Use of a Schizosaccharomyces pombe PKA-repressible reporter to study cGMP metabolising phosphodiesterases.

The Schizosaccharomyces pombe fbp1 gene is transcriptionally repressed by protein kinase A (PKA) that is activated by extracellular glucose via a cAMP-signaling pathway. We previously used an fbp1-ura4 reporter that places uracil biosynthesis under the control of the glucose-sensing pathway to identify mutations in genes of the cAMP pathway. More recently, this reporter has been used in high throughput screens for small molecule inhibitors of heterologously-expressed cyclic nucleotide phosphodiesterases (PDEs) that hydrolyse cAMP to 5' AMP. Here we show that strains lacking the adenylyl cyclase gene respond to either exogenous cAMP or cGMP to activate PKA, thus regulating fbp1-ura4 expression and other PKA-regulated processes such as conjugation and the nuclear export of an Rst2-GFP fusion protein. Expression of cGMP-specific PDEs or ones that hydrolyse both cAMP and cGMP increases the amount of exogenous cGMP required to activate PKA in order to repress fbp1-ura4 expression, creating conditions that allow detection of inhibitors of these PDEs. As proof of this concept, we screened a collection of compounds previously identified as inhibitors of cAMP-specific PDE4 or PDE7 enzymes for their ability to inhibit the mammalian cGMP-specific PDE5A enzyme. We identified compound BC76, which inhibits PDE5A in an in vitro enzyme assay with an IC(50) of 232nM. Further yeast-based assays show that BC76 inhibits PDE1, PDE4, PDE5, PDE8, PDE10 and PDE11, thus demonstrating the utility of this system for detecting and characterising inhibitors of either cAMP- or cGMP-metabolising PDEs.

Surgical management of acute cholecystitis at a tertiary care center in the modern era.

HYPOTHESIS: The advent of laparoscopy has changed the paradigm of surgical training and care delivery for the treatment of patients with acute cholecystitis (AC). DESIGN: Retrospective data collection and analysis. SETTING: Hospital admissions with a primary diagnosis of AC at a tertiary care center from January 1, 2002, to January 1, 2007. PATIENTS: During the study period, 923 patients were admitted with a primary diagnosis of AC. One hundred fourteen patients were excluded from the study because of missing data, medical management, incomplete operative notes or documents, or metastatic gastrointestinal cancer. MAIN OUTCOME MEASURES: Patient demographics, preoperative morbidity, procedures (medical and surgical), and postoperative outcomes were statistically analyzed using chi(2) test, t test, and analysis of variance. RESULTS: Eight hundred nine patients (87.6%) with a primary diagnosis of AC underwent surgery by 44 surgeons. Procedures included 663 laparoscopic cholecystectomies (LCs) (82.0%), 9 open cholecystectomies (1.1%), 51 conversions from LC to open cholecystectomy (6.3%), and 86 cholecystostomy tube placements (10.6%). During the study period, cholecystostomy tube placements increased, while open cholecystectomies and conversions from LC to open cholecystectomy decreased (P < .05). Laparoscopic cholecystectomy was associated with significantly better outcomes, including shorter postsurgical stay (2.2 vs 6.3 days for other modalities) and fewer complications (8.5% vs 17.0%). CONCLUSIONS: Based on 5-year results from a tertiary care center, LC was performed with a low conversion rate to open surgery and was associated with decreased morbidity and mortality compared with other surgical modalities to treat AC. Our data confirm the benefits and widespread use of LC in the modern era, reflecting changes in the training paradigm and learning curve for laparoscopy.

Loss of SR-A and CD36 activity reduces atherosclerotic lesion complexity without abrogating foam cell formation in hyperlipidemic mice.

OBJECTIVE: The scavenger receptors SR-A and CD36 have been implicated in macrophage foam cell formation during atherogenesis and in the regulation of inflammatory signaling pathways, including those leading to lesional macrophage apoptosis and plaque necrosis. To test the impact of deleting these receptors, we generated Apoe(-/-) mice lacking both SR-A and CD36 and fed them a Western diet for 12 weeks. METHODS AND RESULTS: We analyzed atheroma in mice, assessing lesion size, foam cell formation, inflammatory gene expression, apoptosis, and necrotic core formation. Aortic root atherosclerosis in Apoe(-/-)Cd36(-/-)Msr1(-/-) mice, as assessed by morphometry, electron microscopy, and immunohistochemistry, showed no decrease in lesion area or in vivo foam cell formation when compared to Apoe(-/-) mice. However, Apoe(-/-)Cd36(-/-)Msr1(-/-) lesions showed reduced expression of inflammatory genes and morphological analysis revealed a approximately 30% decrease in macrophage apoptosis and a striking approximately 50% decrease in plaque necrosis in aortic root lesions of these mice. CONCLUSIONS: Although targeted deletion of SR-A and CD36 does not abrogate macrophage foam cell formation or substantially reduce atherosclerotic lesion area in Apoe(-/-) mice, loss of these pathways does reduce progression to more advanced necrotic lesions. These data suggest that targeted inhibition of these pathways in vivo may reduce lesional inflammation and promote plaque stability.

Elevated neuronal expression of CD200 protects Wlds mice from inflammation-mediated neurodegeneration.

Axonal damage secondary to inflammation is likely the substrate of chronic disability in multiple sclerosis and is found in the animal model of experimental autoimmune encephalomyelitis (EAE). Wld(s) mice have a triplication of the fusion gene Ube4b/Nmnat and a phenotype of axon protection. Wld(s) mice develop an attenuated disease course of EAE, with decreased demyelination, reduced axonal pathology, and decreased central nervous system (CNS) macrophage and microglial accumulation. We show that attenuated disease in Wld(s) mice was associated with robust constitutive expression of the nonsignaling CD200 molecule on neurons in the CNS compared with control mice. CD200 interacts with its signaling receptor CD200R, which we found to be expressed on microglia, astrocytes, and oligodendrocytes at similar levels in control and Wld(s) mice. Administration of blocking anti-CD200 antibody to Wld(s) mice abrogated disease attenuation and was associated with increased CNS inflammation and neurodegeneration. In vitro, Wld(s) neuronal cultures were protected from microglial-induced neurotoxicity compared with control cultures, but protection was abrogated by anti-CD200 antibody. The CD200-CD200R pathway plays a critical role in attenuating EAE and reducing inflammation-mediated damage in the CNS. Strategies that up-regulate the expression of CD200 in the CNS or molecules that ligate the CD200R may be relevant as neuroprotective strategies in multiple sclerosis.