Albert Y. Wu

The two GAF domains in phosphodiesterase 2A have distinct roles in dimerization and in cGMP binding

Cyclic nucleotide phosphodiesterases (PDEs) regulate all pathways that use cGMP or cAMP as a second messenger. Five of the 11 PDE families have regulatory segments containing GAF domains, 3 of which are known to bind cGMP. In PDE2 binding of cGMP to the GAF domain causes an activation of the catalytic activity by a mechanism that apparently is shared even in the adenylyl cyclase of Anabaena, an organism separated from mouse by 2 billion years of evolution. The 2.9-Å crystal structure of the mouse PDE2A regulatory segment reported in this paper reveals that the GAF A domain functions as a dimerization locus. The GAF B domain shows a deeply buried cGMP displaying a new cGMP-binding motif and is the first atomic structure of a physiological cGMP receptor with bound cGMP. Moreover, this cGMP site is located well away from the region predicted by previous mutagenesis and structural genomic approaches.

Opposing Effects of Reactive Oxygen Species and Cholesterol on Endothelial Nitric Oxide Synthase and Endothelial Cell Caveolae

Synthesis of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) is critical for normal vascular homeostasis. eNOS function is rapidly regulated by agonists and blood flow and chronically by factors that regulate mRNA stability and gene transcription. Recently, localization of eNOS to specialized plasma membrane invaginations termed caveolae has been proposed to be required for maximal eNOS activity. Because caveolae are highly enriched in cholesterol, and hypercholesterolemia is associated with increased NO production, we first studied the effects of cholesterol loading on eNOS localization and NO production in cultured bovine aortic endothelial cells (BAECs). Caveolae-enriched fractions were prepared by OptiPrep gradient density centrifugation. Treatment of BAECs with 30 microgram/mL cholesterol for 24 hours stimulated significant increases in total eNOS protein expression (1.50-fold), eNOS associated with caveolae-enriched membranes (2.23-fold), and calcium ionophore-stimulated NO production (1.56-fold). Because reactive oxygen species (ROS) contribute to endothelial dysfunction in hypercholesterolemia, we next studied the effects of ROS on eNOS localization and caveolae number. Treatment of BAECs for 24 hours with 1 micromol/L LY83583, a superoxide-generating napthoquinolinedione, decreased caveolae number measured by electron microscopy and prevented the cholesterol-mediated increases in eNOS expression. In vitro exposure of caveolae-enriched membranes to ROS (xanthine plus xanthine oxidase) dissociated caveolin more readily than eNOS from the membranes. These results show that cholesterol treatment increases eNOS expression, whereas ROS treatment decreases eNOS expression and the association of eNOS with caveolin in caveolae-enriched membranes. Our data suggest that oxidative stress modulates endothelial function by regulating caveolae formation, eNOS expression, and eNOS-caveolin interactions.

Molecular determinants for cyclic nucleotide binding to the regulatory domains of phosphodiesterase 2A

Binding of cGMP to the GAF-B domain of phosphodiesterase 2A allosterically activates catalytic activity. We report here a series of mutagenesis studies on the GAF-B domain of PDE2A that support a novel mechanism for molecular recognition of cGMP. Alanine mutations of Phe-438, Asp-439, and Thr-488, amino acids that interact with the pyrimidine ring, decrease cGMP affinity slightly but increase cAMP affinity by up to 8-fold. Each interaction is required to provide for cAMP/cGMP specificity. Mutations of any of the residues that interact with the phosphate-ribose moiety or the imidazole ring abolish cGMP binding. Thus, residues that interact with the pyrimidine ring collectively control cAMP/cGMP specificity, whereas residues that bind the phosphate-ribose moiety and imidazole ring are critical for high affinity binding. Similar decreases in binding were found for mutations made in a bacterially expressed GAF-A/B plus catalytic domain construct. Because these constructs had very high catalytic activity, it appears that these mutations did not cause a global denaturation. The affinities of cAMP and cGMP for wild-type GAF-B alone were ∼4-fold greater than for the holoenzyme, suggesting that the presence of neighboring domains alters the conformation of GAF-B. More importantly, the PDE2A GAF-B, GAF-A/B, GAF-A/B+C domains, and holoenzyme all bind cGMP with much higher affinity than has previously been reported. This high affinity suggests that cGMP binding to PDE2 GAF-B activates the enzyme rapidly, stoichiometrically, and in an all or none fashion, rather than variably over a large range of cyclic nucleotide concentrations.

Cadaveric anatomical comparison of the lateral nasal wall after external and endonasal dacryocystorhinostomy.

Purpose: Much literature has accumulated espousing the relative merits of endonasal and external dacryocystorhinostomy (DCR). However, there is comparatively little information on the relative anatomic differences between these 2 approaches. The purpose of this study is to investigate the anatomic relationships of the lateral nasal wall for endonasal and external DCR. Methods: Ten cadaver half heads were used in this study. Half were subject to endonasal and half to external DCR procedures. The lateral nasal wall was then dissected and measurements were taken of ostium and anastomosis size and position relative to other landmarks on the lateral nasal wall. Relationships were compared between the 2 procedures. Results: The dimensions and area of the ostium and the anastomosis were similar between the 2 procedures. The lower portion of the ostium was located more inferiorly in endonasal DCR. Additionally, the ostium was more likely to be found lateral to the axilla of the middle turbinate in endonasal DCR, when compared with anterior for external. External DCR was also more likely to involve opening the anterior ethmoid air cells than endonasal approach. Conclusion: Endonasal and external DCR osteomies appear to be of similar size, with the endonasal opening being located slightly lower and more posterior on the lateral nasal wall.

Bilateral Lacrimal Gland Disease: Clinical Features of 97 Cases

OBJECTIVE Bilateral lacrimal gland (LG) disease is a unique presentation that can result from varied causes. We reviewed the diagnoses, clinical features, and outcomes of 97 patients with this entity. DESIGN Case series. PARTICIPANTS Ninety-seven patients with bilateral LG disease. METHODS Retrospective review and statistical analysis using analysis of variance and the Fisher exact test. MAIN OUTCOME MEASURES Patient demographics, clinical features, diagnostic testing, diagnosis, and treatment. RESULTS Patient age ranging from 8 to 84 years (mean, 46 years). The predominant gender was female (77%), and race included black (49%), white (38%), and Hispanic (12%) patients. Diagnoses fell into 4 categories: inflammatory (n = 51; 53%), structural (n = 20; 21%), lymphoproliferative (n = 19; 20%), and uncommon (n = 7; 7%) entities. The most common diagnoses included idiopathic orbital inflammation (IOI; n = 29; 30%), sarcoidosis (n = 19; 20%), prolapsed LG (n = 15; 15%), lymphoma (n = 11; 11%), lymphoid hyperplasia (n = 8; 8%), and dacryops (n = 5; 5%). Inflammatory conditions were more likely in younger patients (P<0.05) and in those with pain (P<0.001) and mechanical blepharoptosis (P<0.01) at presentation, whereas lymphoma was more common in older patients (P<0.001) without active signs of inflammation at presentation. Black patients were more likely to have sarcoidosis (P<0.01). Laboratory results showed high angiotensin converting enzyme level being significantly more likely in patients with sarcoidosis (P<0.05). However, sensitivity was limited to 45%, with 25% of patients diagnosed with IOI also demonstrating positive results. Corticosteroid therapy was the treatment of choice in 38 cases, corresponding to resolution of symptoms in 29% and improvement in an additional 32%. Overall, chronic underlying disease was found in 71% of patients, among whom 26% achieved a disease-free state, whereas 3% succumbed to their underlying disease. CONCLUSIONS The cause of bilateral lacrimal gland disease most commonly was inflammatory, followed by structural and lymphoproliferative. Patient characteristics and clinical presentations were key features distinguishing between competing possibilities. Despite local control with corticosteroids or radiotherapy, underlying disease continued in 71% of patients and led to death in 3%.

Enucleation versus Evisceration in Ocular Trauma: A Retrospective Review and Study of Current Literature

Abstract Purpose: To compare variables and outcomes from ocular trauma leading to either enucleation or evisceration to better inform surgical decision making. Design: Retrospective chart review. Methods: We reviewed 441 patients between 2001 and 2012 presenting with ocular trauma to a Level 1 trauma center in Queens, New York; of these, there were 16 enucleations and 6 eviscerations. Retrospective chart review noted age, gender, mechanism of injury, initial and final visual acuity, time to surgery, length of follow-up, pain, degree of motility, and complications. A review of literature in the context of our study was performed. Results: 20 patients were male and 2 patients were female; average age was 44 (SD: 20.0, range 18–91). 9/16 patients were enucleated to prevent sympathetic ophthalmia, whereas only 1/5 patient was eviscerated for this indication (p = 0.1619). No cases of sympathetic ophthalmia were reported over an average follow-up of 316 days. Average length of follow-up varied significantly between the two groups, with an average of 370.4 days (SD: 566.9, range 0–1870) for enucleated eyes and 172.7 days (SD: 146.3, range 0–422) for eviscerated eyes (p = 0.42). Medpor implants were preferred in eviscerations (5/6 eviscerations), whereas hydroxyapatite implants were preferred in enucleations (10/16 enucleations, p = 0.04). Conclusions: Surgical decision-making in ocular trauma is largely based on surgeon preference and experience, with minimal evidence in the literature to support either enucleation or evisceration. We recommend evisceration over enucleation in cases of reliable patient follow-up due to the low incidence of sympathetic ophthalmia.

Eyelash resection procedure for severe, recurrent, or segmental cicatricial entropion.

Purpose: To study the safety, efficacy, and cosmetic outcome of the eyelash resection procedure for treatment of severe, recurrent, or segmental cicatricial entropion. Methods: Retrospective consecutive case series of patients with severe, recurrent, or segmental cicatricial entropion treated with eyelash resection at the Moran Eye Center and the University of Vermont. Investigators performed chart reviews of these patients and evaluated effectiveness of the treatment and outcome data, including age, gender, diagnoses, method of repair, recurrence of trichiasis, and cosmetic satisfaction. There were no exclusionary characteristics specified in the study. Results: A total of 26 eyelids were operated on in 5 male and 11 female patients. The mean age was 74 years, with the following diagnoses: idiopathic (6), ocular cicatricial pemphigoid (2), postoperative (2), ocular pseudopemphigoid (drug related) (1), graft-versus-host disease (1), Stevens-Johnson syndrome (1), trachoma (1), linear IgA bullous dermatosis (1), and trauma (1). Mean postoperative follow-up was 13 months. The functional success rate was 90.5%, and the cosmetic success rate was 100%. Conclusion: The eyelash resection procedure is a safe, effective, and cosmetically acceptable procedure for treatment of severe, recurrent, or segmental cicatricial entropion.

Indications for orbital imaging by the oculoplastic surgeon.

Objective: To determine the indications for ordering orbital imaging and the indications for ordering CT versus MRI by oculoplastic surgeons and to assess the correlation between surgeons clinical indications for imaging and the radiologists diagnosis. Design: Retrospective review of imaging requisitions and radiology reports. Participants: Patients of 4 oculoplastic surgeons who required CT or MRI scans. Methods: Imaging requisitions and radiology reports of patients from 4 oculoplastic surgeons were reviewed to determine the indication for ordering a CT or MRI scan between March 2006 and March 2009. The indications were then compared with the radiologists diagnosis. Results: A total of 735 patients were included: 449 (61.1%) female and 286 (38.9%) male, with an average age of 50.1 years and an age range of 7 months to 93 years. Of these patients, a total of 632 CT and 223 MRI scans were ordered, 135 of which were follow-up scans. Conclusions: The most common indication for CT scan was thyroid disease, followed by orbital tumors and then inflammatory disease, while the most common indication for MRI scan was orbital tumors, followed by inflammatory disease and then thyroid disease. CT scans were more commonly ordered than MRI, largely for trauma and to rule out orbital foreign body.

Awareness and Knowledge of Emergent Ophthalmic Disease Among Patients in an Internal Medicine Clinic

IMPORTANCE Emergent ophthalmic disease can lead to permanent visual impairment or blindness if medical attention is delayed. Awareness and knowledge of emergent ophthalmic disease may be important for early medical presentation and maximization of visual prognosis in some cases. OBJECTIVE To assess public awareness and knowledge of 4 emergent ophthalmic diseases. DESIGN, SETTING, AND PARTICIPANTS This cross-sectional study was conducted from June 1 to July 30, 2015, in the waiting rooms of the outpatient internal medicine resident clinic at Mount Sinai Hospital. A written survey was administered to evaluate awareness and knowledge of retinal detachment, acute angle-closure glaucoma, giant cell arteritis, and central retinal artery occlusion. Awareness of each disease was assessed by whether participants knew what the diseases were (yes or no). Knowledge was evaluated by responses to 3 questions for each disease, including 1 question about basic pathophysiologic features, 1 question about basic symptoms, and 1 question about basic treatment options. All English-speaking patients who were physically and cognitively able to fill out the survey without assistance were considered eligible and offered the opportunity to participate during times of survey distribution; 237 completed the survey. Demographic information, including age, sex, race, income, and educational level, was collected. Data were assessed from August 1 to 7, 2015. MAIN OUTCOMES AND MEASURES Awareness of each ophthalmic disease was determined by the proportion of respondents who answered yes, and knowledge was determined by the proportion of aware respondents who answered the knowledge questions correctly. RESULTS Two hundred thirty-seven patients (of 227 who gave complete demographic information, 76 men [33.5%], 151 women [66.5%], and mean [SD] age, 51.3 [16.8] years) completed the survey. Awareness of each of the diseases studied was low; 61 of 220 respondents (27.7%; 95% CI, 21.8%-33.6%) were aware of retinal detachment; 32 of 219 respondents (14.6%; 95% CI, 9.9%-19.3%), acute angle-closure glaucoma; 11 of 216 respondents (5.1%; 95% CI, 2.2%-8.0%), giant cell arteritis; and 10 of 218 respondents (4.6%; 95% CI, 1.8%-7.4%), central retinal artery occlusion. Respondents who were aware and knowledgeable ranged from 29 of 199 (14.6%) for the pathophysiologic features of retinal detachment, 1 of 208 (0.5%) for the symptoms and 2 of 203 (1.0%) for treatment of giant cell arteritis, and 1 of 193 (0.5%) for the pathophysiologic features of central retinal artery occlusion. CONCLUSIONS AND RELEVANCE Levels of awareness and knowledge of emergent ophthalmic diseases are low. These results indicate a need to educate the public about these acutely vision-threatening entities to ensure early medical presentation, to achieve the best possible visual prognosis, and to preserve quality of life.

Accuracy of Biopsy in Subtyping Periocular Basal Cell Carcinoma.

Purpose: To determine the accuracy of initial biopsy in the diagnosis of basal cell carcinoma (BCC) histologic subtype. Methods: Retrospective histopathologic review of patients with a diagnosis of primary periocular BCC from 2006 to 2013 inclusive. Results: A total of 174 primary BCCs were identified. BCCs were classified as nodular, superficial, or aggressive (including mixed cases with an aggressive component). Punch biopsies were used in 41% of cases, while the remaining patients underwent shave or incision biopsies. The final histologic subtypes at excision were nodular (59%), superficial (7%), nodular and superficial (7%), and aggressive (51%). The overall concordance between the BCC subtype identified in the biopsy specimen and the subsequent excision specimen was 54%. In total, there were 51 cases (29%) of BCC, which included aggressive subtypes, of which 52% of initial biopsies failed to detect an aggressive component. There were 45 cases (26%) of mixed BCC, and an aggressive histologic subtype was present in 73% of these cases. Conclusions: The accuracy of initial biopsy for BCC histologic subtype at excision is highest for nodular BCC. For aggressive BCC, biopsy was able to detect the aggressive component in only 48% of cases. This may have implications for choice of treatment modality.