We have cloned and characterized the first human isozyme in a new family of cyclic nucleotide phosphodiesterases, PDE9A. By sequence homology in the catalytic domain, PDE9A is almost equidistant from all eight known mammalian PDE families but is most similar to PDE8A (34% amino acid identity) and least like PDE5A (28% amino acid identity). We report the cloning of human cDNA encoding a full-length protein of 593 amino acids, including a 261-amino acid region located near the C terminus that is homologous to the ϳ270-amino acid catalytic domain of other PDEs. PDE9A is expressed in all eight tissues examined as a ϳ2.0-kilobase mRNA, with highest levels in spleen, small intestine, and brain. The full-length PDE9A was expressed in baculovirus fused to an N-terminal 9-amino acid FLAG tag. Kinetic analysis of the baculovirus-expressed enzyme shows it to be a very high affinity cGMP-specific PDE with a K m of 170 nM for cGMP and 230 M for cAMP. The K m for cGMP makes PDE9A one of the highest affinity PDEs known. The V max for cGMP (4.9 nmol/min/g recombinant enzyme) is about twice as fast as that of PDE4 for cAMP. The enzyme is about twice as active in vitro in 1-10 mM Mn 2؉ than in the same concentration of Mg 2؉ or Ca 2؉ . PDE9A is insensitive (up to 100 M) to a variety of PDE inhibitors including rolipram, vinpocetine, SKF-94120, dipyridamole, and 3-isobutyl-1-methyl-xanthine but is inhibited (IC 50 ‫؍‬ 35 M) by zaprinast, a PDE5 inhibitor. PDE9A lacks a region homologous to the allosteric cGMP-binding regulatory regions found in the cGMP-binding PDEs: PDE2, PDE5, and PDE6.Cyclic nucleotide phosphodiesterases (PDEs), 1 which hydrolyze the intracellular second messengers cAMP and cGMP to their corresponding monophosphates, play an important role in signal transduction by regulating the intracellular concentration of cyclic nucleotides. Eight families of mammalian PDEs have been defined based on sequence similarity, substrate specificity, affinity, sensitivity to cofactors, and sensitivity to inhibitory drugs (1).2 These families are: PDE1, Ca 2ϩ /calmodulin-dependent; PDE2, cGMP-stimulated; PDE3, cGMPinhibited; PDE4, cAMP-specific; PDE5, cGMP-specific; PDE6, photoreceptor cGMP-specific; PDE7, cAMP-specific rolipraminsensitive; and PDE8, cAMP-specific IBMX-insensitive. Within families, there are multiple isozymes and multiple splice variants of those isozymes. PDEs are composed of a catalytic domain of ϳ270 amino acids, an N-terminal regulatory domain responsible for binding cofactors, and in some cases, a C-terminal domain of unknown function. Within the catalytic domain, there is approximately 30% amino acid identity between PDE families and ϳ85-95% identity between isozymes of the same family (e.g. PDE4A versus PDE4B). Furthermore, within a family there is extensive similarity (Ͼ60%) outside the catalytic domain, whereas across families there is little or no sequence similarity. The existence of multiple PDE families, isozymes, and splice variants presents an opportunity for complex regulation of cyclic nucleotide ...

Purpose: Antigenic overlap among circulating endothelial cells (CEC) and progenitors (CEP), platelets, and other blood cells led to the need to develop a reliable standardized method for CEC and CEP quantification. These cells are emerging as promising preclinical/clinical tools to define optimal biological doses of antiangiogenic therapies and to help stratify patients in clinical trials. Experimental Design: We report the experimental validation of a novel flow cytometry method that precisely dissects CEC/CEP from platelets and other cell populations and provides information about CEC/CEP viability.+ CECs, investigated by electron microscopy, were found to be bona fide endothelial cells by the presence of Weibel-Palade bodies. More than 75% of the circulating mRNAs of the endothelial-specific gene,VE-cadherin, found in the blood were present in the sorted population. CECs were 140 F 171/mL in healthy subjects (n = 37) and 951 F1,876/mL in cancer patients (n = 78; P < 0.0001). The fraction of apoptotic/necrotic CECs was 77 F 14% in healthy subjects and 43 F 23% in cancer patients (P < 0.0001). CEPs were 181 F 167/mL in healthy donors and 429 F 507/mL in patients (P = 0.00019). Coefficients of variation were 4 F 4% (intrareader), 17 F 4% (interreader), and 17 F 7% (variability over 0-72 h), respectively. Parallel samples were frozen by a standardized protocol. After thawing, coefficients of variation were 12 F 8% (intrareader), 16 F 10% (interreader), and 26 F 16% (variability over 0-14 days of frozen storage), respectively. Conclusions: This procedure enumerates a truly endothelial cell population with limited intrareader and interreader variability. It appears possible to freeze samples for large-scale CEC enumeration during clinical trials.This approach could be enlarged to investigate other angiogenic cell populations as well.

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WHAT'S KNOWN ON THIS SUBJECT: Recommended management of febrile neonates (#28 days) includes blood, urine, and cerebrospinal fluid cultures with hospital admission for antibiotic therapy. No study has reported adherence to standard recommendations in the management of febrile neonates in US pediatric emergency departments. WHAT THIS STUDY ADDS:There is wide variation in adherence to recommended management of febrile neonates. High rates of serious infections in admitted patients but low return rates for missed infections in discharged patients suggest additional studies needed to understand variation from current recommendations. abstract BACKGROUND: Blood, urine, and cerebrospinal fluid cultures and admission for antibiotics are considered standard management of febrile neonates (0-28 days). We examined variation in adherence to these recommendations across US pediatric emergency departments (PEDs) and incidence of serious infections (SIs) in febrile neonates. METHODS:Cross-sectional study of neonates with a diagnosis of fever evaluated in 36 PEDs in the 2010 Pediatric Health Information System database. We analyzed performance of recommended management (laboratory testing, antibiotic use, admission to hospital), 48-hour return visits to PED, and diagnoses of SI. RESULTS:Of 2253 neonates meeting study criteria, 369 (16.4%) were evaluated and discharged from the PED; 1884 (83.6%) were admitted. Recommended management occurred in 1497 of 2253 (66.4%; 95% confidence interval, 64.5-68.4) febrile neonates. There was more than twofold variation across the 36 PEDs in adherence to recommended management, recommended testing, and recommended treatment of febrile neonates. There was significant variation in testing and treatment between admitted and discharged neonates (P , .001). A total of 269 in 2253 (11.9%) neonates had SI, of whom 223 (82.9%; 95% confidence interval, 77.9-86.9) received recommended management.CONCLUSIONS: There was wide variation across US PEDs in adherence to recommended management of febrile neonates. One in 6 febrile neonates was discharged from the PED; discharged patients were less likely to receive testing or antibiotic therapy than admitted patients. A majority of neonates with SI received recommended evaluation and management. High rates of SI in admitted patients but low return rates for missed infections in discharged patients suggest a need for additional studies to understand variation from the current recommendations.