An investigation of mechanical nociceptive thresholds in dogs with hind limb joint pain compared to healthy control dogs.

This study investigated the effects of osteoarthritis (OA) on somatosensory processing in dogs using mechanical threshold testing. A pressure algometer was used to measure mechanical thresholds in 27 dogs with presumed hind limb osteoarthritis and 28 healthy dogs. Mechanical thresholds were measured at the stifles, radii and sternum, and were correlated with scores from an owner questionnaire and a clinical checklist, a scoring system that quantified clinical signs of osteoarthritis. The effects of age and bodyweight on mechanical thresholds were also investigated. Multiple regression models indicated that, when bodyweight was taken into account, dogs with presumed osteoarthritis had lower mechanical thresholds at the stifles than control dogs, but not at other sites. Non-parametric correlations showed that clinical checklist scores and questionnaire scores were negatively correlated with mechanical thresholds at the stifles. The results suggest that mechanical threshold testing using a pressure algometer can detect primary, and possibly secondary, hyperalgesia in dogs with presumed osteoarthritis. This suggests that the mechanical threshold testing protocol used in this study might facilitate assessment of somatosensory changes associated with disease progression or response to treatment.

Influence of experimental protocol on response rate and repeatability of mechanical threshold testing in dogs.

Mechanical threshold (MT) testing is widely used to measure nociceptive thresholds. However, there has been little research into factors that contribute to the response rate and repeatability (collectively termed 'efficacy') of MT testing protocols. The aim of this study was to investigate whether the efficacy of a protocol using a hand-held algometer to measure MTs (N) in healthy dogs (n = 12) was affected by varying (1) the area over which force was applied (tip diameter), (2) rate of force application, (3) position of dog during testing, and (4) anatomical site of testing. The effect of these factors on MT and the impact of individual dog effects on both efficacy and MT were also investigated. Overall, 3175/3888 tests (82%) resulted in a measurable response. The response rate was reduced by using wider tip diameters, testing at the tibia, and testing when the dog was lying down (compared to sitting upright). Wider tips were associated with higher, more variable MTs (mean ± standard deviation) with values of 4.18 ± 2.55 N for 2 mm diameter tips, 5.54 ± 3.33 for those of 4 mm, and 7.59 ± 4.73 for 8 mm tips. Individual dog effects had the most significant impact on efficacy and MT. The findings indicate that tip diameter, dog position, and anatomical site may affect both protocol efficacy and MTs, and should be taken into account when comparing different studies and in designing protocols to measure MTs in dogs. The predominant effect of the individual dog over other factors indicates that between-subject differences should always be accounted for in future studies.

Dynamic changes in maternal decidual leukocyte populations from first to second trimester gestation.

INTRODUCTION: Decidual leukocytes are critical to the development of the fetomaternal interface, regulating tolerance to the semi-allogeneic fetus and vascular transformation of the uterine spiral arteries. Despite the continuation of these processes beyond the first trimester of pregnancy, the second trimester has largely been unstudied, with investigation focusing on early gestation and term tissues. We sought to characterize changes in decidual leukocyte populations from first to second trimester. METHODS: Multicolor flow cytometry was performed on isolated decidual leukocytes from elective terminations of pregnancy between 6 and 20 weeks of gestation for study of first (6-12 weeks) and second trimesters (13-20 weeks). Specific subpopulations were identified by comparison to isotype and fluorescent-minus-one (FMO) controls. RESULTS: Decidual natural killer cells (CD56(+)CD16(-)CD3(-)) did not change in number, although a population of dNK with decreased CD56 brightness was observed in second trimester decidua. CD14(+)HLA-DR(+) macrophage numbers declined from first to second trimester (p = 0.031), yet a CD163(+)CD206(+) subset designating alternatively activated M2-like macrophages increased during the same period (p = 0.015). Intermediate CD205(+) dendritic cells demonstrated significant decline (p = 0.022), but immature CD209(+) and mature CD83(+) dendritic cells did not differ between trimesters. Total CD3(+) and CD3(+)CD4(+) T lymphocytes increased (p = 0.0079, p = 0.0028); CD3(+)CD8(+) T cells trended towards increase but did not differ significantly. CONCLUSION: Several changes in leukocyte subsets are observed in the second trimester that promote a tolerogenic and angiogenic decidual microenvironment through mid-gestation.

A role for the mitochondrial-associated protein p32 in regulation of trophoblast proliferation.

p32 is a conserved eukaryotic protein which is primarily expressed in the mitochondria and regulates cell proliferation, migration and metabolism in various tissues. In this study, we sought to examine the expression and function of p32 in the human placenta. p32 was highly expressed in the syncytiotrophoblast, the underlying cytotrophoblast (CTB), the vascular endothelium and by a proportion of cells in the villous stroma in first trimester and term placenta. p32 mRNA and protein expression was significantly higher in the first trimester of pregnancy than at term, and expression in the trophoblast was significantly reduced in placentas from women with fetal growth restriction (FGR). Small interfering RNA (siRNA)-mediated knockdown of p32 in term placental explants significantly reduced the number of Ki67-positive CTB, but did not alter CTB apoptosis or necrosis. p32 knockdown increased lactate production, reduced glucose extraction from culture medium and was associated with reduced MitoTracker dye accumulation in trophoblast mitochondria. p32 knockdown was also associated with a significant reduction in expression of the mitochondrial respiratory complexes I and IV. These data suggest that p32 expression is important for CTB proliferation, via a mechanism involving regulation of normal mitochondrial function. As p32 expression is reduced in FGR placentas, this may contribute to some of the observed placental pathology, such as reduced CTB proliferation and mitochondrial dysfunction.

IFPA Meeting 2010 Workshop Report I: Immunology; ion transport; epigenetics; vascular reactivity; epitheliochorial placentation; proteomics.

Workshops are an important part of the IFPA annual meeting. At IFPA Meeting 2010 there were twelve themed workshops, six of which are summarized in this report. 1. The immunology workshop focused on normal and pathological functions of the maternal immune system in pregnancy. 2. The transport workshop dealt with regulation of ion and water transport across the syncytiotrophoblast of human placenta. 3. The epigenetics workshop covered DNA methylation and its potential role in regulating gene expression in placental development and disease. 4. The vascular reactivity workshop concentrated on methodological approaches used to study placental vascular function. 5. The workshop on epitheliochorial placentation covered current advances from in vivo and in vitro studies of different domestic species. 6. The proteomics workshop focused on a variety of techniques and procedures necessary for proteomic analysis and how they may be implemented for placental research.

IFPA Gabor Than Award lecture: Transformation of the spiral arteries in human pregnancy: key events in the remodelling timeline.

During human pregnancy, the uterine spiral arteries are progressively remodelled to form dilated conduits lacking maternal vasomotor control. This phenomenon ensures that a constant supply of blood is delivered to the materno-fetal interface at an optimal velocity for nutrient exchange. Conversion of a tonic maternal arteriole composed of multiple layers of vascular smooth muscle, elastin and numerous other extracellular matrix components, into a highly dilated yet durable vessel, requires tight regulatory control and the coordinated actions of multiple cell types. Initial disruption of the vascular wall, characterised by foci of endothelial cell loss, and separation and misalignment of vascular smooth muscle cells (VSMC), is coincident with an influx of uterine natural killer (uNK) cells and macrophages. uNK cells are a source of angiogenic growth factors and matrix degrading proteases, thus they possess the capacity to initiate changes in VSMC phenotype and instigate extracellular matrix catabolism. However, complete vascular cell loss, mediated in part by apoptosis and dedifferentiation, is only achieved following colonisation of the arteries by extravillous trophoblast (EVT). EVT produce a variety of chemokines, cytokines and matrix degrading proteases, enabling them to influence the fate of other cells within the placental bed and complete the remodelling process. The complex interplay of cell-cell and cell-matrix interactions required for effective vascular transformation will be examined, with a particular focus on the role of (i) uNK cells and (ii) the enzyme matrix metalloproteinase-12 (MMP-12). Parallels with remodelling events occurring in other vascular beds will also be drawn.

Review: Trophoblast-vascular cell interactions in early pregnancy: how to remodel a vessel.

During the first twenty weeks of human pregnancy, extravillous trophoblasts (EVT) colonise the decidua and remodel the uterine spiral arteries as far as the first third of the myometrium. This process leads to an irreversible vasodilatation, ensuring that maximal blood flow is delivered to the materno-fetal interface at an optimal velocity for nutrient exchange. There is accumulating evidence that subtle changes in vascular structure precede EVT colonisation; however, full physiological transformation is only achieved in the presence of trophoblast. This review discusses the mechanisms employed to facilitate arterial dilatation, including recent data regarding the contribution of vascular cell apoptosis, the importance of elastin catabolism and the source of candidate elastases. It also examines how the complex interplay between EVT, endothelial cells, smooth muscle cells and decidual leukocytes (macrophages and uterine natural killer cells) leads to enhanced receptivity to invasion, vascular cell loss and extracellular matrix remodelling.

Plasma from women with preeclampsia inhibits trophoblast invasion.

To assess whether plasma from women with preeclampsia altered trophoblast invasion, SGHPL-4 extravillous trophoblasts were treated with pooled plasma from women with preeclampsia (PE-P; 10%) or with plasma from healthy pregnant controls (C-P). PE-P significantly inhibited SGHPL-4 invasion through Matrigel-coated transwells (P < .01), reduced mitochondrial dehydrogenase activity (P < .01), and increased apoptosis (P < .05); however, invading cells were no more susceptible to PE-P-induced apoptosis than their static counterparts. C-P did not alter rates of invasion, proliferation, or apoptosis. The bioactivity of PE-P was retained after removal of the 6 most abundant plasma proteins using an immunodepletion column (P < .05). Fractionation of PE-P demonstrated that the reduction in invasion was predominantly mediated by factors >100 kd in size. The authors conclude that plasma from women with preeclampsia contains multiple factors that inhibit invasion. These factors do not specifically target invading cells, but instead may reduce the number of cells available to invade.

Adhesion molecules in human trophoblast - a review. II. extravillous trophoblast.

At the tips of anchoring villi, cytotrophoblast (CTB) proliferation leads to a process of multilayering in which cells lose their attachment to the villous basement membrane and develop into columns, within which they adhere to one another using desmosomes, with associated intermediate filament bundles. Non-desmosomal cadherins, tight junction proteins and other adhesion molecules are also present, suggesting that actin-associated adhesions contribute to placental anchorage. In the distal columns, cell-cell interactions diminish, cells upregulate beta1 integrins and bind to a provisional fibrinoid extracellular matrix, eventually detaching to migrate into the decidual stroma and myometrium, where interstitial and endovascular extravillous trophoblast (EVT) populations show distinct repertoires of adhesion molecules.

Adhesion molecules in human trophoblast - a review. I. Villous trophoblast.

In the placental villus, cells attach to basement membrane via integrin alpha6beta4 and adhere both laterally and apically to their neighbours. The most prominent adhesive specialisation seen using the electron microscope is the desmosome, which connects cytotrophoblast cells (CTB) laterally and also contributes to the attachment of CTB to the overlying syncytium. However, numerous cadherins and other junctional proteins are also present in the corresponding plasma membrane domains, indicating a multiplicity of adhesive interactions. Integrins, tight junction components and cadherins are all found in the syncytial microvillous membrane, perhaps reflecting its ability to form intersyncytial bridges. There is a wide gulf to be filled between molecular anatomy and functional studies, with much to be learned about the role of adhesion molecules in regulating villous epithelial integrity, homeostasis and growth.

Trophoblast-derived heparanase is not required for invasion.

To invade the decidua and myometrium, extravillous trophoblast must degrade an assortment of extracellular matrix (ECM) components. The uterine wall is rich in heparan sulphate proteoglycans (HSPG), which interact with collagen, laminin and fibronectin, and bind a variety of growth factors. HSPG are catabolised by heparanase, an enzyme that is highly expressed in the placenta. The aim of this study was to investigate the role of heparanase in first trimester trophoblast invasion. First trimester cytotrophoblasts (CTB) were isolated by trypsin digestion followed by centrifugation on a Percoll gradient. Cells were cultured on Matrigel to promote an extravillous phenotype. Heparanase expression was studied by immunohistochemistry and confocal microscopy. Trophoblast invasion was assessed using an in vitro transwell assay. A high level of heparanase was observed in isolated first trimester trophoblast; however, a function-blocking antibody did not inhibit invasion of primary CTB or the extravillous trophoblast cell line SGHPL-4 at 21% oxygen. In contrast to cancer cells, heparanase expression was not increased following culture at 3% oxygen, and trophoblast invasion was not retarded by the blocking antibody under these conditions. Heparanase expression was observed in stromal cells and vascular endothelium in first trimester parietal decidua. Expression was evident on the cell surface and in the nucleus of trophoblast and decidual cells. In conclusion, trophoblast heparanase is not required for invasion in vitro. Its abundant expression suggests another role during pregnancy, perhaps in controlling the availability of ECM-bound growth factors or acting as a transcription factor.

A re-appraisal of the morphophenotype and basal lamina coverage of cytotrophoblasts in human term placenta.

A recent study of human placental villi [Mori et al., The cytotrophoblast layer of human chorionic villi becomes thinner but maintains its structural integrity during gestation, Biol Reprod 76 (2007) 164-172] concluded that cytotrophoblast (CT) cells occupy 80% of the basal lamina (BL) surface at term and that syncytiotrophoblast (ST) does not make direct contact with the BL. Based on SPINT-1 localisation using immunofluorescence on cryosections, these conclusions run counter to previous light and electron microscopic data suggesting that term CT cells cover no more than about 24% of the BL surface. To resolve these discrepancies, we have undertaken a stereological study of term placenta using transmission electron microscopy (TEM) and a novel immunofluorescence approach. Test line lattices were randomly superimposed on TEM images of villous trophoblast from 13 normal term placentae. Intersections with the test lines were counted to assess the fractional surface of BL occupied by CT cells. After trypsin-mediated removal of syncytium, cells in whole-mounted term and first trimester villi were stained with cytokeratin 7 to identify CT and then visualised by confocal microscopy. CT formed an almost continuous layer in the first trimester. In contrast, term CT cells and their processes were found to cover only 44% (SD 14%) of the BL surface with intervening regions occupied by ST. TEM and confocal images were consistent with the concept of a network of 'octopoid' CT cells with fine processes extending from a central cell body. Our estimates of CT coverage are lower than the recent immunofluorescence estimate but greater than earlier TEM estimates. The former may have been biased by overprojection (section thickness) effects whilst the latter may be underestimates due to failure to include the fine CT cell processes. We conclude that CT cells transform from a cuboidal phenotype early in gestation to flattened cells with multiple interconnecting processes. The CT layer thins but maintains a functional network within which cells intercommunicate without compromising substance transfer via the syncytium.

Regulation of trophoblast invasion - a workshop report.

Trophoblast invasion during placental development helps to establish efficient physiological exchange between maternal and fetal circulatory systems. Trophoblast stem cells differentiate into multiple subtypes, including some that are highly invasive. Signalling to the trophoblast from decidua, uterine natural killer cells and vascular smooth muscle can regulate extravillous trophoblast differentiation. Important questions remain about how these cellular interactions promote trophoblast invasion and the signalling pathways that are involved. New and established biological models are being used to experimentally examine these interactions and the underlying molecular mechanisms.

BeWo cells stimulate smooth muscle cell apoptosis and elastin breakdown in a model of spiral artery transformation.

BACKGROUND: During pregnancy, extravillous trophoblast invades the uterine wall and enters the spiral arteries. Remodelling ensues, with loss of vascular smooth muscle cells (SMCs) to create high flow, low resistance vessels. Pregnancies complicated by pre-eclampsia are characterized by incomplete arterial remodelling. Endovascular trophoblast is not easily accessible for studies to establish the pathogenesis of pre-eclampsia, so we have developed a model appropriate to carry out mechanistic studies of vessel wall transformation. METHODS AND RESULTS: Segments of human spiral artery were perfused with the choriocarcinoma cell line, BeWo; cells invaded the vessel wall and induced apoptosis of vascular SMC. Perfusion of vessels with BeWo-conditioned medium also induced SMC apoptosis, indicating the presence of a soluble apoptotic factor. BeWo express Fas ligand (FasL) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Treatment of BeWo-conditioned medium with antibodies against FasL inhibited vascular SMC apoptosis in vitro. Antibodies that blocked TRAIL receptor function had no effect. Extracellular matrix degradation is also a prerequisite for vascular remodelling; BeWo express matrix metalloproteinase-12 (MMP-12) and BeWo-conditioned medium increased MMP-12 expression in spiral artery SMC. CONCLUSIONS: BeWo induce arterial remodelling via FasL- and MMP-12-dependent mechanisms. BeWo-derived factors up-regulate protease expression in spiral artery SMC to facilitate matrix breakdown.

Traumatic brain injury-induced changes in gene expression and functional activity of mitochondrial cytochrome C oxidase.

Traumatic brain injury (TBI) is documented to have detrimental effects on CNS metabolism, including alterations in glucose utilization and the depression of mitochondrial oxidative phosphorylation. Studies on mitochondrial metabolism have also provided evidence for reduced activity of the cytochrome oxidase complex of the electron transport chain (complex IV) after TBI and an immediate (lhr) reduction in mitochondrial state 3 respiratory rate, which can persist for up to 14 days postinjury. Using differential display methods to screen for differences in gene expression, we have found that cytochrome c oxidase II (COII), a mitochondrial encoded subunit of complex IV, is upregulated following TBI. Since COII carries a binding site for cytochrome c in the respiratory chain, and since it is required for the passage of chain electrons to molecular oxygen, driving the production of ATP, we hypothesized that metabolic dysfunction resulting from TBI alters COII gene expression directly, perhaps influencing the synaptic plasticity that occurs during postinjury recovery processes. To test this hypothesis, we documented COII mRNA expression and complex IV (cytochrome c oxidase) functional activity at 7 days postinjury, focusing on the long-term postinjury period most closely associated with synaptic reorganization. Both central fluid percussion TBI and combined TBI and bilateral entorhinal cortical lesion were examined. At 7 days survival, differential display, RT-PCR, and Northern blot analysis of hippocampal RNA from both TBI and combined insult models showed a significant induction of COII mRNA. This long-term elevation in COII gene expression was supported by increases in COII immunobinding. By contrast, cytochrome oxidase histochemical activity within tissue sections from injured brains suggested a reduction of complex IV activity within the TBI cases, but not within animals subjected to the combined insult. These differences in cytochrome c oxidase activity were supported by in vitro assay of complex IV using cerebral cortical and hippocampal tissues. Our present results support the hypothesis that COII is selectively vulnerable to TBI and that COII differences may indicate the degree of metabolic dysfunction induced by different pathologies. Taken together, such data will better define the role of metabolic function in long-term recovery after TBI.

Secretory phospholipase A2 levels in rat small bowel.

Preliminary studies on ischemia/reperfusion injury in transplanted small bowel grafts showed that secretory phospholipase A2 (sPLA2) may play a substantial role by breaking down membrane phospholipids. This study sought to determine the normal values of sPLA2 in the rat small bowel as a function of site and length as a baseline for future studies. The entire small bowel of male Lewis rats (200 g) was flushed with normal saline to eliminate solid contents. In group 1, the entire small bowel was divided into 5-cm segments (numbered 1-9), which were snap frozen and processed the same day for sPLA2. In group 2, a 25-cm segment of bowel (corresponding to segments 2-6 in group 1) was harvested from each animal, snap frozen, and immediately processed for sPLA2. To assess the effect of bowel storage on enzyme content, group 3 and group 4 grafts were stored for 7 and 14 days, respectively, at -85 degrees C prior to processing. All samples were homogenized in buffer, extracted with H2SO4 and assayed for sPLA2 activity using [1-14C]oleate-labeled autoclaved Escherichia coli as substrate. Results were analyzed statistically by ANOVA. sPLA2 activity rose from 85.46 +/- 14.46% hydrolysis/min fraction-1 in segment 1, to 476.38 +/- 176.75% hydrolysis/min fraction-1 in segment 9. The increase was linear and statistically significant (p < .0001). There was no significant difference in enzymatic activity between groups 2, 3, and 4. Group 2 activity was 263.02 +/- 43.74% hydrolysis/min fraction-1. This value was not statistically different from the mathematically calculated mean of segments 2-6 in group 1 (237.75). The results show that (1) sPLA2 activity increases predictably with distance from the ligament of Treitz (2) storage at -85 degrees C does not affect sPLA2, activity, and (3) 25-cm grafts may be evaluated in toto with reproducible baseline enzyme activity. Given the variability of enzyme activity along the course of the rat small bowel, it is imperative that exact location be identified in any studies evaluating sPLA2 activity.

Human neutrophils store type II 14-kDa phospholipase A2 in granules and secrete active enzyme in response to soluble stimuli.

Although "secretory" type II 14-kDa phospholipase A2 (sPLA2) activity has been described in neutrophils, direct evidence of enzyme secretion has been elusive. We have used immunogold electron microscopy with polyclonal and monoclonal antibodies to sPLA2 to demonstrate localization of the enzyme to granules of resting human neutrophils and translocation to phagolysosomes. Soluble stimuli such as calcium ionophore A23187 stimulate loss of cell-associated enzymatic activity. Supernatant fluids from stimulated neutrophils lack measurable PLA2 but contain proteases which inactivate exogenous sPLA2. The use of alpha-1-antitrypsin as a protease inhibitor permitted this first demonstration of secretion of PLA2 activity from stimulated human neutrophils.

Persistent or recurrent bronchogenic carcinoma: detection with PET and 2-[F-18]-2-deoxy-D-glucose.

PURPOSE: To assess positron emission tomography (PET) with 2-[fluorine-18]-2-deoxy-D-glucose (FDG) in the differentiation of recurrent bronchogenic carcinoma from fibrosis after therapy. MATERIALS AND METHODS: Any patient treated for bronchogenic carcinoma who had a residual chest radiographic abnormality was eligible. Forty-three patients (mean age, 63.5 years) participated. Chest radiographs and thoracic computed tomographic scans helped localize the abnormality prior to PET. Semiquantitative analysis was performed on FDG PET images with calculated standardized uptake ratios (SURs). Sensitivity, specificity, and confidence intervals for recurrent disease were determined. RESULTS: Thirty-five patients had recurrent or persistent tumor (median SUR, 7.6; range, 1.9-18.7). Eight patients had fibrosis but no evidence of disease (SUR, 1.6; range, 0.6-2.4). The sensitivity for detecting recurrent tumor (SUR > 2.5) was 97.1%, and specificity was 100%. The SUR for recurrent tumor was statistically significantly higher than for fibrosis (P = .0001). CONCLUSION: FDG PET accurately helps differentiate recurrent bronchogenic carcinoma from fibrosis.

Sphingolipid metabolism and signal transduction: inhibition of in vitro phospholipase activity by sphingosine.

Sphingosine inhibits protein kinase C activity in vitro and has been used to implicate this enzyme in signal transduction and cell function. We report that sphingosine directly inhibits phospholipases A2 and D. Sphingosine inhibits Ca(2+)-dependent phospholipases A2 from Naja naja, porcine pancreas, Crotalus adamanteus, human disc and neutrophil in a dose-dependent manner with IC50 values ranging from 5-40 microM using [1-14C]oleate-labelled autoclaved E. coli (20 microM) as substrate. Inhibition is comparable using the same concentrations (20 microM) of [1-14C]oleate-labelled C. albicans or E. coli, or aqueous dispersions of 1-acyl-2-[1-14C]linoleoylglycerophosphoethanolamine or -choline. Sphinganine and stearylamine are as inhibitory as sphingosine; monoolein is less inhibitory (IC50 = 70 microM), while octylamine, N-acetylsphingosine, sphingomyelin and ceramide have no effect. Inhibition is relieved by increasing concentrations of substrate phospholipid. The molar ratio of sphingosine to phospholipid required for 50% inhibition ranges from 0.5 to 1.0 with 2-100 microM E. coli phospholipid. In contrast, sphingosine has a biphasic effect on the hydrolysis of E. coli by S. chromofuscus phospholipase D; concentrations less than or equal to 25 microM stimulate activity while concentrations greater than 25 microM are inhibitory. Addition of Triton X-100 eliminates both the stimulatory and inhibitory effects of sphingosine on phospholipase D activity.

[1-14C]oleate-labeled autoclaved yeast: a membranous substrate for measuring phospholipase A2 activity in vitro.

Radiolabeled, autoclaved yeast were tested as a substrate for mammalian phospholipase A2 activity because the only other membranous substrate used for this purpose, autoclaved Escherichia coli, totally lacks a major mammalian phospholipid, phosphatidylcholine. Candida albicans were grown in the presence of [1-14C]oleate and then autoclaved. Sixty three percent of the incorporated label was in yeast phospholipid, and more than 95% of that was in the 2-acyl position. The distribution of label in the yeast phospholipids (phosphatidylcholine and -ethanolamine, -serine + -inositol, and phosphatidic acid corresponded closely to the chemical distribution of phosphorus in those phospholipids. Snake venom (Naja naja) and human synovial fluid phospholipase A2 hydrolyzed yeast phospholipid exclusively to release 14C-labeled fatty acid. When 50-60% of the yeast phospholipid was hydrolyzed, the radioactive fatty acids as determined by gas-liquid chromatographic analysis were predominantly oleate (45%) and linoleate (greater than 54%). Hydrolysis of yeast phospholipid by both enzymes was near-linear with protein and time under conditions of optimal pH (neutral-alkaline) and Ca2- (1-5 mM) previously reported for optimal hydrolysis of autoclaved E. coli phospholipid. N. naja phospholipase A2 showed less preference for phosphatidylethanolamine than -choline as liposomes or yeast phospholipid as compared to human synovial fluid phospholipase A2 which clearly preferred phosphatidylethanolamine to -choline as a liposome or yeast phospholipid. These results illustrate that radiolabeled phospholipids of autoclaved yeast, enriched in phosphatidylcholine, are readily hydrolyzed by snake venom and human nonpancreatic phospholipases A2 and may, therefore, be useful in the measurement of in vitro enzymatic activity.