Are bilateral decubitus views necessary in assessing for vertebral compression fractures using DXA vertebral fracture assessment?

The purpose of this study is to assess the differences in VFA diagnostic accuracy when using bilateral decubitus views and whether diagnostic accuracy is affected by scoliosis. Our findings show that the current practice of performing only one side is valid; however, bilateral views can improve specificity in scoliosis. INTRODUCTION: The diagnostic accuracy of vertebral fracture assessment (VFA) can be influenced by poor patient position and scoliosis. This study aims to assess the differences in VFA diagnostic accuracy for right and left lateral decubitus views and the effect of scoliosis. METHODS: One hundred fourteen postmenopausal women received right and left lateral thoracolumbar spine dual-energy VFA and radiography. Cobb angles were measured from the posteroanterior absorptiometry image, and lumbar spine radiography was the standard reference for vertebral fracture and also provides the levels investigated. McNemar's test was used to compare accuracy between the two decubitus position and Fisher's exact test was used for patients with and without scoliosis. RESULTS: Forty-two vertebral fractures (VFs) were identified. There was no significant difference in sensitivity (p = 0.125) or specificity (p = 0.866) between the left lateral decubitus (64.3, 97.2%) and right lateral decubitus (76.2, 91.1%), respectively, views. Scoliotic patients had a significantly worse specificity (92.7 vs 98.1%, p = 0.003) than patients without scoliosis; however, a combination of both decubitus positions significantly improved specificity (p < 0.001). CONCLUSION: Right and left side lateral decubitus views have excellent agreement with radiography and similar diagnostic accuracy in the detection of VFs. Thus, the current practice of performing only one side is valid. With scoliosis, bilateral decubitus views can improve the specificity of detecting VF; however, this would increase radiation dose.

Newborn hearing screening in prematurity: fate of screening failures and auditory maturation.

OBJECTIVES: The purpose of this study was to identify delayed auditory maturation and the fate of premature infants who failed the newborn hearing screening (NHS) in neonatal intensive care unit. MATERIALS AND METHODS: A total of 1375 neonates underwent NHS using the transient evoked otoacoustic emission (TEOAE) in a tertiary hospital between 2007 and 2010 according to the Joint Committee on Infant Hearing guidelines. In addition, a structured telephone survey was given to caregivers of infants who were lost to follow-up NHS. Auditory steady-state response (ASSR) threshold and the threshold change in diagnostic test failures were analysed. RESULT: Among the 1375 NICU babies, 344 (25.0%) babies, 111 (9.7%) babies and 64 (4.6%) babies failed to pass the first TEOAE, second TEOAE and diagnostic ASSR, respectively. However, at the age of about 5 years, 12 (0.9%) infants showed permanent hearing loss (PHL). The ASSR threshold improved from 69.0 ± 19.7 dB to 52.9 ± 21.6 dB in <4 months (P < 0.001). Premature infants of <29 weeks of gestational age at birth showed higher referral (P = 0.003) rate at the first OAE test compared to the others, and the difference continued until the last follow-up. The odds ratio for the initial ASSR threshold >67.5 dB for PHL was 9.00 (95% confidence interval, 1.7-46.7). CONCLUSION: Most of first TEOAE screening failures (91.3%) showed normal hearing and speech development. Hearing levels in premature infants can improve over time, particularly in neonates with initial ASSR threshold <67.5 dB.

Randomised clinical trial: sodium alginate oral suspension is non-inferior to omeprazole in the treatment of patients with non-erosive gastroesophageal disease.

BACKGROUND: The burden of gastroesophageal reflux disease (GERD) is increasing in the Asia area and the majority of GERD patients have non-erosive reflux disease (NERD). AIM: To evaluate the efficacy and safety of sodium alginate suspension compared to omeprazole in adult subjects with NERD. METHODS: In this 4-week, double-blind, parallel study, 195 NERD subjects were randomised to one of two treatment groups: sodium alginate suspension 20 mL three times a day and omeprazole 20 mg once daily. The primary efficacy endpoint was the percentage of patients achieving adequate heartburn or regurgitation relief at day 28 assessed by patient diary. The secondary efficacy endpoints included percentage of patients achieving adequate heartburn or regurgitation relief, change from baseline of the Reflux Disease Questionnaire total score at day 14 and 28 from baseline, and patients' overall satisfaction. RESULTS: In this study, 183 subjects were included in the intent-to-treat population, and 172 subjects were included in the per-protocol population. Non-inferiority of sodium alginate to omeprazole was demonstrated in the intent-to-treat population [difference, 2.7% (53.3% vs. 50.5%, P = 0.175), 95% lower confidence interval -11.9%, above the preset margin of -19%]. All of the secondary efficacy endpoints were comparable between two groups. The incidence of adverse event was relatively low and there was no difference between the two groups (5.4% vs. 5.5% for sodium alginate vs. omeprazole). No severe adverse event was noted in this study. CONCLUSION: The study showed that sodium alginate was as effective as omeprazole for symptomatic relief in patients with non-erosive reflux disease (Clinicaltrials.gov NCT01338077).

Prognostic significance of the number of examined lymph nodes in node-negative gastric adenocarcinoma.

AIM: In this study, we investigated the prognostic significance of the number of examined lymph nodes in node-negative gastric adenocarcinoma (GC). PATIENTS AND METHODS: A total of 1194 node-positive and 1030 node-negative GC patients undergoing potentially curative gastrectomy was enrolled in this study. Patients were stratified into 3 groups according to the number of examined lymph nodes: group 1, ≤ 15; group 2, 16-25; group 3, >25. RESULTS: Patients with node-negative GC had significantly favorable survival compared with those with node-positive. Among patients with node-negative T2-T4 disease, the percentage of locoregional relapse was higher in those with <25 examined lymph nodes than in those with ≥ 25 examined lymph nodes. The number of examined lymph nodes affected the overall survival rates for patients with node-negative T2-T4 GC but not for patients with T1 lesions. Tumor size, tumor location, the number of examined lymph nodes, T status, and the presence of perineural invasion were significant prognostic factors as determined by multivariate analysis in node-negative GC. CONCLUSIONS: No survival benefit of examining ≥ 15 lymph nodes was noted for patients with node-negative T1 GC. Extensive lymphadenectomy in patients with node-negative T2-T4 lesions in whom the number of examined lymph nodes was >25 had favorable survival.

Potent and selective nonpeptide inhibitors of caspases 3 and 7.

5-Dialkylaminosulfonylisatins have been identified as potent, nonpeptide inhibitors of caspases 3 and 7. The most active compound within this series (34) inhibited caspases 3 and 7 in the 2-6 nM range and exhibited approximately 1000-fold selectivity for caspases 3 and 7 versus a panel of five other caspases (1, 2, 4, 6, and 8) and was at least 20-fold more selective versus caspase 9. Sequence alignments of the active site residues of the caspases strongly suggest that the basis of this selectivity is due to binding in the S2 subsite comprised of residues Tyr204, Trp206, and Phe256 which are unique to caspases 3 and 7. These compounds inhibit apoptosis in three cell-based models: human Jurkat T cells, human chondrocytes, and mouse bone marrow neutrophils.

Potent and selective nonpeptide inhibitors of caspases 3 and 7 inhibit apoptosis and maintain cell functionality.

Caspases have been strongly implicated to play an essential role in apoptosis. A critical question regarding the role(s) of these proteases is whether selective inhibition of an effector caspase(s) will prevent cell death. We have identified potent and selective non-peptide inhibitors of the effector caspases 3 and 7. The inhibition of apoptosis and maintenance of cell functionality with a caspase 3/7-selective inhibitor is demonstrated for the first time, and suggests that targeting these two caspases alone is sufficient for blocking apoptosis. Furthermore, an x-ray co-crystal structure of the complex between recombinant human caspase 3 and an isatin sulfonamide inhibitor has been solved to 2.8-A resolution. In contrast to previously reported peptide-based caspase inhibitors, the isatin sulfonamides derive their selectivity for caspases 3 and 7 by interacting primarily with the S(2) subsite, and do not bind in the caspase primary aspartic acid binding pocket (S(1)). These inhibitors blocked apoptosis in murine bone marrow neutrophils and human chondrocytes. Furthermore, in camptothecin-induced chondrocyte apoptosis, cell functionality as measured by type II collagen promoter activity is maintained, an activity considered essential for cartilage homeostasis. These data suggest that inhibiting chondrocyte cell death with a caspase 3/7-selective inhibitor may provide a novel therapeutic approach for the prevention and treatment of osteoarthritis, or other disease states characterized by excessive apoptosis.

Cloning and characterization of a novel human class I histone deacetylase that functions as a transcription repressor.

Histone acetylation alters chromatin state by modifying lysines on histone and plays an important role in modulating gene transcription. A dynamic balance of histone acetylation/deacetylation is maintained by histone acetyltransferases and histone deacetylases. Emerging evidence suggests that a family of histone deacetylases may exist to regulate diverse cellular functions, including chromatin structure, gene expression, cell cycle progression, and oncogenesis. We describe here a novel human histone deacetylase, named HDAC8, cloned from human kidney. HDAC8 encodes 377 amino acid residues and shares extensive homology to several known HDACs, in particular a histone deacetylase from Arabidopsis thaliana. Northern blot analyses revealed that HDAC8 expression pattern for HDAC8 is distinct from that for HDAC1 and HDAC3, and expression of HDAC8 mRNA occurs in multiple organs including heart, lung, kidney, and pancreas. HDAC8 mRNA was also observed in several cell lines derived from cancerous tissues. When expressed in HEK293 cells, HDAC8 exhibited deacetylase activity toward acetylated histone, indicating that this protein is a bona fide histone deacetylase. Its histone deacetylase activity was inhibited by trichostatin and other known histone deacetylase inhibitors. Furthermore, active recombinant HDAC8 was expressed and purified from Escherichia coli. When ectopically expressed in cells, HDAC8 was found to be localized to the nucleus. Co-transfection experiments demonstrated that expression of HDAC8 repressed a viral SV40 early promoter activity. These results indicate that HDAC8 is a novel member of the histone deacetylase family, which may play a role in the development of a broad range of tissues and potentially in the etiology of cancer.

Beta-lactams SB 212047 and SB 216754 are irreversible, time-dependent inhibitors of coenzyme A-independent transacylase.

The enzyme coenzyme A-independent transacylase (CoA-IT) has been demonstrated to be the key mediator of arachidonate remodeling, a process that moves arachidonate into 1-ether-containing phospholipids. Blockade of CoA-IT by reversible inhibitors has been shown to block the release of arachidonate in stimulated neutrophils and inhibit the production of eicosanoids and platelet-activating factor. We describe novel inhibitors of CoA-IT activity that contain a beta-lactam nucleus. beta-Lactams were investigated as potential mechanism-based inhibitors of CoA-IT on the basis of the expected formation of an acyl-enzyme intermediate complex. Two beta-lactams, SB 212047 and SB 216754, were shown to be specific, time-dependent inhibitors of CoA-IT activity (IC50 = 6 and 20 microM, respectively, with a 10-min pretreatment time). Extensive washing and dilution could not remove the inhibition, suggesting it was irreversible. In stimulated human monocytes, SB 216754 decreased the production of eicosanoids in a time-dependent manner. In an in vivo model of phorbol ester-induced ear inflammation, SB 216754 was able to inhibit indices of both edema and cell infiltration. Taken together, the results support two hypotheses: 1) CoA-IT activity is important for the production of inflammatory lipid mediators in stimulated cells and in vivo and 2) the mechanism by which CoA-IT acts to transfer arachidonate is through an acyl-enzyme intermediate.

Evidence that 85 kDa phospholipase A2 is not linked to CoA-independent transacylase-mediated production of platelet-activating factor in human monocytes.

Platelet-activating factor (PAF) production is carefully controlled in inflammatory cells. The specific removal of arachidonate (AA) from 1-O-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine (GPC), thought to be mediated by CoA-independent transacylase (CoA-IT), is required to generate the PAF precursor 1-O-alkyl-2-lyso-GPC in human neutrophils. Exposure of A23187-stimulated human monocytes to the CoA-IT inhibitors SK&F 98625 and SK&F 45905 inhibited PAF formation (IC50s of 10 and 12 microM, respectively), indicating that these cells also need CoA-IT activity for PAF production. Because CoA-IT activity transfers arachidonate to a 2-lyso phospholipid substrate, its activity is obligated to an sn-2 acyl hydrolase to form the 2-lyso phospholipid substrate. SB 203347, an inhibitor of 14 kDa phospholipase A2 (PLA2), and AACOCF3, an inhibitor of 85 kDa PLA2, both inhibited AA release from A23187-stimulated human monocytes. However, AACOCF3 had no effect on A23187-induced PAF formation at concentrations as high as 3 microM. Further, depletion of 85 kDa PLA2 using antisense (SB 7111, 1 microM) had no effect on PAF production, indicating a lack of a role of 85 kDa PLA2 in PAF biosynthesis. Both SB 203347 and the 14 kDa PLA2 inhibitor scalaradial blocked PAF synthesis in monocytes (IC50s of 2 and 0.5 microM, respectively), suggesting a key role of 14 kDa PLA2 in this process. Further, A23187-stimulated monocytes produced two forms of PAF: 80% 1-O-alkyl-2-acetyl-GPC and 20% 1-acyl-2-acetyl-GPC, which were both equally inhibited by SB 203347. In contrast, inhibition of CoA-IT using SK&F 45905 (20 microM) had a greater effect on the production of 1-O-alkyl (-80%) than of 1-acyl (-14%) acetylated material. Finally, treatment of U937 cell membranes with exogenous human recombinant (rh) type II 14 kDa PLA2, but not rh 85 kDa PLA2, induced PAF production. Elimination of membrane CoA-IT activity by heat treatment impaired the ability of 14 kDa PLA2 to induce PAF formation. Taken together, these results suggest that a 14 kDa PLA2-like activity, and not 85 kDa PLA2, is coupled to monocyte CoA-IT-induced PAF production.

Inhibitors of coenzyme A-independent transacylase induce apoptosis in human HL-60 cells.

ET-18-O-CH3 (1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine) is an antiproliferative agent, blocking the growth of cancer cells both in vitro and in vivo. However, there is controversy regarding the mechanism leading to its antiproliferative effects. CoA-independent transacylase (CoA-IT) is an enzyme that remodels arachidonate between specific phospholipid donor and acceptor molecules in a variety of mammalian cells. ET-18-O-CH3 was found to be a potent inhibitor of CoA-IT (IC50, 0.5 microM), and kinetic analysis revealed that its inhibition was competitive with the lyso-phospholipid substrate. The goal of the current study was to explore the connection between inhibition of CoA-IT and antiproliferative effects using several structurally distinct inhibitors of CoA-IT. ET-18-O-CH3 and other inhibitors of CoA-IT were found to inhibit cell proliferation and thymidine incorporation into the DNA, as well as to induce apoptosis in human HL-60 monocytic leukemia cells. The mechanism of apoptosis induced by ET-18-O-CH3 appeared to be different from that induced by tumor necrosis factor; the former failed to activate NF-kappa B, whereas tumor necrosis factor did. Closer examination of the pharmacology of apoptosis in this model revealed that compounds that were structurally related to CoA-IT inhibitors, but lacked CoA-IT inhibitory activity, also failed to induce apoptosis. In addition, compounds that inhibited other enzymes that participate in arachidonic acid metabolism, cyclooxygenase, 5-lipoxygenase and phospholipase A2, did not induce apoptosis. Taken together, these results demonstrate that inhibition of CoA-IT can be linked to blockade of proliferation and the induction of apoptosis in HL-60 cells.

SB 203347, an inhibitor of 14 kDa phospholipase A2, alters human neutrophil arachidonic acid release and metabolism and prolongs survival in murine endotoxin shock.

Phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 fatty acyl group [predominantly arachidonic acid (AA)] of membrane phospholipids, the products of which are further metabolized, forming a variety of eicosanoids and/or platelet-activating factor. PLA2 activity is significantly enhanced during inflammation and therefore offers an intriguing target in designing anti-inflammatory drugs. SB 203347 (2-[2-[3,5-bis (trifluoromethyl) sulfonamido]-4- trifluoromethylphenoxy] benzoic acid) potently inhibits rh type II 14 kDa PLA2 (IC50 = 0.5 microM) but exhibits a 40-fold weaker inhibition of 85 kDa PLA2 (IC50 = 20 microM) using [3H]-AA E. coli as substrate. A specific interaction with rh type II 14 kDa PLA2 was confirmed both by observing the pH dependence of its IC50 and by demonstrating linear inhibition in a "scooting" kinetic model using radiolabeled phospholipid reporter substrate in a 1,2-dimyristoyl phosphatidylmethanol vesicle. Before evaluating the effect of SB 203347 on AA metabolism in intact human neutrophil, we showed that it fully inhibits PLA2 activity in acid extracted-intact human neutrophil homogenate (IC50 = 4.7 microM). SB 203347 inhibited A23187-induced intact human neutrophil AA mass release in a concentration-dependent manner (IC50 = 1 microM), which coincided with reductions in the biosynthesis of platelet-activating factor (IC50 = 1.5 microM) and leukotriene B4 (IC50 = 2.3 microM). Finally, SB 203347 prolonged survival in a mouse model of endotoxin shock delivered i.p. Taken together, the data support a role of cellular 14 kDa PLA2 in the formation of AA-derived proinflammatory lipid mediator.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of CoA-independent transacylase inhibitors on the production of lipid inflammatory mediators.

The enzyme CoA-independent transacylase (CoA-IT) has been proposed to mediate the movement of arachidonate between specific phospholipid subclasses, and we have shown that two inhibitors of CoA-IT (SK&F 98625 and SK&F 45905) block this movement. In this report, we use these inhibitors to further characterize the role of CoA-IT in the production of lipid mediators. SK&F 98625 (diethyl 7-(3,4,5-triphenyl-2-oxo-2,3-dihydro-imidazol-1-yl)heptane- phosphonate) and SK&F 45905 [2(-)[3-(4-chloro-3-trifluoromethylphenyl)ureido]-4-trifluoromethyl phenoxy]-4,5-dichlorobenzenesulfonic acid) inhibited CoA-IT activity (IC50 values of 9 microM and 6 microM, respectively). Neither compound had any effect on cyclooxygenase, 14-kDa PLA2 or acetyltransferase activities at concentrations below 20 microM. However, SK&F 45905 inhibited 85-kDa PLA2 activity (IC50 = 3 microM), and both compounds inhibited 5-lipoxygenase activity (IC50 values of 2-4 microM). In ionophore-stimulated neurotrophils, SK&F 98625 and SK&F 45905 blocked the liberation of arachidonic acid from phospholipids, which suggests that the movement of arachidonate into specific phospholipid pools is a prerequisite for release. Both compounds also inhibited the production of platelet-activating factor in ionophore-stimulated neutrophils and antigen-stimulated mast cells. This inhibition of platelet-activating factor and arachidonic acid release was not mimicked by an inhibitor of 5-lipoxygenase, zileuton, which indicates that the primary mode of action of SK&F 98625 and SK&F 45905 is via inhibition of CoA-IT. SK&F 98625 and SK&F 45905 were able to decrease prostaglandin production in several inflammatory cells and to block signs of inflammation in ears of phorbol ester-challenged mice. Taken together, these results show that blockade of CoA-IT, which leads to inhibition of arachidonate remodelling between phospholipids, results in the attenuation of platelet-activating factor production, arachidonic acid release and the formation of eicosanoid products.

Inhibitors of CoA-independent transacylase block the movement of arachidonate into 1-ether-linked phospholipids of human neutrophils.

The enzyme CoA-independent transacylase (CoA-IT) has been proposed to mediate the movement of arachidonate between phospholipid subclasses and influence the formation of arachidonic acid metabolites and platelet-activating factor. To substantiate the critical role of CoA-IT, we have developed two structurally diverse inhibitors of CoA-IT activity, SK&F 98625 [diethyl 7-(3,4,5-triphenyl-2-oxo-2,3-dihydro-imidazole-1-yl)heptane phosphonate] and SK&F 45905 [2-[2-(3-4-chloro-3-(trifluoromethyl)phenyl)-ureido]-4- (trifluoromethyl)phenoxy]-4,5-dichlorobenzenesulfonic acid]. These compounds were tested for their capacity to block microsomal CoA-IT activity using two assay systems, the transacylation of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine (GPC) and the transfer of [14C]arachidonate from 1-acyl-2-[14C]arachidonoyl-GPC to lyso-PE. Both SK&F 98625 and SK&F 45905 inhibited CoA-IT activity (IC50s 6-19 microM) in these two assays. In contrast, SK&F 98625 or SK&F 45905 had little or no effect on other lipid-modifying activities, including CoA-dependent acyltransferase or acetyltransferase. Kinetic analysis revealed that both SK&F 98625 and SK&F 45905 interact directly with the enzyme and prevented the acylation of lysophospholipids in a competitive manner. In intact human neutrophils, both SK&F 98625 and SK&F 45905 completely blocked the movement of [3H]arachidonate from 1-acyl-linked phospholipids into 1-alkyl-2-arachidonoyl-GPC and 1-alk-1'-enyl-2-arachidonoyl-GPE. In contrast, these compounds did not inhibit the incorporation of free arachidonic acid into cellular lipids indicating that they did not alter CoA-dependent acyl transferase activities in the intact cell.(ABSTRACT TRUNCATED AT 250 WORDS)

CoA-independent transacylase activity is increased in human neutrophils after treatment with tumor necrosis factor alpha.

CoA-independent transacylase (CoA-IT) appears to play a critical role in lipid mediator generation by rapidly moving arachidonate (AA) between phospholipid pools during cell activation. Tumor necrosis factor-alpha (TNF) pretreatment of human neutrophils increases agonist-induced production of inflammatory mediators. The current study tested if the TNF-induced increase in lipid mediator production may be, in part, due to altered CoA-IT activity. Neutrophils were treated with TNF (250 U/ml, 30 min), homogenates prepared, and CoA-IT activity measured by the ability of these homogenates to acylate 1-[3H]alkyl-2-lyso-sn-glycero-3-phosphocholine (GPC). There was an increased CoA-IT activity, from 9.1 +/- 1.1 to 13.7 +/- 1.4 pmol/mg per min in control vs. TNF-treated samples, respectively. Varying the concentration of 1-alkyl-2-lyso-GPC revealed an increased CoA-IT activity in microsomes that was due to an increased Vmax, from 26 to 54 pmol/mg per min. The ability of TNF to increase CoA-IT activity was concentration-dependent, with maximal response observed at 25 U/ml. This effect on CoA-IT appears to be specific, in that TNF treatment of neutrophils had no effect on CoA-dependent acylation of 1-acyl-2-lyso-sn-glycero-3-phosphocholine, using either AA-CoA or linolenoyl-CoA as substrates. In the intact cell, the movement of [3H]AA from other phospholipids into PE in fMLP-stimulated neutrophils was greatly enhanced after TNF treatment, demonstrating a functional consequence of increased CoA-IT activity. In addition, TNF treatment doubled platelet-activating factor production in response to the chemotactic peptide fMLP, as measured by [3H]acetate incorporation, while the response to A23187 remained unchanged. Taken together, these results provide the first evidence of modulation of CoA-IT activity by a proinflammatory cytokine and suggest that one mechanism for augmented lipid mediator formation is through increases in CoA-IT activity.

Biochemical and pharmacological comparison of a cytosolic, high molecular weight phospholipase A2, human synovial fluid phospholipase A2 and CoA-independent transacylase.

We have compared biochemical and pharmacological characteristics of CoA-independent transacylase (CoA-IT) activity of microsomes from U937 cells to those of two other enzymes involved in arachidonate metabolism, a human type II low molecular weight (M(r) 14,000, LMW) PLA2 isolated from synovial fluid of patients with inflammatory joint disease and a high molecular weight (M(r) 85,000, HMW) PLA2 enzyme isolated from the cytosol of human monocytic U937 cells. Activities of HMW PLA2 and CoA-IT were reduced by treatment with acid, heat or acetonitrile but were not altered by treatment with the sulfhydryl reducing agent dithiothreitol (DTT). In contrast, the activity of LMW PLA2 enzyme was inactivated by DTT, but was insensitive to treatment with acid, heat or acetonitrile. HMW PLA2 activity decreased as NaCl concentration was increased in the assay buffer from 0 to 150 mM, unlike LMW PLA2 and CoA-IT activities, which increased as NaCl increased. Compounds that inhibit LMW PLA2 activity were examined for their effects on HMW PLA2 and CoA-IT activities. The compounds examined (nordihydroguaiaretic acid, manoalide, p-bromophenacyl bromide, arachidonic acid, gossypol, aristologic acid and a mimic of a transition state phospholipid) had different rank orders for inhibition of the three enzymes. Taken together, these data show that these three enzymes, although unpurified, can be biochemically and pharmacologically distinguished.

Effects of scalaradial, a type II phospholipase A2 inhibitor, on human neutrophil arachidonic acid mobilization and lipid mediator formation.

The role of scalaradial (SLD) (or its 12-epi analog), a marine natural product purified from sponge (Cacospongia mollior) in human neutrophil (polymorphonuclear leukocyte, PMN) arachidonic acid metabolism was studied. SLD potently inhibited human recombinant (rh) type II-14 kDa-phospholipase A2 (PLA2) (IC50 = 0.07 microM) but displayed weak inhibition of U937 cell, 85 kDa-PLA2 (IC50 = 20 microM). Sn-2 acylhydrolytic activity expressed in human PMN acid extract, that was completely neutralized by anti-rh type II-14 kDa-PLA2 monoclonal antibody, was inhibited by SLD in a concentration-dependent manner (IC50 = 35 microM). Exposure of human PMN to SLD resulted in a concentration-dependent inhibition of calcium ionophore (A23187)-induced leukotriene B4 release (IC50 = 0.1-0.6 microM). In contrast to the action of selective 5-lipoxygenase inhibitors (WY 50295 or zileuton), SLD decreased A23187-induced PMN liberation of arachidonic acid mass and platelet-activating factor biosynthesis (IC50 = 1-2 microM). PMN acetyltransferase activity was not significantly affected by SLD suggesting that platelet-activating factor inhibition was due, predominantly, to inactivation of PLA2 activity. In vivo, topical application of SLD on mouse ear treated with phorbol ester, not only inhibited edema formation but also the increase in myeloperoxidase activity (an index of cellular infiltration). SLD had little or no effect on arachidonic acid-induced ear edema or myeloperoxidase which is consistent with an action on PLA2. Take together these data suggest that the predominant mechanism of SLD is via inhibition of 14 kDa-like-PLA2 and suggests that this enzyme may participate in PMN arachidonic acid liberation and lipid mediator formation.

Influence of arachidonic acid on indices of phospholipase A2 activity in the human neutrophil.

The present studies were conducted to understand better the regulation of phospholipase A2 (PLA2)-dependent mobilization of lipid mediators by arachidonic acid (C20:4). After stimulation of human neutrophils, g.l.c./m.s. analysis of non-esterified fatty acids indicated that the quantity of C20:4 increased as a function of time after stimulation, from undetectable quantities to > 800 pmol/10(7) cells. In contrast with C20:4, the quantities of other free fatty acids such as oleic and linoleic were high in resting cells and did not change after stimulation. Some 15% of the C20:4 released from cellular lipids remained cell-associated. To examine the effect of C20:4 on its own release, neutrophils were exposed to [2H8]C20:4, to differentiate it by g.l.c./m.s. from naturally occurring C20:4. In A23187-stimulated neutrophils, low concentrations (5-10 microM) of [2H8]C20:4 added just before A23187 increased the quantity of C20:4 produced by the cell, whereas higher concentrations (30-50 microM) decreased the quantity of C20:4 released from phospholipids. As other measures of PLA2 activity, the effects of C20:4 on production of platelet-activity factor (PAF) and leukotriene B4 (LTB4) were assessed. C20:4 treatment just before stimulation of neutrophils blocked PAF and LTB4 production in a concentration-dependent manner (IC50 10-20 microM). The effect of C20:4 was not blocked by the cyclo-oxygenase inhibitor naproxine (10 microM), nor could it be mimicked by 1 microM LTB4, 5-hydroxyeicosa-6,8,11,14-tetraenoic acid (5HETE), 5-hydroperoxyeicosa-6,8,11,14-tetraenoic acid (5HPETE) or 15-hydroxyeicosa-5,8,11,13-tetraenoic acid (15HETE). The 5-lipoxygenase (5LO) inhibitor zileuton induced a concentration-dependent decrease in PAF, with a maximal effect of a 50% decrease at 10-50 microM. The decrease in PAF by the 5LO inhibitor could not be circumvented by addition of 1 microM 5HETE, 5HPETE and LTB4, and may be attributed to the capacity of zileuton to increase the quantity of C20:4 in A23187-treated neutrophils. The inhibitory effect of C20:4 (20-40 microM) on PAF production could be antagonized by the protein kinase C inhibitor staurosporine (30 nM), but not by inhibitors of protein kinase A, tyrosine kinase or calmodulin kinase II. Taken together, these data demonstrate that C20:4 is selectively released from membrane phospholipids of A23187-stimulated neutrophils, and this C20:4 may play an important role in regulating the mobilization of C20:4 by altering PLA2 activity.