Toward Developing a Nurse Endoscopist Role in New Zealand.

The purpose of this exploratory descriptive mixed-method study was to explore the potential role of the nurse endoscopist as a part of the solution in fulfilling the workforce requirements of a bowel screening program, ascertain the possible enablers of a nurse endoscopist role in New Zealand, and determine whether there are endoscopy nurses who would wish to follow the nurse endoscopist/nurse practitioner pathway. A questionnaire with both open- and closed-ended questions gained in-depth information regarding the aspirations of New Zealand endoscopy nurses, their perceived enablers and barriers of a nurse endoscopist role, and statistical information on the New Zealand endoscopy nursing workforce. New Zealand has a highly experienced and educated endoscopy nursing workforce who supports the development of the nurse endoscopist role, some of whom expressed interest in a nurse endoscopist/practitioner pathway. It was concluded that with the addition of a specific education pathway and funding, standardization of training for endoscopists, and specific job description for nurse endoscopists, the future development of this role is possible in New Zealand.

Nursing Considerations in Pediatric Cardiac Critical Care.

OBJECTIVE: The objectives of this review are to describe the education and critical thinking skills that characterize pediatric critical care nursing and how these skills impact patient care and outcomes in pediatric cardiac critical care. DATA SOURCE: MEDLINE and PubMed. CONCLUSIONS: Pediatric cardiac critical care nurses manage complex and vulnerable patients requiring various levels of support. Effective care of these patients requires knowledge about the complex anatomy and physiology associated with congenital and acquired heart disease, as well as the effects of mechanical ventilation, mechanical circulatory support, and vasoactive medications. Strong physical examination skills, accurate interpretation of hemodynamic and laboratory data, active participation in bedside rounds, excellent communication skills, meticulous care of invasive monitoring catheters and tubes, and compassionate support of families are among the skills that distinguish a cardiac critical care nurse.

Functional characterization of mutations in inherited human cPLA2 deficiency.

Group IVA cytosolic phospholipase A(2) (cPLA(2)α) catalyzes the first step in the arachidonic acid cascade leading to the synthesis of important lipid mediators, the prostaglandins and leukotrienes. We previously described a patient deficient in cPLA(2)α activity, which was associated with mutations in both alleles encoding the enzyme. In this paper, we describe the biochemical characterization of each of these mutations. Using saturating concentrations of calcium, we showed that the R485H mutant was nearly devoid of any catalytic activity, that the S111P mutation did not affect the enzyme activity, and that the known K651R polymorphism was associated with activity slightly higher than that of the wild type. Using MDCK cells, we showed that translocation to the Golgi in response to serum activation was impaired for the S111P mutant but not for the other mutants. Using immortalized mouse lung fibroblasts lacking endogenous cPLA(2)α activity, we showed that both mutations S111P and R485H/K651R caused a profound defect in the enzyme catalytic activity in response to cell stimulation with serum. Taken together, our results show that the S111P mutation hampers calcium binding and membrane translocation without affecting the catalytic activity, and that the mutation R485H does not affect membrane translocation but blocks catalytic activity that leads to inactivation of the enzyme. Interestingly, our results show that the common K651R polymorphism confers slightly higher activity to the enzyme, suggesting a role of this residue in favoring a catalytically active conformation of cPLA(2)α. Our results define how the mutations negatively influence cPLA(2)α function and explain the inability of the proband to release arachidonic acid for eicosanoid production.

The nursing perspective on monitoring hemodynamics and oxygen transport.

Maintenance of adequate systemic oxygen delivery requires careful clinical assessment integrated with hemodynamic measurements and calculations to detect and treat conditions that may compromise oxygen delivery and lead to life-threatening shock, respiratory failure, or cardiac arrest. The bedside nurse constantly performs such assessments and measurements to detect subtle changes and trends in patient condition. The purpose of this editorial is to highlight nursing perspectives about the hemodynamic and oxygen transport monitoring systems summarized in the Pediatric Cardiac Intensive Care Society Evidence- Based Review and Consensus Statement on Monitoring of Hemodynamics and Oxygen Transport Balance. There is no substitute for the observations of a knowledgeable and experienced clinician who understands the patient's condition and potential causes of deterioration and is able to evaluate response to therapy.

Activation of group IVC phospholipase A(2) by polycyclic aromatic hydrocarbons induces apoptosis of human coronary artery endothelial cells.

Exposure to environmental pollutants, such as polycyclic aromatic hydrocarbons (PAHs) found in coal tar mixtures and tobacco sources, is considered a significant risk factor for the development of heart disease in humans. The goal of this study was to determine the influence of PAHs present at a Superfund site on human coronary artery endothelial cell (HCAEC) phospholipase A(2) (PLA(2)) activity and apoptosis. Extremely high levels of 12 out of 15 EPA high-priority PAHs were present in both the streambed and floodplain sediments at a site where an urban creek and its adjacent floodplain were extensively contaminated by PAHs and other coal tar compounds. Nine of the 12 compounds and a coal tar mixture (SRM 1597A) activated group IVC PLA(2) in HCAECs, and activation of this enzyme was associated with histone fragmentation and poly (ADP) ribose polymerase (PARP) cleavage. Genetic silencing of group IVC PLA(2) inhibited both (3)H-fatty acid release and histone fragmentation by PAHs and SRM 1597A, indicating that individual PAHs and a coal tar mixture induce apoptosis of HCAECs via a mechanism that involves group IVC PLA(2). Western blot analysis of aortas isolated from feral mice (Peromyscus leucopus) inhabiting the Superfund site showed increased PARP and caspase-3 cleavage when compared to reference mice. These data suggest that PAHs induce apoptosis of HCAECs via activation of group IVC PLA(2).

Resuscitation and extracorporeal life support during cardiopulmonary resuscitation following the Norwood (Stage 1) operation.

The success of extracorporeal support in providing cardiopulmonary support for a variety of patients has led to use of Extracorporeal Life Support, also known as ECLS, as a rescue for patients failing conventional resuscitation. The use of Extracorporeal Life Support in circumstances of cardiac arrest has come to be termed "Extracorporeal Life Support during Cardiopulmonary Resuscitation" or "ECPR". Although Extracorporeal Life Support during Cardiopulmonary Resuscitation was originally described in patients following repair of congenital cardiac defects who suffered a sudden arrest, it has now been used in a variety of circumstances for patients both with and without primary cardiac disease. Multiple centres have reported successful use of Extracorporeal Life Support during Cardiopulmonary Resuscitation in adults and children. However, because of the cost, the complexity of the technique, and the resources required, Extracorporeal Life Support during Cardiopulmonary Resuscitation is not offered in all centres for paediatric patients with refractory cardiac arrest. The increasing success and availability of Extracorporeal Life Support during Cardiopulmonary Resuscitation in post-operative cardiac patients, coupled with the fact that patients undergoing the Norwood (Stage 1) operation can have rapid, unpredictable cardiac deterioration and arrest, has led to a steady increase in the use of Extracorporeal Life Support during Cardiopulmonary Resuscitation in this population. For Extracorporeal Life Support during Cardiopulmonary Resuscitation to be most successful, it must be deployed rapidly while the patient is undergoing excellent cardiopulmonary resuscitation. Early activation of the team that will perform cannulation could possibly shorten the duration of cardiopulmonary resuscitation and might improve survival and outcome. More research needs to be done to refine the populations and circumstances that offer the best outcome with Extracorporeal Life Support during Cardiopulmonary Resuscitation, to evaluate the ratios of cost to benefit, and establish the long-term neurodevelopmental outcomes in survivors.

Regulation of cytosolic phospholipase A2alpha by hsp90 and a p54 kinase in okadaic acid-stimulated macrophages.

In resident mouse peritoneal macrophages, group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) mediates arachidonic acid (AA) release and eicosanoid production in response to diverse agonists such as A23187, phorbol myristate acetate, zymosan, and the enterotoxin, okadaic acid (OA). cPLA(2)alpha is regulated by phosphorylation and by calcium that binds to the C2 domain and induces translocation from the cytosol to membranes. In contrast, OA activates cPLA(2)alpha-induced AA release and translocation to the Golgi in macrophages without an apparent increase in calcium. Inhibitors of heat shock protein 90 (hsp90), geldanamycin, and herbimycin blocked AA release in response to OA but not to A23187, PMA, or zymosan. OA, but not the other agonists, induced activation of a cytosolic serine/threonine 54-kDa kinase (p54), which phosphorylated cPLA(2)alpha in in-gel kinase assays and was associated with cPLA(2)alpha in immunoprecipitates. Activation of the p54 kinase was inhibited by geldanamycin. The kinase coimmunoprecipitated with hsp90 in unstimulated macrophages, and OA induced its loss from hsp90, concomitant with its association with cPLA(2)alpha. The results demonstrate a role for hsp90 in regulating cPLA(2)alpha-mediated AA release that involves association of a p54 kinase with cPLA(2)alpha upon OA stimulation.

Chlamydophila pneumoniae myopericarditis in a child.

An 11-year-old boy with serologically confirmed Chlamydophila pneumoniae infection presented with clinical, laboratory, and echocardiographic changes consistent with myopericarditis. No reports on C. pneumoniae myopericarditis in children are found in the medical literature. The boy, previously healthy, presented with fever, rash, constitutional symptoms, elevated acute phase reactants, elevated cardiac enzymes, and high brain natriuretic peptide levels. Hemodynamic instabilities, including hypotension and mild hypoxia, were noted. Two-dimensional echocardiographic findings showed mildly depressed left ventricular systolic function and small pericardial effusion. Requiring inotropic support, the boy was treated with azithromycin 10 mg/kg once daily for 7 days and a single dose of intravenous immunoglobulin 2 g/kg. He recovered fully with improved left ventricular systolic function before hospital discharge. An early definitive diagnosis is essential to knowing the etiology of pediatric myocarditis. Specific therapy may play role in the management and prognosis of this disorder.

Properties of the Group IV phospholipase A2 family.

The Group IV phospholipase A2 family is comprised of six intracellular enzymes commonly called cytosolic phospholipase A2 (cPLA2) alpha, cPLA2beta, cPLA2gamma, cPLA2delta, cPLA2epsilon and cPLA2zeta. They are most homologous to phospholipase A and phospholipase B/lysophospholipases of filamentous fungi particularly in regions containing conserved residues involved in catalysis. However, a number of other serine acylhydrolases (patatin, Group VI PLA2s, Pseudomonas aeruginosa ExoU and NTE) contain the Ser/Asp catalytic dyad characteristic of Group IV PLA2s, and recent structural analysis of patatin has confirmed its structural similarity to cPLA2alpha. A characteristic of all these serine acylhydrolases is their ability to carry out multiple reactions to varying degrees (PLA2, PLA1, lysophospholipase and transacylase activities). cPLA2alpha, the most extensively studied Group IV PLA2, is widely expressed in mammalian cells and mediates the production of functionally diverse lipid products in response to extracellular stimuli. It has PLA2 and lysophospholipase activities and is the only PLA2 that has specificity for phospholipid substrates containing arachidonic acid. Because of its role in initiating agonist-induced release of arachidonic acid for the production of eicosanoids, cPLA2alpha activation is important in regulating normal and pathological processes in a variety of tissues. Current information available about the biochemical properties and tissue distribution of other Group IV PLA2s suggests they may have distinct mechanisms of regulation and functional roles.

Academic Performance in Primary School Children With Common Emotional and Behavioral Problems.

BACKGROUND: Many emotional and behavioral problems first emerge in primary school and are the forerunners of mental health problems occurring in adolescence. However, the extent that these problems may be associated with academic failure has been explored less. We aimed to quantify the association between emotional and behavioral problems with academic performance. METHODS: A stratified random sample of 8- to 9-year-olds (N = 1239) were recruited from schools in Australia. Data linkage was performed with a national assessment of academic performance to assess reading and numeracy. Parent report assessed emotional and behavioral problems with students dichotomized into "borderline/abnormal" and "normal" categories. RESULTS: One in 5 grade 3 students fell in the "borderline/abnormal" category. Boys with total difficulties (ß = -47.8, 95% CI: -62.8 to -32.8), conduct problems, and peer problems scored lower on reading. Numeracy scores were lower in boys with total difficulties (ß = -37.7, 95% CI: -53.9 to -21.5) and emotional symptoms. Children with hyperactivity/inattention scored lower in numeracy. Girls with peer problems scored lower in numeracy. CONCLUSIONS: Boys with emotional and behavioral problems in mid-primary school were 12 months behind their peers. Children with emotional and behavioral problems are at high risk for academic failure, and this risk is evident in mid-primary school.

Harnessing Teams and Technology to Improve Outcomes in Infants With Single Ventricle.

Infants with single ventricle require staged cardiac surgery, with stage I typically performed shortly after birth, stage II at 4 to 6 months of age, and stage III at 3 to 5 years of age. There is a high risk of interstage mortality and morbidity after infants are discharged from the hospital between stages I and II. Traditional home monitoring requires caregivers to record measurements of weight and oxygen saturation into a binder and requires families to assume a surveillance role. We have developed a tablet PC-based solution that provides secure and nearly instantaneous transfer of patient information to a cloud-based server, with the capacity for instant alerts to be sent to the caregiver team. The cloud-based IT infrastructure lends itself well to being able to be scaled to multiple sites while maintaining strict control over the privacy of each site. All transmitted data are transferred to the electronic medical record daily. The system conforms to recently released Food and Drug Administration regulation that pertains to mobile health technologies and devices. Since this platform was developed in March 2014, 30 patients have been monitored. There have been no interstage deaths. The experience of care providers has been unanimously positive. The addition of video has added to the use of the monitoring program. Of 30 families, 23 expressed a preference for the tablet PC over the notebook, 3 had no preference, and 4 preferred the notebook to the tablet PC.

Establishment of Pediatric Cardiac Intensive Care Advanced Practice Provider Services.

The addition of advanced practice providers (APPs; nurse practitioners and physician assistants) to a pediatric cardiac intensive care unit (PCICU) team is a health care innovation that addresses medical provider shortages while allowing PCICUs to deliver high-quality, cost-effective patient care. APPs, through their consistent clinical presence, effective communication, and facilitation of interdisciplinary collaboration, provide a sustainable solution for the highly specialized needs of PCICU patients. In addition, APPs provide leadership, patient and staff education, facilitate implementation of evidence-based practice and quality improvement initiatives, and the performance of clinical research in the PCICU. This article reviews mechanisms for developing, implementing, and sustaining advance practice services in PCICUs.

Low temperature induces expression of nitrate reductase in tomato that temporarily overrides circadian regulation of activity.

Overnight low-temperature exposure inhibits photosynthesis in chilling-sensitive species, such as tomato and cucumber, by as much as 60%. Earlier work showed that low temperature stalled the endogenous rhythm controlling transcription of certain nuclear-encoded genes in chilling-sensitive plants causing the synthesis of the corresponding transcripts and proteins to be mistimed upon rewarming. The activity of nitrate reductase (NR), the first and rate-limiting step in the assimilation of nitrate into amino acids in leaves, is subjected to a varied range of regulatory influences including a robust circadian rhythm. We show here that although NR regulation is disrupted by low temperatures, the change is transient and does not alter the phase of the NR endogenous rhythm following the chill. There is a temporary induction of de novo transcription of NR causing an increase in both NR protein and activity. This occurs regardless of the time in the circadian cycle that the chilling episode is initiated thereby decoupling the normally closely coordinated processes of carbon and nitrogen assimilation. This decoupling would be expected to deplete cellular reductant and carbon skeleton reserves as well as allow accumulation of cytotoxic intermediates of nitrogen assimilation thereby contributing to the low temperature induced disruption of metabolism that takes place in photosynthetic cells of chilling sensitive plant species.

Hemodynamics and cerebral oxygenation following repair of tetralogy of Fallot: the effects of converting from positive pressure ventilation to spontaneous breathing.

PURPOSE: Following corrective surgery for tetralogy of Fallot (TOF), approximately one-third of these patients develop low cardiac output (CO) due to right ventricular (RV) diastolic heart failure. Extubation is beneficial in these patients because the fall in intrathoracic pressure that occurs with conversion from positive pressure breathing to spontaneous breathing improves venous return, RV filling and CO. We hypothesized that if CO were to increase but remain limited following extubation, the obligatory increase in perfusion to the respiratory pump that occurs with loading of the respiratory musculature may occur at the expense of other vital organs, including the brain. MATERIALS AND METHODS: We conducted a retrospective analysis of all patients undergoing repair of TOF and monitoring of cerebral oxygenation using near infrared spectroscopy. We evaluated the following parameters two hours prior to and following extubation: mean and systolic arterial blood pressure (MBP, SBP), right atrial pressure (RAP), heart rate (HR) and cerebral oxygenation. RESULTS: The study included 22 patients. With extubation, MBP and SBP increased significantly from 67.3 ± 6.5 to 71.1 ± 8.4 mm Hg (P= 0.004) and from 87.2 ± 8.6 to 95.9 ± 10.9 mm Hg (P= 0.001), respectively, while the HR remained unchanged (145 vs. 146 bpm). The RAP remained unchanged following extubation (11.9 vs. 12.0 mm Hg). Following extubation, cerebral oxygen saturations increased significantly from 68.5 ± 8.4 to 74.2 ± 7.9% (P < 0.0001). Cerebral oxygen saturations increased by ≥5% in 11 of 22 patients and by ≥10% in 5 of 22 patients. CONCLUSION: We conclude that converting from positive pressure ventilation to spontaneous negative pressure breathing following repair of TOF significantly improves arterial blood pressure and cerebral oxygenation.

Role of phosphorylation and basic residues in the catalytic domain of cytosolic phospholipase A2alpha in regulating interfacial kinetics and binding and cellular function.

Group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) is regulated by phosphorylation and calcium-induced translocation to membranes. Immortalized mouse lung fibroblasts lacking endogenous cPLA(2)alpha (IMLF(-/-)) were reconstituted with wild type and cPLA(2)alpha mutants to investigate how calcium, phosphorylation, and the putative phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding site regulate translocation and arachidonic acid (AA) release. Agonists that elicit distinct modes of calcium mobilization were used. Serum induced cPLA(2)alpha translocation to Golgi within seconds that temporally paralleled the initial calcium transient. However, the subsequent influx of extracellular calcium was essential for stable binding of cPLA(2)alpha to Golgi and AA release. In contrast, phorbol 12-myristate 13-acetate induced low amplitude calcium oscillations, slower translocation of cPLA(2)alpha to Golgi, and much less AA release, which were blocked by chelating extracellular calcium. AA release from IMLF(-/-) expressing phosphorylation site (S505A) and PIP(2) binding site (K488N/K543N/K544N) mutants was partially reduced compared with cells expressing wild type cPLA(2)alpha, but calcium-induced translocation was not impaired. Consistent with these results, Ser-505 phosphorylation did not change the calcium requirement for interfacial binding and catalysis in vitro but increased activity by 2-fold. Mutations in basic residues in the catalytic domain of cPLA(2)alpha reduced activation by PIP(2) but did not affect the concentration of calcium required for interfacial binding or phospholipid hydrolysis. The results demonstrate that Ser-505 phosphorylation and basic residues in the catalytic domain principally act to regulate cPLA(2)alpha hydrolytic activity.

Activation of cytosolic phospholipase A2alpha in resident peritoneal macrophages by Listeria monocytogenes involves listeriolysin O and TLR2.

Eicosanoid production by macrophages is an early response to microbial infection that promotes acute inflammation. The intracellular pathogen Listeria monocytogenes stimulates arachidonic acid release and eicosanoid production from resident mouse peritoneal macrophages through activation of group IVA cytosolic phospholipase A2 (cPLA2alpha). The ability of wild type L. monocytogenes (WTLM) to stimulate arachidonic acid release is partially dependent on the virulence factor listeriolysin O; however, WTLM and L. monocytogenes lacking listeriolysin O (DeltahlyLM) induce similar levels of cyclooxygenase 2. Arachidonic acid release requires activation of MAPKs by WTLM and DeltahlyLM. The attenuated release of arachidonic acid that is observed in TLR2-/- and MyD88-/- macrophages infected with WTLM and DeltahlyLM correlates with diminished MAPK activation. WTLM but not DeltahlyLM increases intracellular calcium, which is implicated in regulation of cPLA2alpha. Prostaglandin E2, prostaglandin I2, and leukotriene C4 are produced by cPLA2alpha+/+ but not cPLA2alpha-/- macrophages in response to WTLM and DeltahlyLM. Tumor necrosis factor (TNF)-alpha production is significantly lower in cPLA2alpha+/+ than in cPLA2alpha-/- macrophages infected with WTLM and DeltahlyLM. Treatment of infected cPLA2alpha+/+ macrophages with the cyclooxygenase inhibitor indomethacin increases TNFalpha production to the level produced by cPLA2alpha-/- macrophages implicating prostaglandins in TNFalpha down-regulation. Therefore activation of cPLA2alpha in macrophages may impact immune responses to L. monocytogenes.

Function, activity, and membrane targeting of cytosolic phospholipase A(2)zeta in mouse lung fibroblasts.

Group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) initiates eicosanoid production; however, this pathway is not completely ablated in cPLA(2)alpha(-/-) lung fibroblasts stimulated with A23187 or serum. cPLA(2)alpha(+/+) fibroblasts preferentially released arachidonic acid, but A23187-stimulated cPLA(2)alpha(-/-) fibroblasts nonspecifically released multiple fatty acids. Arachidonic acid release from cPLA(2) alpha(-/-) fibroblasts was inhibited by the cPLA(2)alpha inhibitors pyrrolidine-2 (IC(50), 0.03 microM) and Wyeth-1 (IC(50), 0.1 microM), implicating another C2 domain-containing group IV PLA(2). cPLA(2) alpha(-/-) fibroblasts contain cPLA(2)beta and cPLA(2)zeta but not cPLA(2)epsilon or cPLA(2)delta. Purified cPLA(2)zeta exhibited much higher lysophospholipase and PLA(2) activity than cPLA(2)beta and was potently inhibited by pyrrolidine-2 and Wyeth-1, which did not inhibit cPLA(2)beta. In contrast to cPLA(2)beta, cPLA(2)zeta expressed in Sf9 cells mediated A23187-induced arachidonic acid release, which was inhibited by pyrrolidine-2 and Wyeth-1. cPLA(2)zeta exhibits specific activity, inhibitor sensitivity, and low micromolar calcium dependence similar to cPLA(2)alpha and has been identified as the PLA(2) responsible for calcium-induced fatty acid release and prostaglandin E(2) production from cPLA(2) alpha(-/-) lung fibroblasts. In response to ionomycin, EGFP-cPLA(2)zeta translocated to ruffles and dynamic vesicular structures, whereas EGFP-cPLA(2)alpha translocated to the Golgi and endoplasmic reticulum, suggesting distinct mechanisms of regulation for the two enzymes.

Group IVC cytosolic phospholipase A2gamma is farnesylated and palmitoylated in mammalian cells.

Cytosolic phospholipase A(2)gamma (cPLA(2)gamma) is a member of the group IV family of intracellular phospholipase A(2) enzymes, but unlike the well-studied cPLA(2)alpha, it is constitutively bound to membrane and is calcium independent. cPLA(2)gamma contains a C-terminal CaaX sequence and is radiolabeled by mevalonic acid when expressed in cPLA(2)alpha-deficient immortalized lung fibroblasts (IMLF(-/-)). The radiolabel associated with cPLA(2)gamma was identified as the farnesyl group. The protein farnesyltransferase inhibitor BMS-214662 prevented the incorporation of [(3)H]mevalonic acid into cPLA(2)gamma and partially suppressed serum-stimulated arachidonic acid release from IMLF(-/-) and undifferentiated human skeletal muscle (SkMc) cells overexpressing cPLA(2)gamma, but not from cells overexpressing cPLA(2)alpha. However, BMS-214662 did not alter the amount of cPLA(2)gamma associated with membrane. These results were consistent in COS cells expressing the C538S cPLA(2)gamma prenylation mutant. cPLA(2)gamma also contains a classic myristoylation site and several potential palmitoylation sites and was found to be acylated with oleic and palmitic acids but not myristoylated. Immunofluorescence microscopy revealed that cPLA(2)gamma is associated with mitochondria in IMLF(-/-), SkMc cells, and COS cells.

Control of nitrate reductase by circadian and diurnal rhythms in tomato.

Nitrate reductase (NR, EC 1.6.6.1) is a key regulatory enzyme in the assimilation of nitrate into amino acids in plant leaves. NR activity is intricately controlled by multifarious regulatory mechanisms acting at different levels ranging from transcription to protein degradation. It is among the few enzymes known to have a robust circadian rhythm of enzyme activity in many plant species. Although many aspects of NR regulation have been studied in depth, how these different types of control interact in a plant to deliver integrated control of activity in leaves over the course of the day has not been systematically investigated. This work documents that NR in young tomato (Lycopersicon esculentum Mill.) leaves has an endogenous rhythm in mRNA and protein level, which in nearly all circumstances are in phase with the rhythm in NR enzyme activity. Our data show that the diurnal control of NR activity in tomato leaves rests primarily with circadian regulation of Nia gene expression. The accompanying oscillations in protein level in tomato are made possible by a short half-life of NR protein that is approx. 6 h under normal conditions and approx. 2.5 h when plants are darkened during mid-day. NR post-transcriptional regulation via phosphorylation and subsequent 14-3-3 protein binding has a physiologically vital but secondary regulatory role in tomato of rapidly deactivating NR in response to changes in light intensity that cannot be anticipated by circadian timing. The post-translational reactivation of phosphorylated NR appears to have its primary physiological role in tomato leaves in reversing the down regulation of NR following transient shading events. Although there is a significant steady-state pool of apparently inactive NR throughout the diurnal, our data indicate that tomato leaves are unable to draw on this reserve to compensate for NR protein that is degraded during shading.

Role of cytosolic phospholipase A(2) in prostaglandin E(2) production by lung fibroblasts.

Prostaglandin (PG)E2 acts in an autocrine fashion to suppress proliferation of lung fibroblasts and production of collagen, and may negatively regulate pulmonary fibrosis. The role of Group IVA cytosolic phospholipase A2 alpha (cPLA2 alpha) in PGE2 production was investigated by comparing lung fibroblasts from wild-type and cPLA2 alpha-deficient mice. Arachidonic acid release from wild-type mouse lung fibroblasts (MLF+/+) was stimulated by serum, A23187 plus phorbol-myristate acetate (PMA), and lysophosphatidic acid (LPA) plus platelet-derived growth factor, but was > or = 80% lower from cPLA2 alpha-deficient MLF (MLF-/-). Transforming growth factor-beta increased cyclooxygenase-2 (COX2) expression to similar levels in MLF+/+ and MLF-/-, but MLF+/+ produced an order of magnitude more PGE2 than MLF-/- in response to A23187/PMA or platelet-derived growth factor/LPA. MLF+/+ synthesized less collagen than MLF-/-, supporting a role for PGE2 in suppressing collagen production. An SV40 immortalized line developed from MLF+/+ released arachidonic acid and expressed COX2 to levels similar to those of primary fibroblasts but produced 30-fold lower amounts of PGE2. Unlike primary fibroblasts, immortalized cells were deficient in microsomal PGE synthase (mPGES) but expressed slightly higher levels of cytosolic PGES. The results demonstrate a primary role for cPLA2 alpha in providing arachidonic acid for PGE2 production in mouse lung fibroblasts and support a role for this pathway in regulating collagen production.