray printing technology is used to prepare spotted arrays of analyte binding sites, as well as, to deliver samples, secondary antibodies and other signal development reagents directly to these sites in a parallel fashion are called as overprint immunoassays. A micro-ELISA is demonstrated based upon the use of Protein A as a universal microarray. All components of the assay (capture antibody, antigen, and signal development reagents) were site-specifically dispensed in parallel fashion to the surface in nanoliter volumes. This represents a 1000-fold reduction in reagent consumption from that used in a conventional 96-well microtiter plate assay. Overprinting nanoliter volumes directly onto 200-300 microm spots yields similar levels of sensitivity achieved with the bulk dispensing of microliter volumes into wells.

Objective: To evaluate the effect of ruboxistaurin, an orally administered protein kinase C beta (PKC beta) isozyme-selective inhibitor, on vision loss in patients with diabetes., Design: Thirty-six-month, randomized, double-masked, placebo-controlled, parallel, multicenter trial., Participants: Six hundred eighty-five patients randomized at 70 clinical sites., Methods: Ophthalmologic examination was performed at screening and at each 3-month visit. Retinopathy status was assessed every 6 months with Early Treatment Diabetic Retinopathy Study (ETDRS) standard 7-field 30 degrees color stereoscopic fundus photography. Levels of diabetic retinopathy and diabetic macular edema were determined by 2 independent graders masked to site and treatment assignment, with additional independent adjudication as required. Eligible patients had a best-corrected visual acuity (VA) score of > or =45 letters, retinopathy level > or = 47A and < or = 53E, and no prior panretinal photocoagulation in at least one eye., Main Outcome Measure: Effect of oral ruboxistaurin (32 mg/day) on reduction of sustained moderate visual loss (> or =15-letter decrease in ETDRS VA score maintained > or = 6 months) in patients with moderately severe to very severe nonproliferative diabetic retinopathy., Results: Sustained moderate visual loss occurred in 9.1% of placebo-treated patients versus 5.5% of ruboxistaurin-treated patients (40% risk reduction, P = 0.034). Mean VA was better in the ruboxistaurin-treated patients after 12 months. Baseline-to-end point visual improvement of > or =15 letters was more frequent (4.9% vs. 2.4%) and > or =15-letter worsening was less frequent (6.7% vs. 9.9%) in ruboxistaurin-treated patients relative to placebo (P = 0.005). When clinically significant macular edema was >100 microm from the center of the macula at baseline, ruboxistaurin treatment was associated with less frequent progression of edema to within 100 microm (68% vs. 50%, P = 0.003). Initial laser treatment for macular edema was 26% less frequent in eyes of ruboxistaurin-treated patients (P = 0.008)., Conclusion: Oral ruboxistaurin treatment reduced vision loss, need for laser treatment, and macular edema progression, while increasing occurrence of visual improvement in patients with nonproliferative retinopathy.

Purpose: To evaluate the effect of the multivitamin Centrum on the development and progression of age-related lens opacities., Design: Clinic-based prospective cohort study., Participants: Four thousand five hundred ninety individuals with at least one natural lens and photographic follow-up (median, 6.3 years) were assessed for development or progression of lens opacities., Main Outcome Measures: Progression of "any" lens opacity or type-specific opacity was ascertained from lens photographs taken at baseline and at annual visits beginning at year 2., Methods: The Age-Related Eye Disease Study (AREDS) showed no statistically significant effect of a high-dose antioxidant formulation on progression of lens opacities. Centrum also was provided to approximately two thirds of the study participants. Because Centrum use was elective, a logistic regression model of baseline characteristics was used to generate a propensity score for Centrum use. Repeated-measures logistic regression, adjusted for propensity score and other covariates, was used to evaluate associations of Centrum use and lens opacity., Results: Centrum use, adjusted for propensity score and other covariates, was associated with a reduction in "any" lens opacity progression (odds ratio [OR] = 0.84, 95% confidence interval [CI] = 0.72-0.98, P = 0.025). Results for individual lens opacity types suggested that Centrum use was protective for nuclear opacity events (OR = 0.75, 95% CI = 0.61-0.91, P = 0.004)., Conclusion: Observational data from the AREDS and other studies suggest that use of a multivitamin may delay the progression of lens opacities. A National Eye Institute-sponsored clinical trial scheduled for completion in 2007 will provide additional data on Centrum use and cataract development.

Background: Little is known about the association between dietary carbohydrates and cataract in nondiabetic persons., Objective: The aim was to test whether recent dietary carbohydrate intakes or glycemic index (GI; a measure of carbohydrate intake quality) was associated with the presence of cortical or nuclear opacities., Design: A modified Block food-frequency questionnaire was used to obtain dietary information from 3377 participants (aged 60-80 y; 56% were women) in the Age-Related Eye Disease Study (AREDS). Lens status was evaluated by using the AREDS System for Classifying Cataracts. Associations were examined for eyes with only a single, or pure, type of lens opacity by using the generalized estimating approach to logistic regression to account for the lack of independence between the eyes of a person., Results: For participants in the highest quartile, dietary GI was associated with a higher prevalence of all pure nuclear opacities [grade >2; odds ratio (OR): 1.29; 95% CI: 1.04, 1.59; P for trend = 0.02] and moderate nuclear opacities (grade > or =4; OR: 1.43; 95% CI: 0.96, 2.14; P for trend = 0.052). The OR in a comparison of the highest with the lowest quartile of intake was 1.27 (95% CI: 0.99, 1.63; P for trend = 0.09) for cortical opacities of any severity (>0% of area opaque), and the OR increased somewhat for moderate cortical opacities (>5% of area opaque; OR: 1.71; 95% CI: 1.00, 2.95; P for trend = 0.056)., Conclusions: Results from the cross-sectional analysis of AREDS baseline data suggest that dietary glycemic quality and dietary carbohydrate quantity may be associated with prevalent nuclear and cortical opacities, respectively.

Objective: We investigated whether age-related macular degeneration risk factors are associated with high-sensitivity C-reactive protein (CRP) and homocysteine (HCY), systemic biomarkers for cardiovascular disease., Methods: Subjects with a range of age-related macular maculopathies or no maculopathy at two centers in the United States were evaluated. Risk factors and biomarkers were assessed by questionnaire, direct measurement, or analyses of blood specimens., Results: Higher levels of serum antioxidants vitamin C and lutein/zeaxanthin and higher fish intake were associated with lower serum CRP levels, whereas serum vitamin E, smoking, and increased body mass index were associated with increased CRP. Serum vitamin E, serum alpha-carotene, and dietary intake of antioxidants and vitamin B6 were associated with lower levels of plasma HCY, whereas hypertension was associated with increased HCY., Conclusions: C-reactive protein and HCY levels are related to traditional dietary and behavioral factors associated with age-related macular degeneration.

Purpose: To assess the relationship between plasma levels of homocysteine and age-related macular degeneration (AMD)., Design: Cross-sectional, case-control study., Methods: Fasting plasma homocysteine levels were measured at two centers in 934 individuals who were participating in an ancillary study of the Age-Related Eye Disease Study. There were 547 cases and 387 control subjects, who were determined by fundus photography. Conditional logistic regression analyses were conducted to assess the association of homocysteine with AMD., Results: Median values of homocysteine were higher among advanced AMD cases (9.51 mmol/l) compared with persons with no AMD (8.81 mmol/l; P = .01). Values of >12 mmol/l vs < or =12 mmol/l were also associated with an increased risk of AMD (P = .023), when controlled for other covariates., Conclusion: Results are consistent with a possible small, independent association between higher homocysteine levels and AMD. Homocysteine may be a modifiable risk factor for AMD.

Objective: To develop a simplified clinical scale defining risk categories for development of advanced age-related macular degeneration (AMD)., Methods: Following development of a detailed scale for individual eyes based on gradings of fundus photographs in the Age-Related Eye Disease Study, rates of progression to advanced AMD were assessed in cross-tabulations of presence or absence in each eye of 2 easily identified retinal abnormalities, drusen and pigment abnormalities. Large drusen and any pigment changes were particularly predictive of developing advanced AMD., Results: The scoring system developed for patients assigns to each eye 1 risk factor for the presence of 1 or more large (> or = 125 microm, width of a large vein at disc margin) drusen and 1 risk factor for the presence of any pigment abnormality. Risk factors are summed across both eyes, yielding a 5-step scale (0-4) on which the approximate 5-year risk of developing advanced AMD in at least one eye increases in this easily remembered sequence: 0 factors, 0.5%; 1 factor, 3%; 2 factors, 12%; 3 factors, 25%; and 4 factors, 50%. For persons with no large drusen, presence of intermediate drusen in both eyes is counted as 1 risk factor., Conclusion: This simplified scale provides convenient risk categories for development of advanced AMD that can be determined by clinical examination or by less demanding photographic procedures than used in the Age-Related Eye Disease Study.

Objective: To develop a fundus photographic severity scale for age-related macular degeneration (AMD)., Methods: In the Age-Related Eye Disease Study, stereoscopic color fundus photographs were taken at baseline, at the 2-year follow-up visit, and annually thereafter. Photographs were graded for drusen characteristics (size, type, area), pigmentary abnormalities (increased pigment, depigmentation, geographic atrophy), and presence of abnormalities characteristic of neovascular AMD (retinal pigment epithelial detachment, serous or hemorrhagic sensory retinal detachment, subretinal or sub-retinal pigment epithelial hemorrhage, subretinal fibrous tissue). Advanced AMD was defined as presence of 1 or more neovascular AMD abnormalities, photocoagulation for AMD, or geographic atrophy involving the center of the macula. We explored associations among right eyes of 3212 participants between severity of drusen characteristics and pigmentary abnormalities at baseline and development of advanced AMD within 5 years of follow-up., Results: A 9-step severity scale that combines a 6-step drusen area scale with a 5-step pigmentary abnormality scale was developed, on which the 5-year risk of advanced AMD increased progressively from less than 1% in step 1 to about 50% in step 9. Among the 334 eyes that had at least a 3-step progression on the scale between the baseline and 5-year visits, almost half showed stepwise progression through intervening severity levels at intervening visits. Replicate gradings showed agreement within 1 step on the scale in 87% of eyes., Conclusions: The scale provides convenient risk categories and has acceptable reproducibility. Progression along it may prove to be useful as a surrogate for progression to advanced AMD.

Purpose: To describe the association of demographic, behavioral, medical, and nonretinal ocular factors with the incidence of neovascular age-related macular degeneration (AMD) and central geographic atrophy (CGA) in the Age-Related Eye Disease Study (AREDS), a randomized trial of antioxidants and zinc supplementation prophylaxis for development of advanced AMD., Design: Clinic-based prospective cohort study., Participants: Of individuals with early or intermediate AMD at baseline with a median follow-up of 6.3 years, 788 were at risk of developing advanced AMD in one eye (the fellow eye had advanced AMD), and 2506 were at risk in both eyes., Methods: The incidence of neovascular AMD and CGA was assessed from stereoscopic color fundus photographs taken at baseline and at annual visits beginning at year 2., Main Outcome Measures: Neovascular AMD was defined as photocoagulation for choroidal neovascularization, or photographic documentation at the reading center of any of the following: nondrusenoid retinal pigment epithelial detachment, serous or hemorrhagic retinal detachment, hemorrhage under the retina or the retinal pigment epithelium, and subretinal fibrosis. Central geographic atrophy was defined as geographic atrophy involving the center of the macula., Results: In multivariable models, in persons at risk of advanced AMD in both eyes, while controlling for age, gender, and AREDS treatment group, the following variables were statistically significantly associated with the incidence of neovascular AMD: race (odds ratio [OR], white vs. black, 6.77; 95% confidence interval [CI], 1.24-36.9) and larger amount smoked (OR, >10 vs. < or =10 pack-years [a pack-year is an average of 1 pack of cigarette smoked per day for a year], 1.55; 95% CI, 1.15-2.09). The following were statistically significantly associated with the incidence of CGA: less education (OR, high school graduate or less vs. college graduate, 1.75; 95% CI, 1.10-2.78), greater body mass index (BMI) (OR, obese vs. nonobese, 1.93; 95% CI, 1.25-2.65), larger amount smoked (OR, >10 pack-years vs. < or =10 pack-years, 1.82; 95% CI, 1.25-2.65), and antacid use (OR, 0.29; 95% CI, 0.09-0.91). In persons at risk of developing advanced AMD in one eye, the incidence of neovascular AMD was associated with diabetes (OR, 1.88; 95% CI, 1.07-3.31), and the incidence of CGA was associated with use of antiinflammatory medications (OR, 0.22; 95% CI, 0.08-0.59)., Conclusions: Results suggest that, among persons with early or intermediate AMD, smoking and BMI are modifiable factors associated with progression to advanced AMD, and suggest other associations (e.g., use of antacids and antiinflammatory medications) that warrant further study. This article contains additional online-only material available at http://www.ophsource.org/periodicals/ophtha. .

Context: C-reactive protein (CRP) is a systemic inflammatory marker associated with risk for cardiovascular disease (CVD). Some risk factors for CVD are associated with age-related macular degeneration (AMD), but the association between CRP and AMD is unknown., Objective: To test the hypothesis that elevated CRP levels are associated with an increased risk for AMD., Design, Setting, and Participants: A total of 930 (91%) of 1026 participants at 2 centers in the Age-Related Eye Disease Study (AREDS), a multicenter randomized trial of antioxidant vitamins and minerals, were enrolled in this case-control study. There were 183 individuals without any maculopathy, 200 with mild maculopathy, 325 with intermediate disease, and 222 with advanced AMD (geographic atrophy or neovascular AMD). The AMD status was assessed by standardized grading of fundus photographs, and stored fasting blood specimens drawn between January 1996 and April 1997 were analyzed for high-sensitivity CRP levels., Main Outcome Measure: Association between CRP and AMD., Results: The CRP levels were significantly higher among participants with advanced AMD (case patients) than among those with no AMD (controls; median values, 3.4 vs 2.7 mg/L; P =.02). After adjustment for age, sex, and other variables, including smoking and body mass index, CRP levels were significantly associated with the presence of intermediate and advanced stages of AMD. The odds ratio (OR) for the highest vs the lowest quartile of CRP was 1.65 (95% confidence interval [CI], 1.07-2.55; P for trend =.02). The OR for CRP values at or above the 90th percentile (10.6 mg/L) was 1.92 (95% CI, 1.20-3.06), and the OR for CRP values at or above the mean plus 2 SDs (16.8 mg/L) was 2.03 (95% CI, 1.03-4.00). A trend for an increased risk for intermediate and advanced AMD with higher levels of CRP was seen for smokers (OR, 2.16; 95% CI, 1.33-3.49) and those who never smoked (OR, 2.03; 95% CI, 1.19-3.46) with the highest level of CRP., Conclusion: Our results suggest that elevated CRP level is an independent risk factor for AMD and may implicate the role of inflammation in the pathogenesis of AMD.

Objective: To estimate the potential public health impact of the findings of the Age-Related Eye Disease Study (AREDS) on reducing the number of persons developing advanced age-related macular degeneration (AMD) during the next 5 years in the United States., Methods: The AREDS clinical trial provides estimates of AMD progression rates and of reduction in risk of developing advanced AMD when a high-dose nutritional supplement of antioxidants and zinc is used. These results are applied to estimates of the US population at risk, to estimate the number of people who would potentially avoid advanced AMD during 5 years if those at risk were to take a supplement such as that used in AREDS., Results: An estimated 8 million persons at least 55 years old in the United States have monocular or binocular intermediate AMD or monocular advanced AMD. They are considered to be at high risk for advanced AMD and are those for whom the AREDS formulation should be considered. Of these people, 1.3 million would develop advanced AMD if no treatment were given to reduce their risk. If all of these people at risk received supplements such as those used in AREDS, more than 300,000 (95% confidence interval, 158,000-487,000) of them would avoid advanced AMD and any associated vision loss during the next 5 years., Conclusion: If people at high risk for advanced AMD received supplements such as those suggested by AREDS results, the potential impact on public health in the United States would be considerable during the next 5 years.

Objective: To examine the association between cigarette smoking and the incidence of nuclear and non-nuclear lens opacities in members of the Framingham Eye Study Cohort., Participants and Methods: Eye examinations were conducted on surviving members of the Framingham Heart Study Cohort from 1973 to 1975 (Framingham Eye Study I) and again from 1986 to 1989 (Framingham Eye Study II). Smoking data, collected biennially since 1948 in the Heart Study, were used to examine the relationship between cigarette smoking and the incidence of lens opacities. Two thousand six hundred seventy-five persons were examined in the Framingham Eye Study I. Our analysis included 660 persons, aged 52 to 80 years, who were free of lens opacities at the first eye examination., Results: During the approximately 12.5 years between eye examinations, lens opacities developed in a total of 381 persons, with nuclear opacities constituting the most frequent type. In logistic regression analyses that controlled for age, sex, education, and diabetes, a significant positive association with increasing duration of smoking and number of cigarettes smoked daily was found for nuclear lens opacities, alone or in combination (test for trend, P < or = .002), but not for nonnuclear opacities (test for trend, P = .62). Among the heavier smokers (persons who smoked > or = 20 cigarettes per day according to 6 or more biennial Framingham Heart Study examinations), 77% were still smoking at the time of the first eye examination. Persons who smoked 20 or more cigarettes per day at the time of the first eye examination were at substantially increased risk for the development of nuclear opacities than nonsmokers (odds ratio, 2.84; 95% confidence interval, 1.46-5.51). There was no apparent excess risk for persons with nonnuclear lens opacities (odds ratio, 1.42; 95% confidence interval, 0.65-3.07)., Conclusion: This study provides further evidence that cigarette smokers have an increased risk of developing nuclear lens opacities. The risk was greatest for heavier smokers, who tended to be current smokers and who smoked more cigarettes and for a longer duration.

Purpose: To examine the distribution of cortical opacities across the lens in the Italian-American Natural History Study of Age-Related Cataract and to study the association between an index of sunlight exposure and the location of cortical cataract within the lens., Methods: Lens photographs of one eye of 731 persons with cortical opacities (503 with pure and 228 with mixed types of opacity) were included in the analysis. A radial grid superimposed on the photographs was used to assess presence, location, and severity of wedge-shaped cortical opacities., Results: Both the prevalence and the extent of cortical opacities were highest in the inferior-nasal quadrant and lowest in the superior-nasal quadrant of the lens. In polychotomous logistic regression, persons with the greatest excess areal involvement in the inferior half of the lens were more likely to have high exposure to sunlight, as measured by a sunlight index, than persons with excess involvement in the superior half of the lens (odds ratio, 1.73; 95% confidence interval 1.03, 2.93). Excess areal involvement of the inferior lens also was associated with the pure type of cortical cataract and with the total extent of the opacity., Conclusions: Age-related cortical opacities occur more frequently inferiorly than superiorly and, to a lesser extent, nasally than temporally. Possibly higher exposure of these lens segments to sunlight may explain this preferential location of cortical opacities.

The purpose of this study was to characterize the clinical course of myopia in a selected pediatric population. The computerized records of a health maintenance organization provided longitudinal data on 501 children aged 5 to 15 years. We examined the influence of age at diagnosis, gender, race, and initial refraction on progression of myopia. Mean follow-up was 34 months, with 75% of children having follow-up longer than 16 months. The mean rate of myopic progression was greater for children whose myopia was diagnosed at a younger age (5-7 years, -0.56 diopters/year versus 11-15 years, -0.28 diopters/year; p < 0.0001). Children with more than one diopter of myopia at first diagnosis progressed faster than children with less than or equal to one diopter of myopia (mean rate -0.48 diopter/year versus -0.41 diopter/year; p = 0.05). Cumulative event rate curves suggest a gender effect in the prepuberty years of 8 to 10, with myopia progressing faster in girls than boys (p = 0.003). Progression of myopia did not differ between white and non-white children. More rapidly progression of myopia is associated with younger age at initial diagnosis and greater severity of initial myopic refraction.

Objective: To evaluate associations with biochemical indicators of nutritional and other risk factors in the Lens Opacities Case-Control Study., Design: Case-control study., Setting and Participants: The Lens Opacities Case-Control Study determined risk factors for cortical, nuclear, and posterior subcapsular opacities among 1380 participants aged 40 to 79 years., Data Collection: Vitamin E, selenium, and biochemistry profile determinations were performed on all patients; red blood cell enzymes and amino acids were measured in systematic samples of about 25% of the Lens Opacities Case-Control Study population., Outcome: Laboratory test values in cases and controls were compared and expressed as odds ratios and 95% confidence intervals., Results: In polychotomous logistic regression analyses controlling for age and sex, the risk of opacities was reduced to less than one half in persons with higher levels of vitamin E (odds ratio, 0.44 for nuclear opacities), albumin-globulin ratio (odds ratio, 0.41 for mixed opacities), or iron (odds ratio, 0.43 for cortical opacities); higher uric acid levels increased risk (odds ratio, 1.74 for mixed opacities). Persons with opacities were twice as likely to have high glutathione reductase activity (with flavin adenine dinucleotide), which suggests low riboflavin status (odds ratio, 2.13). Most odds ratios for amino acids were under unity and were significantly decreased for glycine (0.36) and aspartic acid (0.31)., Conclusions: Lens opacities were associated with lower levels of riboflavin, vitamin E, iron, and protein nutritional status. Higher levels of uric acid increased risk of mixed opacities. The findings for riboflavin, vitamin E, iron, and uric acid are compatible with the dietary intake and medical history results of the Lens Opacities Case-Control Study.

Mössbauer spectroscopic studies on a heme peptide (HP) derived from cytochrome c and on the HP recombined non-covalently with the remaining cleaved section are reported. The results suggest that the environment of the heme site in the known crystal structure of cytochrome c may differ in detail from the environment of the heme in the working protein.

To explore the existence of a dose-response relationship between sunlight exposure and risk of age-related cataracts, we analyzed data collected from 1008 patients with cataracts and 469 control subjects enrolled in the Italian-American Case-Control Study of Age-Related Cataracts. Fourteen variables related to sunlight exposure history were included in the questionnaire administered to the study participants. A sunlight index was constructed and its relationship to the presence of cataracts was modeled by logistic regression. After adjustments for potential confounding variables and for age and sex, a significant dose-response effect (P = 0.01) was detected between the sunlight exposure index and the presence of pure cortical cataracts. With the exception of corticonuclear cataracts, all the other mixed types of opacity also showed a dose-response association with the sunlight index. These data support the hypothesis that sunlight exposure is a risk factor in the development of cortical cataracts, and demonstrate the existence of a dose-response relationship in this association.

Ten Sloan letters were used in the visual acuity charts developed for use in the Early Treatment Diabetic Retinopathy Study. We used the data from the 3,710 Early Treatment Diabetic Retinopathy Study subjects to investigate the relative difficulty of the ten Sloan letters and to evaluate whether the position of a letter on a line affected its relative difficulty. In general, our findings were consistent with those of the previous study. The four letters with curved contours (C, O, S, and D) were more difficult to discern at threshold than the six letters (Z, N, H, V, R, and K) composed of straight lines. Our data demonstrate that under these test conditions, letters at the end of a line are more likely to be read incorrectly than letters at the beginning of the line. This finding indicates that these data are probably not useful for evaluating possible crowding phenomena.

Objective: To determine whether the vitamin/mineral supplements used in two cancer intervention trials affected the risk of developing age-related cataracts., Design: Two randomized, double-masked trials with a duration of 5 to 6 years and end-of-trial eye examinations., Setting: Rural communes in Linxian, China., Participants: In trial 1, 2141 participants aged 45 to 74 years, and, in trial 2, 3249 participants aged 45 to 74 years., Interventions: Multivitamin/mineral supplement or matching placebo in trial 1; factorial design to test the effect of four different vitamin/mineral combinations in trial 2 (retinol/zinc, riboflavin/niacin, ascorbic acid/molybdenum, and selenium/alpha-tocopherol/beta carotene)., Main Outcome Measures: Prevalence of nuclear, cortical, and posterior subcapsular cataracts in treatment groups at end of trials., Results: In the first trial, there was a statistically significant 36% reduction in the prevalence of nuclear cataract for persons aged 65 to 74 years who received the supplements. In the second trial, the prevalence of nuclear cataract was significantly lower in persons receiving riboflavin/niacin compared with persons not receiving these vitamins. Again, persons in the oldest group, 65 to 74 years, benefited the most (44% reduction in prevalence). No treatment effect was noted for cortical cataract in either trial. Although the number of posterior subcapsular cataracts was very small, there was a statistically significant deleterious effect of treatment with riboflavin/niacin., Conclusions: Findings from the two trials suggest that vitamin/mineral supplements may decrease the risk of nuclear cataract. Additional research is needed in less nutritionally deprived populations before these findings can be translated into general nutritional recommendations.

Homogeneous pre-pro-GnRH/GAP protein was recently synthesized in 100 mg quantities by solid-phase methods and surprisingly, the synthetic pre-pro-protein, which normally does not escape the endoplasmic reticulum, was found to inhibit the release of prolactin from cultured pituitary cells. This is the first demonstration of significant biological activity associated with a precursor protein and provides the rationale for its further study. We now report the results of our initial examination of the conformational properties of pre-pro-GnRH/GAP protein as a prelude to solving its solution phase conformation by homonuclear 1H-NMR protocols. Thermal and pH titration fluorescence and circular dichroism spectroscopies reveal that the protein is resistant to thermal-induced conformational changes but is particularly sensitive to pH-induced conformational changes; while Asp/Glu and Arg residues may contribute to structural stability, His and Lys residues predominate. Pre-pro-GnRH/GAP is about 30% helix in the range of 2-40 degrees C; however, even at 90 degrees C, the peptide retains nearly 50% of its helix character. There is no evidence for a cooperative transition; for this reason, differential scanning calorimetry failed to yield a defined transition thermogram. Pre-pro-GnRH/GAP apparently does not pass through a transition state as a function of temperature but appears to flex and retain a high percentage of helix structure, resulting in subtle changes in secondary structure. There is no discernible isodichroic point. On either side of the neutral pH range, however, there are dramatic changes in structure that result in nonreversible denaturation of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

The human gonadotropin-releasing hormone precursor protein, pHGnRH (Met-23-Ile69) (preproGnRH), and three of its fragment peptides, pHGnRH (Asp14-Ile69) (gonadotropin-releasing hormone associated peptide--GAP), pHGnRH (Phe38-Ile69), and pHGnRH (Ser47-Ile69), were assembled in a stepwise solid-phase cosynthesis employing Boc/Bzl tactics and an optimized acylation schedule which included recoupling steps with hexafluoro-2-propanol to help overcome the aggregation of the pendant peptide chains of the peptidoresin during difficult couplings. Reversed-phase high-performance liquid chromatography (HPLC) purification yielded products which were characterized by analytical reversed-phase HPLC, ion-exchange chromatography, capillary zone electrophoresis, SDS-polyacrylamide gel electrophoresis, and ion-spray mass spectrometry to reveal a high degree of homogeneity. Biological characterization demonstrated that only GAP stimulated luteinizing hormone and follicle-stimulating hormone release from primary cultures of rat anterior pituitary cells, while GAP, pHGnRH (Phe38-Ile69), and preproGnRH all inhibited prolactin release, with the latter being the most potent at concentrations comparable to bromocryptine. However, only GAP and pHGnRH (Phe38-Ile69) were able to displace a labeled gonadotropin-releasing hormone agonist from binding to rat pituitary membrane preparations. This first demonstration of significant biological activity with a precursor protein also suggests that the gonadotropin-releasing and prolactin release-inhibiting functions of GAP are not mediated through the same pituitary receptors.

The D and L forms of the enzyme HIV-1 protease have been prepared by total chemical synthesis. The two proteins had identical covalent structures. However, the folded protein-enzyme enantiomers showed reciprocal chiral specificity on peptide substrates. That is, each enzyme enantiomer cut only the corresponding substrate enantiomer. Reciprocal chiral specificity was also evident in the effect of enantiomeric inhibitors. These data imply that the folded forms of the chemically synthesized D- and L-enzyme molecules are mirror images of one another in all elements of the three-dimensional structure. Enantiomeric proteins are expected to display reciprocal chiral specificity in all aspects of their biochemical interactions.

All non-mammalian vertebrates as well as marsupial mammals have two or more forms of gonadotropin-releasing hormone (GnRH) in the brain. Goldfish brain and pituitary contains two molecular forms of GnRH, salmon GnRH ([Trp7, Leu8]m-GnRH; s-GnRH) and chicken GnRH-II ([His5, Trp7, Tyr8]m-GnRH; cII-GnRH). Both sGnRH and cII-GnRH stimulate gonadotropin (GtH) as well as growth hormone (GH) release from the goldfish pituitary. The purpose of the present study was to study the activity of the five known forms of GnRHs as well as analogs of mammalian GnRH (m-GnRH) with variant amino acid residues in positions 5, 7 and 8 in terms of binding to GnRH receptors, and release of GTH and GH from the perifused fragments of goldfish pituitary in vitro. All five vertebrate GnRH peptides stimulated both GtH and GH release in a dose-dependent manner, although their potencies were very different. cII-GnRH was somewhat more active than s-GnRH in releasing GtH, whereas s-GnRH tended to have a greater potency than cII-GnRH in terms of GH release. Both chicken GnRH-I (cI-GnRH) and lamprey GnRH (l-GnRH) were significantly less potent than mGnRH, s-GnRH and cII-GnRH in releasing GtH and GH. cII-GnRH binds with higher affinity for the high affinity binding sites compared to all other native peptides. The activity of [Trp7]-GnRH was similar to both s-GnRH and cII-GnRH in releasing GtH and GH. Substitution of His5 resulted in a significant decrease in GtH releasing potencies compared to mGnRH, sGnRH and cII-GnRH. [His5]-GnRH also had lower GH releasing potency than mGnRH and sGnRH. Tyr8, His8 and Leu8 substitutions caused significant decreases in GtH releasing potencies compared to mGnRH, s-GnRH and cII-GnRH, but did not cause a significant change in GH releasing potency. The combination of [His5, Trp7]-GnRH had GtH and GH releasing activities similar to m-GnRH, s-GnRH and cII-GnRH.(ABSTRACT TRUNCATED AT 400 WORDS)