The HysNiche trial: hysteroscopic resection of uterine caesarean scar defect (niche) in patients with abnormal bleeding, a randomised controlled trial.

BACKGROUND: A caesarean section (CS) can cause a defect or disruption of the myometrium at the site of the uterine scar, called a niche. In recent years, an association between a niche and postmenstrual spotting after a CS has been demonstrated. Hysteroscopic resection of these niches is thought to reduce spotting and menstrual pain. However, there are no randomised trials assessing the effectiveness of a hysteroscopic niche resection. METHODS/DESIGN: We planned a multicentre randomised trial comparing hysteroscopic niche resection to no intervention. We study women with postmenstrual spotting after a CS and a niche with a residual myometrium of at least 3 mm during sonohysterography. After informed consent is obtained, eligible women will be randomly allocated to hysteroscopic resection of the niche or expectant management for 6 months. The primary outcome is the number of days with postmenstrual spotting during one menstrual cycle 6 months after randomisation. Secondary outcomes are menstrual characteristics, menstruation related pain and experienced discomfort due to spotting or menstrual pain, quality of life, patient satisfaction, sexual function, urological symptoms, medical consultations, medication use, complications, lost productivity and medical costs. Measurements will be performed at baseline and at 3 and 6 months after randomisation. A cost-effectiveness analysis will be performed from a societal perspective at 6 months after randomisation. DISCUSSION: This trial will provide insight in the (cost)effectiveness of hysteroscopic resection of a niche versus expectant management in women who have postmenstrual spotting and a niche with sufficient residual myometrium to perform a hysteroscopic niche resection. TRIAL REGISTRATION: Dutch Trial Register NTR3269 . Registered 1 February 2012. ZonMw Grant number 80-82305-97-12030.

Prevalence, potential risk factors for development and symptoms related to the presence of uterine niches following Cesarean section: systematic review.

OBJECTIVE: To review systematically the medical literature reporting on the prevalence of a niche at the site of a Cesarean section (CS) scar using various diagnostic methods, on potential risk factors for the development of a niche and on niche-related gynecological symptoms in non-pregnant women. METHODS: The PubMed and EMBASE databases were searched. All types of clinical study reporting on the prevalence, risk factors and/or symptoms of a niche in non-pregnant women with a history of CS were included, apart from case reports and case series. RESULTS: Twenty-one papers were selected for inclusion in the review. A wide range in the prevalence of a niche was found. Using contrast-enhanced sonohysterography in a random population of women with a history of CS, the prevalence was found to vary between 56% and 84%. Nine studies reported on risk factors and each study evaluated different factors, which made it difficult to compare studies. Risk factors could be classified into four categories: those related to closure technique, to development of the lower uterine segment or location of the incision or to wound healing, and miscellaneous factors. Probable risk factors are single-layer myometrium closure, multiple CSs and uterine retroflexion. Six out of eight studies that evaluated niche-related symptoms described an association between the presence of a niche and postmenstrual spotting. CONCLUSIONS: The reported prevalence of a niche in non-pregnant women varies depending on the method of detection, the criteria used to define a niche and the study population. Potential risk factors can be categorized into four main categories, which may be useful for future research and meta-analyses. The predominant symptom associated with a niche is postmenstrual spotting.

Ultra-wide-field autofluorescence imaging in non-traumatic rhegmatogenous retinal detachment.

PURPOSE: Rhegmatogenous retinal detachment (RRD) affects the function of the retina before and after surgical repair. We investigated ultra-wide-field autofluorescence (UAF) abnormalities in patients with acute RRD to improve our understanding of the functional changes in the retina before and after surgery. METHODS: In this retrospective study, we present the UAF imaging findings of 16 patients with acute, non-traumatic RRD. Imaging was obtained with the Optos 200 Tx (Optos) in 14 eyes preoperatively and in 12 eyes postoperatively. Twelve eyes had RRDs that involved the macula (group A), whereas four eyes had macula-sparing RRDs (group B). RESULTS: All patients (100%) with bullous retinal detachments demonstrated hypofluorescence over the area of retinal detachment. A hyperfluorescent leading edge (HLE) to the retinal detachment was observed preoperatively in 100% of eyes in group A and 75% of eyes in group B. Preoperative UAF through the fovea of group A eyes was normal (30%), hypofluorescent (50%) or hyperfluorescent (20%). In all patients with a HLE preoperatively, the HLE resolved by the 1-month postoperative visit. A residual line of demarcation remained in 8 of the 12 eyes (67%). In group A eyes, postoperative granular autofluorescent changes were present in four of the nine (44%) eyes, and were associated with worse preoperative (P=0.04) and postoperative (P=0.09) visual acuity. CONCLUSION: UAF imaging reveals abnormalities in RRDs that allow excellent demarcation of the extent of the retinal detachment and assist in preoperative characterization of the detachment and postoperative counselling.

2-Aryl-2,2-difluoroacetamide FKBP12 ligands: synthesis and X-ray structural studies.

[structure: see text] 2-Aryl-2,2-difluoroacetamido-proline and pipecolate esters are high affinity FKBP12 ligands whose rotamase inhibitory activity is comparable to that seen for the corresponding ketoamides. X-ray structural studies suggest that the fluorine atoms participate in discrete interactions with the Phe36 phenyl ring and the Tyr26 hydroxyl group, with the latter resembling a moderate-to-weak hydrogen bond.

The membrane-bound intestinal enzymes of waxwings and thrushes: adaptive and functional implications of patterns of enzyme activity.

Cedar waxwings (Bombycilla cedrorum) feed predominantly on fruits that are rich in simple sugars and low in nitrogen, supplementing this diet with arthropod prey during the summer months as well as flowers and tree sap in springtime. In contrast, thrushes feed extensively on fatty, protein-rich invertebrate prey, supplemented with sugary and lipid-rich fruits. Simple sugars and fats are digested and/or absorbed by distinctly different physiological mechanisms, which suggests the possibility of contrasting digestive strategies in animals specialized to diets containing one of these two energy sources. In this study, we quantified enzymatic activity of three membrane-bound intestinal enzymes of cedar waxwings and five species of thrushes to explore this aspect of their digestive physiology. These enzymes catalyze the final steps in the digestion of carbohydrates (sucrase-isomaltase and maltase-glucoamylase) and protein (aminopeptidase-N). The two carbohydrases are homologous enzymes with overlapping functions; both enzymes catalyze the hydrolysis of maltase and isomaltase. The membrane-bound digestive enzyme systems that we described for cedar waxwings and thrushes can be explained by the particular nutrients contained within their respective natural diets. Consistent with previous work, cedar waxwings displayed intestinal sucrase activity, whereas thrushes did not. Correspondingly, cedar waxwings eat some foods containing sucrose, whereas thrushes do not. Sucrase-isomaltase conferred all maltase and isomaltase activity in cedar waxwings. In contrast, all maltase and isomaltase activity in thrushes was necessarily sucrase independent, which indicated the presence of maltase-glucoamylase. The absence of sucrase-independent maltase activity in cedar waxwings suggests that sucrase-isomaltase obviates the need for maltase-glucoamylase. Indeed, total maltase and isomaltase activities were much higher in cedar waxwings than in thrushes. Neither waxwings nor thrushes eat starchy foods; sucrase-isomaltase in waxwings and maltaseglucoamylase in thrushes probably function in digesting glycogen in animal foods. We suggest that digestive traits associated with specialization to monosaccharide-rich fruits (lack of a grinding gizzard) by frugivorous waxwings and thrushes may prevent utilization of starchy seeds. Total aminopeptidase-N activity in cedar waxwings was indistinguishable from the allometric pattern among thrush species, but the distribution of this enzyme along the intestines of waxwings and thrushes was distinctly different, which demonstrates that total enzyme activity can be insufficient as a descriptor of the functional activity of brush border enzymes. Aminopeptidase-N activity peaked in the anterior part of the intestines of thrushes and in the terminal portion of the intestines of waxwings, which suggests contrasting strategies for protein digestion from fatty versus sugary diets, respectively.

BMS-229724 is a tight-binding inhibitor of cytosolic phospholipase A2 that acts at the lipid/water interface and possesses anti-inflammatory activity in skin inflammation models.

Cytosolic phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. BMS-229724 (4-[4-[2-[2-[bis(4-chlorophenyl)methoxy]ethyl-sulfonyl]ethoxy]phenyl]-1,1,1-trifluoro-2-butanone) was found to be a selective inhibitor of cPLA2 (IC50 = 2.8 microM) in that it did not inhibit secreted phospholipase A2 in vitro, nor phospholipase C and phospholipase D in cells. The compound was active in inhibiting arachidonate and eicosanoid production in U937 cells, neutrophils, platelets, monocytes, and mast cells. With a synthetic covesicle substrate system, the dose-dependent inhibition could be defined by kinetic equations describing competitive inhibition at the lipid/water interface. The apparent equilibrium dissociation constant for the inhibitor bound to the enzyme at the interface (K(I)*(app)) was determined to be 1. 10(-5) mol% versus an apparent dissociation constant for the arachidonate-containing phospholipid of 0.35 mol%. The unit of concentration in the interface is mole fraction (or mol%), which is related to the surface concentration of substrate, rather than bulk concentration that has units of molarity. Thus, BMS-229724 represents a novel inhibitor of cPLA2, which partitions into the phospholipid bilayer and competes with phospholipid substrate for the active site. This potent inhibition of the enzyme translated into anti-inflammatory activity when applied topically (5%, w/v) to a phorbol ester-induced chronic inflammation model in mouse ears, inhibiting edema and neutrophil infiltration, as well as prostaglandin and leukotriene levels in the skin. In hairless guinea pigs, BMS-229724 was active orally (10 mg/kg) in a UVB-induced skin erythema model in hairless guinea pigs.

HIV-1 integrase inhibitors that compete with the target DNA substrate define a unique strand transfer conformation for integrase.

Diketo acids such as L-731,988 are potent inhibitors of HIV-1 integrase that inhibit integration and viral replication in cells. These compounds exhibit the unique ability to inhibit the strand transfer activity of integrase in the absence of an effect on 3' end processing. To understand the reasons for this distinct inhibitory profile, we developed a scintillation proximity assay that permits analysis of radiolabeled inhibitor binding and integrase function. High-affinity binding of L-731,988 is shown to require the assembly of a specific complex on the HIV-1 long terminal repeat. The interaction of L-731,988 with the complex and the efficacy of L-731, 988 in strand transfer can be abrogated by the interaction with target substrates, suggesting competition between the inhibitor and the target DNA. The L-731,988 binding site and that of the target substrate are thus distinct from that of the donor substrate and are defined by a conformation of integrase that is only adopted after assembly with the viral end. These results elucidate the basis for diketo acid inhibition of strand transfer and have implications for integrase-directed HIV-1 drug discovery efforts.

X-ray structure of simian immunodeficiency virus integrase containing the core and C-terminal domain (residues 50-293)--an initial glance of the viral DNA binding platform.

The crystal structure of simian immunodeficiency virus (SIV) integrase that contains in a single polypeptide the core and the C-terminal deoxyoligonucleotide binding domain has been determined at 3 A resolution with an R-value of 0.203 in the space group P2(1)2(1)2(1). Four integrase core domains and one C-terminal domain are found to be well defined in the asymmetric unit. The segment extending from residues 114 to 121 assumes the same position as seen in the integrase core domain of avian sarcoma virus as well as human immunodeficiency virus type-1 (HIV-1) crystallized in the absence of sodium cacodylate. The flexible loop in the active site, composed of residues 141-151, remains incompletely defined, but the location of the essential Glu152 residue is unambiguous. The residues from 210-218 that link the core and C-terminal domains can be traced as an extension from the core with a short gap at residues 214-215. The C(alpha) folding of the C-terminal domain is similar to the solution structure of this domain from HIV-1 integrase. However, the dimeric form seen in the NMR structure cannot exist as related by the non-crystallographic symmetry in the SIV integrase crystal. The two flexible loops of the C-terminal domain, residues 228-236 and residues 244-249, are much better fixed in the crystal structure than in the NMR structure with the former in the immediate vicinity of the flexible loop of the core domain. The interface between the two domains encompasses a solvent-exclusion area of 1500 A(2). Residues from both domains purportedly involved in DNA binding are narrowly distributed on the same face of the molecule. They include Asp64, Asp116, Glu152 and Lys159 from the core and Arg231, Leu234, Arg262, Arg263 and Lys264 from the C-terminal domain. A model for DNA binding is proposed to bridge the two domains by tethering the 228-236 loop of the C-terminal domain and the flexible loop of the core.

Inhibitors of strand transfer that prevent integration and inhibit HIV-1 replication in cells.

Integrase is essential for human immunodeficiency virus-type 1 (HIV-1) replication; however, potent inhibition of the isolated enzyme in biochemical assays has not readily translated into antiviral activity in a manner consistent with inhibition of integration. In this report, we describe diketo acid inhibitors of HIV-1 integrase that manifest antiviral activity as a consequence of their effect on integration. The antiviral activity of these compounds is due exclusively to inhibition of one of the two catalytic functions of integrase, strand transfer.

Purification, solution properties and crystallization of SIV integrase containing a continuous core and C-terminal domain.

The C-terminal two-thirds segment of integrase derived from the simian immunodeficiency virus has been cloned, expressed in Escherichia coli, and purified to greater than 95% homogeneity. The protein encompasses amino-acid residues 50-293 and contains a F185H substitution to enhance solubility. In dilute solutions at concentrations below 1 mg ml(-1), the enzyme is predominantly dimeric. At the higher concentrations (>10 mg ml(-1)) required to enable crystallization, the enzyme self-associates to form species with molecular weights greater than 200 kDa. Despite the apparent high aggregation in solution, the enzyme crystallizes from a 8%(v/v) polyethylene glycol (molecular weight 6000) solution in a form suitable for X-ray diffraction studies. The resulting single crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 79.76, b = 99.98, c = 150.2 A, alpha = beta = gamma = 90 degrees and Z = 4. Under X-ray irradiation generated with a rotating-anode generator, the crystals diffract to 2.8 A resolution and allow collection of a native 3 A resolution diffraction data set.

Efficiency and fidelity of full-site integration reactions using recombinant simian immunodeficiency virus integrase.

Full-site integration by recombinant wild-type and mutant simian immunodeficiency virus (SIV) integrase (IN) was investigated with linear retrovirus-like DNA (469 bp) as a donor substrate and circular DNA (2,867 bp) as a target substrate. Under optimized conditions, recombinant SIV IN produced donor-target products consistent with full-site (two donor ends) and half-site (one donor end) reactions with equivalent frequency. Restriction enzyme analysis of the 3.8-kbp full-site reaction products confirmed the concerted insertion of two termini from separate donors into a single target molecule. Donor ends carrying the viral U5 termini were preferred over U3 termini for producing both half-site and full-site products. Bacterial genetic selection was used to isolate individual donor-target recombinants, and the donor-target junctions of the cloned products were characterized by sequencing. Analysis of 149 recombinants demonstrated approximately 84% fidelity for the appropriate simian retrovirus 5-bp host duplication. As seen previously in similar reactions with human immunodeficiency virus type 1 (HIV-1) IN from lysed virions, approximately 8% of the donor-target recombinants generated with recombinant SIV IN incurred specific 17- to 18- or 27- to 29-bp deletions. The efficiency and fidelity of the full-site integration reaction mediated by the purified, recombinant SIV IN is comparable to that of HIV-1 IN from virions. These observations suggest that a purified recombinant lentivirus IN is itself sufficient to recapitulate the full-site integration process.

Competitive, reversible inhibition of cytosolic phospholipase A2 at the lipid-water interface by choline derivatives that partially partition into the phospholipid bilayer.

Cytosolic phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. When assaying the human recombinant cPLA2 using membranes isolated from [3H]arachidonate-labeled U937 cells as substrate, 2-(2'-benzyl-4-chlorophenoxy)ethyl-dimethyl-n-octadecyl-ammonium chloride (compound 1) was found to inhibit the enzyme in a dose-dependent manner (IC50 = 5 microM). It was over 70 times more selective for the cPLA2 as compared with the human nonpancreatic secreted phospholipase A2, and it did not inhibit other phospholipases. Additionally, it inhibited arachidonate production in N-formyl-methionyl-leucyl-phenylalanine-stimulated U937 cells. To further characterize the mechanism of inhibition, an assay in which the enzyme is bound to vesicles of 1,2-dimyristoyl-sn -glycero-3-phosphomethanol containing 6-10 mol % of 1-palmitoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine was employed. With this substrate system, the dose-dependent inhibition could be defined by kinetic equations describing competitive inhibition at the lipid-water interface. The apparent equilibrium dissociation constant for the inhibitor bound to the enzyme at the interface (KI*app) was determined to be 0.097 +/- 0.032 mol % versus an apparent dissociation constant for the arachidonate-containing phospholipid of 0.3 +/- 0.1 mol %. Thus, compound 1 represents a novel structural class of inhibitor of cPLA2 that partitions into the phospholipid bilayer and competes with the phospholipid substrate for the active site. Shorter n-alkyl-chained (C-4, C-6, C-8) derivatives of compound 1 were shown to have even smaller KI*app values. However, these short-chained analogs were less potent in terms of bulk inhibitor concentration needed for inhibition when using the [3H]arachidonate-labeled U937 membranes as substrate. This discrepancy was reconciled by showing that these shorter-chained analogs did not partition into the [3H]arachidonate-labeled U937 membranes as effectively as compound 1. The implications for in vivo efficacy that result from these findings are discussed.

The size and curvature of anionic covesicle substrate affects the catalytic action of cytosolic phospholipase A2.

Cytosolic phospholipase A2 (cPLA2) is normally located in the cytosol, but in response to cellular activation the enzyme binds to the membrane at the lipid/water interface where it catalyzes the hydrolysis of the sn-2 ester of arachidonate-containing phospholipids. Synthetic phospholipid vesicle systems have been used in kinetic and mechanistic analyses of cPLA2, but these systems result in a rapid loss of enzyme activity. In the present research, covesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) containing

Ecological and evolutionary implications of energy and protein requirements of avian frugivores eating sugary diets.

To assess how the high-sugar/low-protein content of fruit diets affects digestive function and nutrition of frugivorous birds, I compared intake, passage rate, sugar utilization, protein requirements, and mass changes of cedar waxwings (Bombycilla cedrorum), American robins (Turdus migratorius), and wood thrushes (Hylocichla mustelina) fed synthetic diets simulating the range of sugar (6.6%, 12.4%, and 22.0% solutes) and protein (4.5%, 3.0%, and 1.5% of dry matter) content of bird-dispersed fruits. The dietary emphasis on sugary fruits by cedar waxwings suggests the potential for digestive and physiological specializations to this food type. All birds increased volumetric food intake and passage rates as sugar concentration declined. Birds completely (22.0%-12.4% sugar solute concentration) or incompletely (12.4%-6.6% sugar solute concentration) compensated for dietary dilution. Cedar waxwings consumed each diet at higher rates than did thrushes, as they do when eating sugary fruits, demonstrating that interspecific differences in ingestion rates of sugary fruits are a consequence of nutrient composition, rather than seed bulk or secondary compounds of fruits. Passage rate was not responsible for interspecific differences in short-term food intake rate, implicating gut morphology as the key functional feature limiting intake. Most sugary fruits are nutritionally deficient in apparent protein for thrushes but are nutritionally adequate in protein for cedar waxwings because of this species' relatively high intake rates and low protein requirements. The digestive systems of frugivorous birds respond flexibly to dietary sugar concentration, but protein content of fruits can present a nutritional limitation, potentially influencing the proportions of fruit and animal foods in birds' diets.

A beta-lactam inhibitor of cytosolic phospholipase A2 which acts in a competitive, reversible manner at the lipid/water interface.

Cytosolic phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. When assaying the human recombinant cPLA2 using membranes isolated from [3H]arachidonate-labeled U937 cells as substrate, 3,3-Dimethyl-6-(3-lauroylureido)-7-oxo-4-thia-1-azabicyclo[3,2,0] heptane-2-carboxylic acid (1) was found to inhibit the enzyme in a dose-dependent manner (IC50 = 72 microM). This beta-lactam did not inhibit other phospholipases, including the human nonpancreatic secreted phospholipase A2. The inhibition of cPLA2 was found not to be time-dependent. This, along with the observation that the degradation of the inhibitor was not catalyzed by the enzyme, demonstrates that the inhibition does not result from the formation of an acyl-enzyme intermediate with the active site serine residue. Moreover, the ring-opened form of 1 is also able to inhibit cPLA2 with near-equal potency. To further characterize the mechanism of inhibition, an assay in which the enzyme is bound to vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol containing 6-10 mole percent of 1-palmitoyl-2-[1-14C]-arachidonoyl-sn-glycero-3-phosphocholine was employed. With this substrate system, the dose-dependent inhibition was defined by kinetic equations describing competitive inhibition at the lipid/water interface. The apparent dissociation constant for the inhibitor bound to the enzyme at the interface (KI*app) was determined to be 0.5 +/- 0.1 mole% versus an apparent dissociation constant for the arachidonate-containing phospholipid of 0.4 +/- 0.1 mole%. Thus, 1 represents a novel structural class of inhibitors of cPLA2 which partitions into the phospholipid bilayer and competes with the phospholipid substrate for the active site.

Presence of glycerol masks the effects of phosphorylation on the catalytic efficiency of cytosolic phospholipase A2.

Cytosolic phospholipase A2 catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. The enzymatic activity of cPLA2 is affected by several mechanisms, including substrate presentation and the phosphorylation state of the enzyme. Using covesicles of 1-palmitoy1-2-arachidonoyl-[arachidonoyl-1-14C]-8n-glycero-3 -phosphocholine and 1,2-dimyristoyl-phosphatidylmethanol as substrate, the effects of phosphorylation on the interfacial binding and catalytic constants were investigated. Phosphorylated and dephosphorylated enzyme forms were shown to have identical values of 2.6 microM for KMapp, an equilibrium dissociation constant which consists of the intrinsic dissociation constant from the lipid/water interface (Ks) and the dissociation constant for phospholipid from the active site (KM*). Moreover, the values of KM* for phosphorylated and dephosphorylated enzyme did not differ significantly (0.4 +/- 0.1 and 0.2 +/- 0.1, respectively). However, dephosphorylation of the enzyme reduced the value of kcat by 39%. The phosphorylation state of the enzyme had no effect on either the cooperativity shown by this enzyme or the thermal stability of the enzyme. Surprisingly, the presence of glycerol (4 M) masks the effect of phosphorylation on kcat. Instead, glycerol increased the value of kcat by 440% for the phosphorylated enzyme and by 760% for the dephosphorylated form. Moreover, addition of glycerol had only small effects on KMapp. the increase in the kcat upon addition of glycerol results from a substantial decrease in the activation energy from 29.4 to 14.8 kcal. mol-1. To determine whether the effects of phosphorylation of the enzyme or addition of glycerol are unique to this artificial substrate, membranes from U937 cells were isolated and used as substrate. With these membranes, the dephosphorylated enzyme was only 21% less active than the phosphorylated enzyme. In the presence of glycerol, there was no detectable difference the two enzyme forms, and the rate of hydrolysis was increased by 300-390% over that measured in the absence of glycerol. These results suggest that the catalytic efficiency of the phosphorylated enzyme is not particularly relevant to its activation in vivo. Moreover, it may be that glycerol is mimicking the effect of some unidentified factor which greatly enhances the catalytic efficiency of the enzyme.

Cooperativity and binding in the mechanism of cytosolic phospholipase A2.

Cytosolic phospholipase A2 (cPLA2) hydrolyzes the sn-2 ester of phospholipids and is believed to be responsible for the receptor-regulated release of arachidonic acid from phospholipid pools. The enzyme was assayed using vesicles containing arachidonate-containing phospholipid substrate, such as 1-palmitoyl-2-arachidonoylphosphatidylcholine (PAPC) or 1-stearoyl-2-arachidonoylphosphatidylinositol (SAPI), dispersed within vesicles of 1,2-dimyristoylphosphatidylmethanol (DMPM). We report here that the enzyme shows an apparent cooperative effect with respect to the mole fraction of arachidonate-containing phospholipids within these covesicles. The data can be fit to a modified Hill equation yielding Hill coefficients, n, of 2-3. This effect is unusual in that it is dependent on the nature of the sn-2 ester as opposed to the phosphoglycerol head group. This cooperativity is independent of both the concentration of glycerol, which greatly increases enzyme activity and stability, and the concentration of calcium, which facilitates the fusion of the covesicles. Surprisingly, 1-palmitoyl-2-arachidonoylphosphatidylethanolamine (PAPE) does not show the same cooperative effect, although the rate at which it is hydrolyzed is much greater when PAPC is present. Moreover, PAPE has a dissociation constant from the active site (KD* = 0.7 mol %) which is comparable to that of PAPC and SAPI (KD* values of 0.3 and 0.3 mol %, respectively). These results are consistent with the presence of an allosteric site that, when occupied, induces a change in the enzyme which facilitates enzymatic hydrolysis. If so, PAPC and SAPI, but not PAPE, must be able to bind to this allosteric site. Alternatively, this effect may result from changes in the physical nature of the bilayer which result upon increasing the bilayer concentration of arachidonate-containing phospholipids. This previously unobserved effect may represent another mechanism by which cells can regulate the activity of cPLA2.

Active recombinant human cytosolic phospholipase A2 is expressed in Escherichia coli.

The cDNA encoding human cytosolic phospholipase A2 (cPLA2) has been subcloned into a prokaryotic pET16b expression vector which also encodes an amino-terminal deca-histidine affinity tag to facilitate purification of the recombinant enzyme. Soluble, active fusion protein, designated His-cPLA2, has been obtained reproducibly from this expression system using the E. coli strain BL21 (DE3). The protein has been purified to homogeneity in four steps and the mass confirmed by electrospray mass spectrometry. His-cPLA2 was characterized by kinetic analysis which demonstrated that the enzyme is similar to native cPLA2 in all respects investigated. Specifically, the enzyme binds to anionic vesicles containing substrate, and acts processively on these vesicles. Enzymatic activity is supported by the presence of Ca2+ and several other divalent metal ions, and is inhibited by several transition metal ions. Finally, the enzyme demonstrates lysophospholipase activity and exhibits a high selectivity for sn-2 arachidonyl esters. This prokaryotic expression system yields moderate amounts of unmodified recombinant His-cPLA2 and is advantageous for rapid production of protein and mutational analyses.