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.