Measurement Of Phospholipase Research Articles

Microfluidic sperm sorting selects a subpopulation of high-quality sperm with a higher potential for fertilization.

Is a microfluidic sperm sorter (MSS) able to select higher quality sperm compared to conventional methods? The MSS selects sperm with improved parameters, lower DNA fragmentation, and higher fertilizing potential. To date, the few studies that have compared microfluidics sperm selection with conventional methods have used heterogeneous study population and have lacked molecular investigations. The efficiency of a newly designed MSS in isolating high-quality sperm was compared to the density-gradient centrifugation (DGC) and swim-up (SU) methods, using 100 semen samples in two groups, during 2023-2024. Semen specimens from 50 normozoospermic and 50 non-normozoospermic men were sorted using MSS, DGC, and SU methods to compare parameters related to the quality and fertilizing potential of sperm. The fertilizing potential of sperm was determined by measurement of phospholipase C zeta (PLCζ) and post-acrosomal sheath WW domain-binding protein (PAWP) expression using flow cytometry, and the chromatin dispersion test was used to assess sperm DNA damage. In both normozoospermic and non-normozoospermic groups, the MSS-selected sperm with the highest progressive motility, PLCζ positive expression and PLCζ and PAWP fluorescence intensity the lowest non-progressive motility, and minimal DNA fragmentation, compared to sperm selected by DGC and SU methods (P < 0.05). The major limitations of our study were the low yield of sperm in the MSS chips and intentional exclusion of severe male factor infertility to yield a sufficient sperm count for molecular experiments; thus testing with severe oligozoospermic semen and samples with low count and motility is still required. In addition, due to ethical considerations, at present, it was impossible to use the sperm achieved from MSS in the clinic to assess the fertilization rate and further outcomes. Our research presents new evidence that microfluidic sperm sorting may result in the selection of high-quality sperm from raw semen. This novel technology might be a key to improving clinical outcomes of assisted reproduction in infertile patients. The study is funded by the Iran University of Medical Sciences and no competing interest exists. N/A.

Chemical fixation to arrest phospholipid signaling for chemical cytometry

Chemical cytometry is a powerful tool for measuring biological processes such as enzymatic signaling at the single cell level. Among these technologies, single-cell capillary zone electrophoresis (CZE) has emerged as a powerful tool to assay a wide range of cellular metabolites. However, analysis of dynamic processes within cells remains challenging as signaling pathways are rapidly altered in response to changes in the cellular environment, including cell manipulation and storage. To address these limitations, we describe a method for chemical fixation of cells to stop the cellular reactions to preserve the integrity of key signaling molecules or reporters within the cell and to enable the cell to act as a storage reservoir for the reporter and its metabolites prior to assay by single-cell CZE. Fluorescent phosphatidylinositol 4,5-bisphosphate reporters were loaded into cells and the cells were chemically fixed and stored prior to analysis. The reporter and its metabolites were electrophoretically separated by single-cell CZE. Chemical fixation parameters such as fixative, fixation time, storage solution, storage duration, and extraction solution were optimized. When cells were loaded with a fluorescent C6- or C16-PIP2 followed by glutaraldehyde fixation and immediate analysis, 24±2% and 139±12% of the lipid was recoverable, respectively, when compared to an unfixed control. Storage of the cells for 24h yielded recoverable lipid of 61±3% (C6-PIP2) and 55±5% (C16-PIP2) when compared to cells analyzed immediately after fixation. The metabolites observed with and without fixation were identical. Measurement of phospholipase C activity in single leukemic cells in response to an agonist demonstrated the capability of chemical fixation coupled to single-cell CZE to yield an accurate snapshot of cellular reactions with the probe. This methodology enables cell assay with the reporter to be separated in space and time from reporter metabolite quantification while preserving assay integrity.

Characterisation of human recombinant somatostatin receptors. 4. Modulation of phospholipase C activity

Total [3H]phosphoinositide (IPx) accumulation, a measure of phospholipase C (PLC) activity, induced by somatostatin (somatotropin release-inhibiting factor, SRIF) and cortistatin (CST) analogues was studied at human somatostatin receptor subtypes 1-5 (hsst1-5) recombinantly expressed in CCL39 (Chinese hamster lung fibroblast) cells. SRIF14 (10 microM) stimulated total [3H]-IPx production 200% and 1070% over basal levels, and increased intracellular Ca2+ ([Ca2+]i) 1600% and 2790%, in cells expressing hsst3 and hsst5 receptors, respectively. The SRIF14-stimulated IPx production was partly blocked by 100 ng/ml pertussis toxin (PTX) (30% and 15% inhibition, respectively). At hsst1, hsst2, and hsst4 receptors, only weak or no stimulation of PLC activity was found (Emax = 114%, 122%, and 102%, respectively). Consequently, hsst3 and hsst5 receptors were subjected to more detailed studies to establish pharmacological profiles of PLC stimulation. At hsst3 receptors, the relative efficacies of most ligands were in the same range (maximum response Emax = 218-267%). At hsst5 receptors Emax varied over a broad range, seglitide, CST17, SRIF28 displaying almost full agonism compared to SRIF14, whereas octreotide and BIM 23052 showed very low partial agonism. BIM 23056 behaved as an antagonist on SRIF14-induced total [3H]-IPx accumulation with a pKB (negative logarithm of antagonist binding constant) of 6.74 at hsst3 receptors, and of 6.94 at hsst5 receptors. The putative cycloantagonist SA showed weak antagonist activity on SRIF14-induced total [3H]-IPx levels at hsst3 (pKB = 5.85), but not at hsst5 receptors. The [3H]-IPx accumulation profiles at sst3/sst5 receptors were compared to their respective radioligand binding ([125I]LTT-SRIF28, [125I][Tyr10]CST14, [125I]CGP 23996, [125I][Tyr3]octreotide binding), to [35S]GTPgammaS binding, and to forskolin-stimulated adenylate cyclase (FSAC) inhibition profiles determined previously in CCL39 cells. The different affinity profiles correlated relatively well at both receptor subtypes with PLC activation (sst3: r = 0.90-0.97; sst5: r = 0.80-0.87). However, [35S]GTPgammaS binding correlated only minimally with stimulation of [3H]-IPx levels at sst5 receptors (r = 0.59), but rather well at sst3 receptors (r = 0.80). A moderate correlation was also observed between inhibition of FSAC activity and stimulation of PLC activity for hsst3 and hsst5 receptors with correlation coefficients of 0.85 and 0.70, respectively. In summary, most SRIF analogues behave as full agonists at hsst3 receptors and agonist-induced phosphoinositide turnover correlates well with radioligand binding, [35S]GTPgammaS binding and inhibition of adenylate cyclase activity, all measured in CCL39 cells. By contrast, at hsst5 receptors, most SRIF analogues behave as intermediate or very low partial agonists (although receptor levels are comparatively high, 7000 vs. 400 fmol/mg), and the agonist-induced phosphoinositide turnover correlates rather poorly with radioligand binding, [35S]GTPgammaS binding or inhibition of adenylate cyclase activity, all measured in the same cell line. Agonist-induced phosphoinositide turnover, [35S]GTPgammaS binding and inhibition of adenylate cyclase activity, show differences both in the rank orders of potency and relative efficacy at hsst3 and markedly at hsst5 receptors, suggesting either that PLC activity is functionally irrelevant or, more probably, that agonist-dependent receptor trafficking is taking place in CCL39 cells.

Measurement of phospholipase D activity.

Measurement of phospholipase D activity.

Role of G-protein beta gamma subunits in the augmentation of P2Y2 (P2U)receptor-stimulated responses by neuropeptide Y Y1 Gi/o-coupled receptors.

Neuropeptide Y (NPY) significantly potentiates the constrictor actions of noradrenaline and ATP on blood vessels via a pertussis toxin (PTX)-sensitive mechanism involving Gi/o (alpha beta gamma) protein subunits (Gi/o, GTP-binding proteins sensitive to PTX). In Chinese hamster ovary K1 (CHO K1) cells expressing specific receptors for these neurotransmitters, stimulation of Gi/o protein-coupled receptors for NPY and other neurotransmitters can augment the Gq/11-coupled (Gq/11, GTP-binding proteins insensitive to PTX) alpha 1B adrenoceptor- or ATP receptor-induced arachidonic acid (AA) release and inositol phosphate (IP) production (early events which may precede vasoconstriction). In this study, we have assessed the role of G beta gamma subunits in the synergistic interaction between Gi/o- (NPY Y1, 5-hydroxytryptamine 5-HT1B, adenosine A1) and Gq/11- [ATP P2Y2 (P2U)]-coupled receptors on AA release by using the specific abilities of regions of the beta-adrenergic receptor kinase (beta ARK1 residues 495-689) and the transducin alpha subunit to associate with G-protein beta gamma subunit dimers and to act as G beta gamma subunit scavengers. Transient expression of beta ARK1(495-689) in CHO K1 cells heterologously expressing NPY Y1 receptors had no significant effect on the PTX-insensitive ability of ATP to stimulate AA release. Stimulation of NPY Y1 receptors (as well as the endogenous 5-hydroxytryptamine 5-HT1B receptor and the transiently expressed human adenosine A1 receptor) resulted in a PTX-sensitive augmentation of ATP-stimulated AA release, which was inhibited by expression of both G beta gamma subunit scavengers. Expression of beta ARK1(495-689) similarly inhibited NPY Y1 receptor augmentation of ATP-stimulated IP production (a measure of phospholipase C activity), a step thought to precede the NPY Y1 receptor-augmented protein kinase C-dependent AA release previously observed in these cells. These experiments demonstrate that G beta gamma subunits, as inhibited by two different G beta gamma scavengers, significantly contribute to the synergistic interaction between NPY Y1 Gi/o- and Gq/11-coupled receptor activity, and are required for the augmentation of IP production and AA release observed in this model cell system.

Measurement of phospholipase A 2 and 1-alkylglycerophosphocholine acetyltransferase activities in stimulated alveolar macrophages by HPLC analysis of NBD-labeled ether lipids

The importance of phospholipases in cellular signaling and 1-alkylglycerophosphocholine acetyltransferase in the formation of platelet-activating factor (PAF) has stimulated demand for methods to measure these enzyme activities in inflammatory cells. Most of the assays currently used rely on radiolabeled substrates. We have synthesized NBD-labeled ether lipids as substrates for measuring enzyme activities of the PAF cycle and of lysosomal phospholipase A 2 (PLA 2). The fluorescent lipids were incubated with homogenates of stimulated bovine alveolar macrophages. The generated products were separated from the substrates by HPLC on a normal phase and monitored with a fluorescence detector. NBD-lyso-PAF was well accepted by acetyl- and acyltransferases of the cell-free preparations, which metabolized the substrate into NBD-PAF and NBD-alkyl-acylglycerophosphocholines. Homogenates of stimulated cells showed an enhanced production of NBD-PAF. The increased formation of the biological mediator was dependent on the nature of the stimuli and the time of stimulation. Lysosomal PLA 2 was measured with 1-0-(12-NBD-aminododecyl)-2-acyl- sn-glycero-3-phosphocholine as substrate. By varying the pH and the calcium concentration, it was possible to distinguish between the cytosolic PLA 2 and the lysosomal PLA 2 activity. Optimal conditions for the determination of the lysosomal PLA 2 were obtained at pH 4.5 and in the presence of EDTA. Stimulation with particulate agonists induced an enhancement of the lysosomal PLA 2 activity in macrophages.

Amyloid beta protein (25–35) stimulation of phospholipases A, C and D activities of LA-N-2 cells

[ 3H]Myristic acid prelabeled LA-N-2 cells were exposed to varying concentrations of amyloid beta protein (25–35), from 20 to 250 μg/ml, and the activation of phospholipases A and D estimated. A progressive increase in phosphatidylethanol formation, a measure of phospholipase D activity, and of free fatty acid release, a measure of phospholipase A activity, was observed over a time-course of 60 min. [ 3H]Inositol prelabeled LA-N-2 cells were exposed to varying concentrations of AβP, from 20 to 125 μg/ml, and phospholipase C activation was measured. There was an increased release of inositol phosphates in the presence of amyloid beta protein as a function of incubation time. The effects of adrenergic, metabotropic amino acid and bombesin antagonists on the AβP mediated stimulation of phospholipase C activity was investigated. Propranolol, a β adrenergic antagonist, 7-chlorokynurenic acid, a metabotropic amino acid antagonist, and [Tyr 4- d-Phe 12]bombesin, a bombesin antagonist, blunted the AβP stimulation of phospholipase C activity in [ 3H]inositol prelabeled L-AN-2 cells. This suggests that amyloid beta protein activation of phospholipase C may be receptor mediated. The phospholipase C inhibitor U 71322 prevented the activation of phospholipase C by AβP. However, this activation was not effected by tocopherol, propylgallate, or vitamin C.

The role of protein kinase C in arachidonic acid release and prostaglandin E production from CHO cells transfected with EGF receptors

Arachidonic acid release and prostaglandin production are stimulated by both phorbol esters and growth factors in various cell types. Whereas phorbol esters activate and transmit a signal via protein kinase C, this pathway is not necessarily involved in growth factor signal transduction. We investigated the involvement of protein kinase C in the pathway of arachidonic acid metabolism by CHO cells transfected with full-length EGF receptor (CHOwt). Two isoforms of protein kinase C were identified in CHOwt cells, α and ζ. On downregulation, the parallel loss of phorbol ester-stimulated arachidonic acid release and the α-isoform suggests a possible involvement of this isoform in phospholipase A 2 activation in these cells. In addition, we propose that the ζ-isoform may be separately involved in prostaglandin production as residual phorbol ester-stimulation of PGE production occurs in downregulated cells where PKCζ is the sole remaining isoform. EGF stimulation of arachidonic acid release, as a measure of phospholipase A 2 activation, and subsequent prostaglandin production are unaffected by inhibition of protein kinase C in CHOwt cells. Indeed one such inhibitor, staurosporine, augmented the EGF effect. These results suggest that PKC is not required for EGF activation of phospholipase A 2 in these cells.

Calcium and the Endothelin-1 and α1-Adrenergic Stimulated Phosphatidylinositol Cycle in Cultured Rat Cardiomyocytes

Cultured neonatal rat cardiac myocytes have been utilized as a model for the study of the effect of variations in cytoplasmic free Ca2+ on the activity of phospholipase C, a key enzyme in agonist-stimulated signal transduction through the phosphoinositide pathway. Cells prelabelled with [3H]inositol were exposed to various agents in an attempt to modulate the cytoplasmic free Ca2+ concentration and the formation of [3H]inositolphosphates (15-30 min) in the presence of Li+ was taken as a measure of phospholipase C activity. Not the basal but the endothelin-1 (10-8 M) induced [3H]inositolphosphate production (15 min) was stimulated 1.54- and 1.43-fold by A23187 (10-8 μM external Ca2+) and 50 mM K+ (1.3 mM external Ca2+) treatment of cells, respectively. The phenylephrine (10-4 M)induced response was also stimulated (1.35-fold) by A23187, however it was 43% inhibited by high K+. Ouabain (10-8 μM) treatment of cells did not affect either or agonist stimulated phosphoinositide turnover. On the other hand, total removal of external free Ca2+ by addition of 50 μM ethylene glycol bis (β-aminoethyl ether) (N,N,N′,N′-tetraacetic acid strongly inhibited (75%) the endothelin-1 induced but not the basal phospholipase C activity. Endothelin-1 binding to its receptor was shown not to be inhibited by the absence of external Ca2+ while resynthesis of [3H]phosphatidylinositol 4,5-bisphosphate was not rate-limiting under this condition. The lack of external Ca2+ eventually resulted in total standstill of the ET-1 induced PtdIns turnover after 30 min. Although not always as predicted, effects on basal and agonist-activated phospholipase C were observed too when cells were treated with low Ca2+ medium, Ca2+ entry blocker nifedipine (1 μM) or Ca2+-channel agonist Bay K8644 (1 μM) but most of these effects were only seen after 90 min incubation. Fluorometric (fura-2) measurements showed that total removal of external free Ca2+ for a short period decreased, while short exposure to high K+ increased cytoplasmic free Ca2+ but neither Ca2+ free buffer or nifedipine nor Bay K8644 had any effect. Furthermore, in saponin-permeabilized cardiomyocytes we could demonstrate that basal as well as GTPγS (30 μM) stimulated phospholipase C activity was strongly activated by free Ca2+ in the concentration range of 0.1-10 μM. We conclude that in the intact cardiomyocyte the signalling pathway through phospholipase C/phosphatidylinositol 4,5-bisphosphate, stimulated by agonist-receptor interaction that activates GTP-binding proteins as does GTPγS, is likely be a Ca2+ dependent process.

Cholecystokinin JMV-180 and caerulein effects on the pancreatic acinar cell cytoskeleton.

We previously demonstrated that the supramaximally effective concentrations of caerulein caused marked changes in the apical cytoskeleton of the rat pancreatic acinar cell. These changes included ablation of microvilli, the terminal actin web, and intermediate filament bands. The present study was designed to elucidate part of the intracellular signalling mechanism mediating these changes. For these studies we used a cholecystokinin (CCK) analogue, CCK-JMV-180, that has been previously demonstrated not to inhibit enzyme secretion and to prevent the inhibition caused by caerulein. We investigated the effects of CCK-JMV-180 alone and in combination with supramaximal concentrations of caerulein on the morphology of the apical structures, on 1,2-diacylglycerol production (a measure of phospholipase C activity), and on amylase secretion in rat pancreatic acini. Supramaximally effective concentrations of caerulein caused inhibition of enzyme secretion. CCK-JMV-180 had no effect on the ultrastructure of the apical region of the acinar cell and it prevented the ablation of apical cytoskeleton induced by a supramaximal concentration of caerulein (10 nM). CCK-JMV-180 inhibited the increase in 1,2-diacylglycerol formation and the inhibition of amylase release caused by 10 nM caerulein. Mimicking the effect of 1,2-diacylglycerol on activation of protein kinase C with phorbol 12-myristate 13-acetate and reproducing changes in [Ca2+]i caused by 10 nM caerulein with 100 nM bombesin did not alter the apical cytoskeleton. These results suggest that the cytoskeletal changes observed with inhibitory concentrations of caerulein are caused by the phospholipase C effects of caerulein on membrane phospholipids.

Purification and properties of an antivenom factor from the plasma of the South American rattlesnake (Crotalus durissus terrificus)

Purification and properties of an antivenom factor from the plasma of the South American rattlesnake ( Crotalus durissus terrificus). Toxicon 29, 997–1008, 1991.—The lethal toxicity of Crotalus durissus terrificus (Crotalinae, Viperidae) can be attributed mainly to the presence of a neurotoxic protein, crotoxin, which also shows phospholipase A 2 activity. It has been previously demonstrated that both lethal and phospholipase A 2 activities of crotoxin can be neutralized by an alpha 1-globulin factor that is present in the homologous blood. Crotalus durissus terrificus plasma also renders some degree of protection to mice against the lethal toxicity of heterologous venoms from snakes of the genus Bothrops (Crotalinae, Viperidae), but not of the genus Micrurus (Elapinae, Elapidae). An anti-toxic factor was purified to homogeneity from C.d. terrificus plasma after three chromatographic steps (DEAE-Sephacel anion exchange, CM-Sepharose cation exchange and Pro-RPC reverse phase chromatography); it is named CNF for Crotalus neutralizing factor. The purification process was accompanied by polyacrylamide gel electrophoresis in the presence of SDS and by measurements of phospholipase A 2 inhibition. After the first two purification steps, an 86-fold increase of the inhibitory activity of CNF was observed; however, the third step caused an apparent inactivation of the factor. The inactive CNF was shown to correspond to the previously active plasma material and to be homogeneous on electrophoresis, immunoelectrophoresis and partial amino-terminal sequence. The mol. wt of CNF was estimated as 23,600 by SDS-PAGE.

Treatment with dexamethasone down-regulates IgE-receptor-mediated signals and up-regulates adenosine-receptor-mediated signals in a rat mast cell (RBL-2H3) line.

As reported by other workers, the treatment of rat basophilic leukemia RBL-2H3 cells with dexamethasone resulted in a marked decrease in responsiveness to Ag-stimulation. All responses measured, which included hydrolysis of inositol phospholipids, increase in concentration of cytosolic Ca2+, release of arachidonic acid and the secretion of serotonin, were suppressed, but once the cells were permeabilized the inhibitory actions of dexamethasone were no longer apparent. This suggested that all the necessary components of the stimulatory cascade were intact in the dexamethasone-treated cells. The measurement of phospholipase C activity in cell extracts and studies with phorbol ester also indicated that the cells contained a normal complement of phospholipase C and protein kinase C activity. We had previously shown that both Ag and the adenosine analog, 5'-(N-ethylcarboxamide)-adenosine, can activate phospholipase C, but they do so through different G proteins. Interestingly, the activation of phospholipase C by 5'-(N-ethylcarboxamide)-adenosine and the ensuing stimulatory events were markedly enhanced in dexamethasone-treated cells. The treatment with dexamethasone thus did not result in direct suppression of effector systems, but instead resulted in the selective modulation of the coupling between receptors and the effector systems by mechanisms that require soluble cytosolic factors.

The measurement of phospholipase A 2 activity in human myometrium: Physiological and pathological implications

Phospholipase A 2 activity was measured in human myometrium obtained at hysterectomy in a group of 41 patients using a double isotope ratio assay based on the liberation of [14-C] oleic acid from 1-palmitoyl-2-[14-C] oleoyl phosphatidylcholine. The enzyme was shown to be calcium independent and to have an optimum pH of 7. There was no significant difference (Mann Whitney U test) in myometrial phospholipase A 2 activity between proliferative and secretory phases of the menstrual cycle (ranges: 3.88 – 30.8 and 0.47 – 25.85 nmol/mg protein per h respectively) but there was a significant (P < 0.01) increase in activity in myometrium from uteri with fibroids (median 11.33, range 2.18–30.88 nmol/mg protein per h) compared to those without fibroids (median 6.94, range 0.31 – 25.85 nmol/mg protein per h). Myometrial phospholipase A 2 activity was significantly lower (P < 0.001) in the 33–40 age group (median 4.71, range 0.31 – 6.94) compared to the 41–50 age group (median 11.35, range 2.18–30.88 nmol/mg protein per h). In the 51–55 age group phospholipase A 2 activity (median 8.71, range 2.5–17.71 nmol/mg protein per h) was not significantly different from that of the other two groups. The increase in activity in the 41–50 age group was not due to the increased incidence of uterine fibroids. These findings suggest that myometrial phospholipase A 2 may be important in the pathophysiology of the uterus.

The role of phospholipase A activity in rat liver microsomal lipid peroxidation.

The involvement of phospholipase(s) A in lipid peroxidation of rat liver microsomes was investigated by: (a) determining the effects of phospholipase A inhibitors (p-bromophenylacyl bromide, chlorpromazine, mepacrine) on the accumulation of thiobarbituric acid reactivity or on levels of oxidized phospholipids in response to selected oxidative stimuli and (b) measurement of phospholipase A activities in response to these agents. Lipid peroxidation in response to various peroxidation systems was inhibited completely by exposure of microsomes to p-bromophenylacyl bromide (250 microM). The effectiveness of p-bromophenylacyl bromide was dependent on the presence of glutathione (200 microM) in preincubation mixtures. Chlorpromazine (100 microM) and mepacrine (100 microM) also effectively inhibited peroxidation, and their potency was independent of glutathione. The accumulation of oxidized phospholipids in response to the potent peroxidation stimulus alloxan/ferrous ion was similarly inhibited by p-bromophenylacyl bromide, although the level of oxidized phospholipid in response to the initiator ADP/ferrous ion was not affected. Microsomal phospholipase A1 activity, assessed using a liposomal substrate, was substantially enhanced by promoters of lipid peroxidation. Phospholipase A2 activity was not detected using a liposomal substrate but was evident using radiolabeled microsomes as endogenous substrate and was enhanced by oxidative stimuli. We conclude that phospholipase A activity may play an integral role in the microsomal lipid peroxidation mechanism. Based on this study, we hypothesize a role for phospholipases in facilitating propagation reactions.

Immunologic and biochemical evaluation of the potency of whole insect body extracts

Recent studies have indicated that currently available whole body extracts have little potency and are ineffective for diagnosis and treatment of stinging insect allergy. Pure venom is a potent effective allergen but is difficult to obtain in sufficient quantities from all Hymenoptera species. In these studies, an attempt was made to prepare a potent whole body extract. Whole bee body extracts were prepared with different extraction periods and at cold and room temperatures. Potency was examined biochemically by measurements of phospholipase A (PLA) activity and immunologically by PLA and bee venom radioallergosorbent test (RAST) inhibition experiments and gel diffusion studies with the use of rabbit antisera. All extracts prepared in the laboratory had some potency, indicating that it is possible to make a whole body extract containing small quantities of PLA or bee venom. However, the potency of these extracts was minimal as compared with bee venom. Three commercial extracts were almost devoid of detectable immunologic activity. While further attempts may be made to prepare a potent whole body insect extract, these results suggest that it is necessary to obtain venom in relatively pure form for the diagnosis and treatment of stinging insect allergy.