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Candida albicans, a polymorphic yeast, is a physiological component of the human and animal commensal microbiome. It is an etiological factor of candidiasis, which is treated by azole antifungals. Growing resistance to azoles is a reason to look for other alternative treatment options. The pharmacotherapeutic use of plant extracts and essential oils has become increasingly important. In our experiment, C. albicans showed susceptibility to four observed plant extracts and essential oils from peppermint ( Mentha piperita), thyme ( Thymus vulgaris), sage ( Salvia officinalis), and oregano ( Origanum vulgare). Oregano plant extract and essential oil showed the highest antifungal activity, at MIC values of 4.9 mg/mL and 0.4 mg/mL respectively. Therefore, it was subjected to further research on the influence of virulence factors - biofilm formation, extracellular phospholipase production and germ tube formation. Oregano plant extract and essential oil showed an inhibitory effect on the observed C. albicans virulence factors at relatively low concentrations. The extract inhibited the adherence of cells at MIC 12.5 mg/mL and essential oil at MIC 0.25 mg/mL. Degradation of the formed biofilm was detected at MIC 14.1 mg/mL for plant extract and at MIC 0.4 mg/mL for essential oil. Extracellular phospholipase production was most effectively inhibited by the essential oil. In particular, the number of isolates with intensive extracellular phospholipase production decreased significantly. Of the 12 isolates intensively producing extracellular phospholipase, only 1 isolate (4.5%) retained intense production. Essential oil caused up to a 100 % reduction in germ tubes formation and plant extract reduced their formation depending on the concentration as follows: 2.6% (0.8 mg/mL), 21.2 % (6.25 mg/mL), and 64.5 % (12.5 mg/mL) compared to the control., (© 2024 The Authors. This is an open access article under the CC BY-NC-ND 4.0 license.)

Platelets assume a pivotal role in the cardiovascular diseases (CVDs). Thus, targeting platelet activation is imperative for mitigating CVDs. Ginkgetin (GK), from Ginkgo biloba L, renowned for its anticancer and neuroprotective properties, remains unexplored concerning its impact on platelet activation, particularly in humans. In this investigation, we delved into the intricate mechanisms through which GK influences human platelets. At low concentrations (0.5-1 μM), GK exhibited robust inhibition of collagen and arachidonic acid (AA)-induced platelet aggregation. Intriguingly, thrombin and U46619 remained impervious to GK's influence. GK's modulatory effect extended to ATP release, P-selectin expression, intracellular calcium ([Ca 2+ ]i) levels and thromboxane A 2 formation. It significantly curtailed the activation of various signaling cascades, encompassing phospholipase Cγ2 (PLCγ2)/protein kinase C (PKC), phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3β and mitogen-activated protein kinases. GK's antiplatelet effect was not reversed by SQ22536 (an adenylate cyclase inhibitor) or ODQ (a guanylate cyclase inhibitor), and GK had no effect on the phosphorylation of vasodilator-stimulated phosphoprotein Ser157 or Ser239 . Moreover, neither cyclic AMP nor cyclic GMP levels were significantly increased after GK treatment. In mouse studies, GK notably extended occlusion time in mesenteric vessels, while sparing bleeding time. In conclusion, GK's profound impact on platelet activation, achieved through inhibiting PLCγ2-PKC cascade, culminates in the suppression of downstream signaling and, ultimately, the inhibition of platelet aggregation. These findings underscore the promising therapeutic potential of GK in the CVDs., (© 2024 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background and Aims: Macrophage-derived foam cells play a causal role during the pathogenesis of atherosclerosis. P2Y6 receptor (P2Y6R) highly expressed has been considered as a disease-causing factor in atherogenesis, but the detailed mechanism remains unknown. This study aims to explore P2Y6R in regulation of macrophage foaming, atherogenesis, and its downstream pathways. Furthermore, the present study sought to find a potent P2Y6R antagonist and investigate the feasibility of P2Y6R-targeting therapy for atherosclerosis., Methods: The P2Y6R expression was examined in human atherosclerotic plaques and mouse artery. Atherosclerosis animal models were established in whole-body P2Y6R or macrophage-specific P2Y6R knockout mice to evaluate the role of P2Y6R. RNA sequencing, DNA pull-down experiments, and proteomic approaches were performed to investigate the downstream mechanisms. High-throughput Glide docking pipeline from repurposing drug library was performed to find potent P2Y6R antagonists., Results: The P2Y6R deficiency alleviated atherogenesis characterized by decreasing plaque formation and lipid deposition of the aorta. Mechanically, deletion of macrophage P2Y6R significantly inhibited uptake of oxidized low-density lipoprotein through decreasing scavenger receptor A expression mediated by phospholipase Cβ/store-operated calcium entry pathways. More importantly, P2Y6R deficiency reduced the binding of scavenger receptor A to CALR, accompanied by dissociation of calreticulin and STIM1. Interestingly, thiamine pyrophosphate was found as a potent P2Y6R antagonist with excellent P2Y6R antagonistic activity and binding affinity, of which the pharmacodynamic effect and mechanism on atherosclerosis were verified., Conclusions: Macrophage P2Y6R regulates phospholipase Cβ/store-operated calcium entry/calreticulin signalling pathway to increase scavenger receptor A protein level, thereby improving foam cell formation and atherosclerosis, indicating that the P2Y6R may be a potential therapeutic target for intervention of atherosclerotic diseases using P2Y6R antagonists including thiamine pyrophosphate., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Phospholipases A 2 (PLA 2 ) may be involved in α 1 -adrenergic contraction by formation of thromboxane A 2 in different smooth muscle types. However, whether this mechanism occurs with α 1 -adrenergic contractions of the prostate, is still unknown. While α 1 -adrenoceptor antagonists are the first line option for medical treatment of voiding symptoms in benign prostatic hyperplasia (BPH), improvements are limited, probably by nonadrenergic contractions including thromboxane A 2 . Here, we examined effects of PLA 2 inhibitors on contractions of human prostate tissues., Methods: Prostate tissues were obtained from radical prostatectomy. Contractions were induced by electric field stimulation (EFS) and by α 1 -adrenergic agonists in an organ bath, after application of the cytosolic PLA 2 inhibitors ASB14780 and AACOCF3, the secretory PLA 2 inhibitor YM26734, the leukotriene receptor antagonist montelukast, or of solvent to controls., Results: Frequency-dependent contractions of human prostate tissues induced by EFS were inhibited by 25% at 8 Hz, 38% at 16 Hz and 37% at 32 Hz by ASB14780 (1 µM), and by 32% at 16 Hz and 22% at 32 Hz by AACOCF3 (10 µM). None of both inhibitors affected contractions induced by noradrenaline, phenylephrine or methoxamine. YM26734 (3 µM) and montelukast (0.3 and 1 µM) neither affected EFS-induced contractions, nor contractions by α 1 -adrenergic agonists, while all contractions were substantially inhibited by silodosin (100 nM)., Conclusions: Our findings suggest presynaptic PLA 2 functions in prostate smooth muscle contraction, while contractions induced by α 1 -adrenergic agonists occur PLA 2 -independent. Lacking sensitivity to montelukast excludes an involvement of PLA 2 -derived leukotrienes in promotion of contractile neurotransmission., (© 2023 The Authors. Neurourology and Urodynamics published by Wiley Periodicals LLC.)

Mitophagy, which selectively eliminates the dysfunctional and excess mitochondria by autophagy, is crucial for cellular homeostasis under stresses such as hypoxia. Dysregulation of mitophagy has been increasingly linked to many disorders including neurodegenerative disease and cancer. Triple-negative breast cancer (TNBC), a highly aggressive breast cancer subtype, is reported to be characterized by hypoxia. However, the role of mitophagy in hypoxic TNBC as well as the underlying molecular mechanism is largely unexplored. Here, we identified GPCPD1 (glycerophosphocholine phosphodiesterase 1), a key enzyme in choline metabolism, as an essential mediator in hypoxia-induced mitophagy. Under the hypoxic condition, we found that GPCPD1 was depalmitoylated by LYPLA1, which facilitated the relocating of GPCPD1 to the outer mitochondrial membrane (OMM). Mitochondria-localized GPCPD1 could bind to VDAC1, the substrate for PRKN/PARKIN-dependent ubiquitination, thus interfering with the oligomerization of VDAC1. The increased monomer of VDAC1 provided more anchor sites to recruit PRKN-mediated polyubiquitination, which consequently triggered mitophagy. In addition, we found that GPCPD1-mediated mitophagy exerted a promotive effect on tumor growth and metastasis in TNBC both in vitro and in vivo . We further determined that GPCPD1 could serve as an independent prognostic indicator in TNBC. In conclusion, our study provides important insights into a mechanistic understanding of hypoxia-induced mitophagy and elucidates that GPCPD1 could act as a potential target for the future development of novel therapy for TNBC patients. Abbreviations : ACTB: actin beta; 5-aza: 5-azacytidine; BNIP3: BCL2 interacting protein 3; BNIP3L: BCL2 interacting protein 3 like; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; ChIP: chromatin immunoprecipitation; co-IP: co-immunoprecipitation; CQ: chloroquine; CsA: cyclosporine; DOX: doxorubicin; FIS1: fission, mitochondrial 1; FUNDC1: FUN14 domain containing 1; GPCPD1: glycerophosphocholine phosphodiesterase 1; HAM: hydroxylamine; HIF1A: hypoxia inducible factor 1 subunit alpha; HRE: hypoxia response element; IF: immunofluorescence; LB: lysis buffer; LC3B/MAP1LC3B: microtubule associated protein 1 light chain 3 beta; LC-MS: liquid chromatography-mass spectrometry; LYPLA1: lysophospholipase 1; LYPLA2: lysophospholipase 2; MDA231: MDA-MB-231; MDA468: MDA-MB-468; MFN1: mitofusin 1; MFN2: mitofusin 2; MKI67: marker of proliferation Ki-67; OCR: oxygen consumption rate; OMM: outer mitochondrial membrane; OS: overall survival; PalmB: palmostatin B; PBS: phosphate-buffered saline; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; SDS: sodium dodecyl sulfate; TOMM20: translocase of outer mitochondrial membrane 20; TNBC: triple-negative breast cancer; VBIT-4: VDAC inhibitor; VDAC1: voltage dependent anion channel 1; WT: wild type.

The involvement of Trypanosoma congolense sialidase alongside phospholipase A 2 has been widely accepted as the major contributing factor to anemia during African animal trypanosomiasis. The enzymes aid the parasite in scavenging sialic acid and fatty acids necessary for survival in the infected host, but there are no specific drug candidates against the two enzymes. This study investigated the inhibitory effects of β-sitosterol on the partially purified T. congolense sialidase and phospholipase A 2 . Purification of the enzymes using DEAE cellulose column led to fractions with highest specific activities of 8016.41 and 39.26 µmol/min/mg for sialidase and phospholipase A 2 , respectively. Inhibition kinetics studies showed that β-sitosterol is non-competitive and an uncompetitive inhibitor of sialidase and phospholipase A 2 with inhibition binding constants of 0.368 and 0.549 µM, respectively. Molecular docking of the compound revealed binding energies of - 8.0 and - 8.6 kcal/mol against the sialidase and phospholipase A 2 , respectively. Furthermore, 100 ns molecular dynamics simulation using GROMACS revealed stable interaction of β-sitosterol with both enzymes. Hydrogen bond interactions between the ligand and Glu284 and Leu102 residues of the sialidase and phospholipase A 2 , respectively, were found to be the major stabilizing forces. In conclusion, β-sitosterol could serve as a dual inhibitor of T. congolense sialidase and phospholipase A 2; hence, the compound could be exploited further in the search for newer trypanocides., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The phospholipase A and acyltransferase (PLAAT) family is composed of three isoforms in mice (PLAAT1, 3, and 5), all of which function as phospholipid-metabolizing enzymes exhibiting phospholipase A 1 /A 2 and acyltransferase activities. Plaat3-deficient (Plaat3 -/- ) mice were previously reported to show lean phenotype and remarkable hepatic fat accumulation under high-fat diet (HFD) feeding, while Plaat1 -/- mice have not been analyzed. In the present study, we generated Plaat1 -/- mice and investigated the effects of PLAAT1 deficiency on HFD-induced obesity, hepatic lipid accumulation, and insulin resistance. After HFD treatment, PLAAT1 deficiency caused a lower body weight gain compared to wild-type mice. Plaat1 -/- mice also showed reduced liver weight with negligible hepatic lipid accumulation. In accordance with these findings, PLAAT1 deficiency improved HFD-induced hepatic dysfunction and lipid metabolism disorders. Lipidomics analysis in the liver revealed that in Plaat1 -/- mice, the levels of various glycerophospholipids tended to increase, while all classes of lysophospholipids examined tended to decrease, suggesting that PLAAT1 functions as phospholipase A 1 /A 2 in the liver. Interestingly, the HFD treatment of wild-type mice significantly increased the mRNA level of PLAAT1 in the liver. Furthermore, the deficiency did not appear to elevate the risk of insulin resistance in contrast to PLAAT3 deficiency. These results suggested that the suppression of PLAAT1 improves HFD-induced overweight and concomitant hepatic lipid accumulation., (© 2023 Federation of American Societies for Experimental Biology.)

Pseudomonas aeruginosa is an important nosocomial pathogen with a capacity of resistance to multiple antibiotics and production of various extracellular and cell-associated virulence factors that clearly contribute to its pathogenicity. The objective of this study was to investigate the antibiotic susceptibility, virulence factors, and clonal relationship among clinical isolates of P. aeruginosa. Different clinical specimens from hospitalized patients were investigated for P. aeruginosa. Susceptibility of the isolates was evaluated by disc diffusion and broth microdilution methods, as described by the Clinical and Laboratory Standards Institute (CLSI) guideline. A total of 97 P. aeruginosa isolates were recovered from clinical specimens. The percentage of isolates resistant to antimicrobials was imipenem 25.77%, meropenem 15.46%, gentamicin 16.49%, tobramycin 15.46%, amikacin 16.49%, ciprofloxacin 20.61%, levofloxacin 24.74, ceftazidime 20.61%, piperacillin 15.46%, piperacillin/tazobactam 12.37%, colistin 9.27%, and polymyxin B 11.34%. Of isolates, 87.62% possessed β-hemolytic activity, 78.35% lecithinase, 59.8% elastase, 37.11% DNase, and 28.86% twitching motility. The frequency of virulence genes in isolates was lasB 82.47%, plcH 82.47%, exoA 58.76%, exoS 56.7%, and pilA 10.3%. ERIC-PCR typing clustered P. aeruginosa isolates to 19 common types (CT1-CT19) containing isolates from different hospitals and 43 single types (ST1-ST43). Colistin and polymyxin B were the most effective agents against the majority of P. aeruginosa isolates, emphasizing the effort to maintain their antibacterial activity as last-line therapy. The frequency of some virulence factors and genes was noticeably high, which is alarming. In addition, more effective strategies and surveillance are necessary to confine and prevent the inter-hospital and/or intra-hospital dissemination of P. aeruginosa between therapeutic centers., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Vaginal candidiasis is a medical condition characterized by the overgrowth of Candida spp. in the vaginal cavity with complex recurrent pathogenicity as well as tolerance to antifungal therapy and hence is awaiting more safe and effective treatments. This work aimed to assess the potential antifungal activity of galloylquinic acid compounds (GQAs) from Copaifera lucens leaves against vaginal Candida albicans. The antifungal susceptibility test was performed against 20 isolates of multidrug-resistant (MDR) C. albicans using agar diffusion and broth microdilution assays. The results showed that GQAs exhibited strong antagonistic activity against the test isolates, with inhibition zone diameters ranging from 26 to 38 mm and low MICs (1 to 16 μg/mL) as well as minimum fungicidal concentrations (2 to 32 μg/mL). The MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay confirmed the safety of GQAs against the Vero cell line, showing a 50% inhibitory concentration (IC 50 ) of 168.17 mg/mL. A marked difference in the growth pattern of the treated and untreated pathogens was also observed, where a concentration-dependent reduction in the growth rate occurred. Moreover, a pronounced fungicidal effect was demonstrated 6 h after treatment with 1× the minimum fungicidal concentration (MFC), as evidenced by time-kill assays, where the number of survivors was decreased a 6-fold. GQAs effectively inhibited and eradicated about 80% of C. albicans biofilm at 6 μg/mL and 32 μg/mL, respectively. Interestingly, GQAs disturbed the fungal membrane integrity, induced cell lysis, and reduced the virulence factors (proteinase and phospholipase) as well as the catalase activity. Moreover, the ergosterol content in the plasma membrane decreased in a concentration-dependent manner. Additionally, the altered mitochondrial membrane potential was associated with an increased release of cytochrome c from mitochondria to the cytosol, suggesting the initiation of early apoptosis in GQA-treated cells. Transcriptional analysis revealed that all test genes encoding virulence traits, including SAP1, PLB1, LIP1, HWP1 , and ALS1 , were markedly downregulated in GQA-treated cells compared to the control. The in vivo murine model of vaginal candidiasis further confirmed the therapeutic activity of GQAs (4 mg/kg of body weight) against C. albicans. This work comprehensively evaluated the antifungal, antivirulence, and antibiofilm activities of GQAs against C. albicans isolates using in vitro and in vivo models, providing molecular-level insights into the antifungal mechanism of action and experimental evidence that supports the potential use of GQAs for the treatment of vaginal candidiasis. IMPORTANCE Our work presents a new perspective on the potential use of GQAs as safe and highly effective phytochemicals against MDR C. albicans. This microorganism colonizes the human vaginal epithelium, causing vaginal candidiasis, a condition characterized by recurrent pathogenicity and tolerance to traditional antifungal therapy. Based on the results of in vitro tests, our study reports GQAs antifungal modes of action. These compounds exhibited an anticandidal effect by deactivating the fungal hydrolytic enzymes, reducing ergosterol content in the plasma membrane, altering the potential of the mitochondrial membrane, and inducing apoptosis. Additionally, GQAs showed high activity in eradicating the biofilm formed by the fungus via the downregulation of HWP1, ALS, SAP, PLB , and LIP genes, which are constitutively expressed in the biofilm. In an in vivo murine model of vaginal candidiasis, GQAs further demonstrated strong evidence of their effectiveness as an antifungal therapy. In this regard, our findings provide novel insights into the potential therapeutic use of these phytoactive molecules for vaginal candidiasis treatment.

Plastic wastes deposited in canals running through Thiruvananthapuram city have created stagnant waters providing breeding sites for mosquitoes. In the present study, plastic waste-derived bisphenol A (BPA) was quantified from four mosquito breeding sites. During summer rain, the concentration of BPA in the stagnant water samples was found to be between 0.86 and 1.14 mg/L, and hence 1 mg/L BPA was considered as the environmentally relevant concentration. In the present study, the effect of BPA on the life cycle and metamorphosis of filarial vector, Culex quinquefasciatus Say was elucidated by rearing larvae in water added with BPA at and above the environmentally relevant concentration viz., 1, 2, and 4 mg/L. The duration required for adult emergence was reduced from 10 to 8.5 days, when the concentration of BPA was increased from 1 to 4 mg/L respectively. Our study revealed that embryonic and larval developments were shortened by BPA treatment. BPA also caused a dose-dependent advancement of 20-hydroxyecdysone (20-E) peaks; phospholipase A 2 induction; and upregulation of ecdysone receptor gene, EcRA, and ecdysone inducible gene E75A, which culminated in early pupation. No significant difference in sanguivory and fecundity was observed in adult mosquitoes treated with 1 mg/L of BPA. Our study reveals that BPA is a developmental agonist of C. quinquefasciatus., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Robust biofilms and the complex airway environment with thick sputum, local hypoxia and persistent inflammation induce the intractability of chronic pulmonary infections caused by Pseudomonas aeruginosa (P. aeruginosa). Herein, we proposed a type of antibiotic-adjuvant liposomes (NANO@PS-LPs), co-incorporating azithromycin (AZI), adjuvant (2-nitroimidazole derivative, 6-NIH) and biofilm dispersant (nitric oxide donor, DETA NONOate). NANO@PS-LPs possessing negatively-charged surface and good hydrophilicity could easily penetrate through the sputum layer, then disassembled triggered by overexpressed phospholipase A 2 (PLA 2 ) in the microenvironment around biofilms. Nitric oxide produced by DETA NONOate promoted P. aeruginosa biofilms dispersal. 6-NIH was reduced to 2-aminomidazole derivative (6-AIH) under a hypoxic condition, and hence acted as an AZI adjuvant to enhance the antibacterial activity of AZI. It was found that NANO@PS-LPs could significantly eliminate mature P. aeruginosa biofilms, effectively kill dispersed bacteria, inhibit the metabolism of survivors and prevent P. aeruginosa adherence to airway epithelial cells, accordingly restrain recurrent infections. Additionally, NANO@PS-LPs performed a remarkable advantage in killing AZI-resistant P. aeruginosa and removing their biofilms. In summary, NANO@PS-LPs present a potential nano-strategy to treat stubborn pseudomonal pulmonary infections and overcome correlative drug resistance., (Copyright © 2022 Elsevier B.V. All rights reserved.)

We studied an amorphous solid dispersion of berberine with absorption enhancer sodium caprate (Huang-Gui solid dispersion preparations, HGSD). A therapeutic effect of HGSD was revealed in mice with type 2 diabetes mellitus and palmitate-induced injury to MIN6 β-cells. HGSD treatment (150 mg/kg) improved glucose metabolism and decreased β-cell apoptosis in diabetic mice. Furthermore, the effective component of HGSD berberine significantly attenuated the palmitate-induced decrease in MIN6 β-cells viability and insulin secretion. Moreover, molecular docking analysis and Western blotting showed that berberine decreased cell apoptosis and expression of group VIA phospholipase A 2 (iPLA 2 ), p38 mitogen-activated protein kinase (p38 MAPK), and caspase-3. These data suggest that HGSD treatment protected β-cells via inhibiting the iPLA 2 /p38 MAPK pathway., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: Phosphoinositide-specific phospholipases C-γ1 (PLC-γ1) signaling has been shown to modulate osteoarthritis (OA) chondrocyte metabolism. However, the role of PLC-γ1 in OA osteoblasts remains unclear. Herein, whether and how PLC-γ1 was involved in mineralization in OA subchondral bone osteoblasts were investigated., Methods: Primary non-OA and OA osteoblasts of human and rat isolated from the subchondral bone or the calvaria were cultured in vitro, as well as mouse pre-osteoblastic cell line MC3T3-E1 cells. Rat knee OA model was induced by anterior cruciate ligament transection (ACLT), in which bone canal was carried out from the surface of lateral epicondyle of femur using micro-electric drill. Morphological characteristics of subchondral bone structure and articular cartilage were assessed using CT, micro-CT, and Safranin O/Fast green staining, respectively. Mineralization was measured by alizarin red staining. The expression and production of genes involved in osteoblastic phenotype and mineralization were evaluated by qPCR, western blotting, and immunohistochemistry assays, respectively. The inhibitions were performed using inhibitors and ShRNAs., Results: The decreased relative bone density and thickness in the early stage of OA and the increased one in the late stage of OA were observed in subchondral bone of ACLT-rat model. Decreased ALP and OCN levels and absorbance values of ARS content were observed in in vitro osteoblasts isolated from 2 w post-ACLT rat model, as well as IL-1β-treated (for maintaining and mimicking inflammatory status) human OA and rat osteoblasts. Decreased Atg7 level and LC3BII/I ratio in combination with an increase in the P62 level, was concomitant with decreased ALP and OCN mRNA levels and absorbance values of ARS content in OA or IL-1β-treated osteoblasts. Specific inhibition of PLC-γ1 by ShRNAs or inhibitor (U73122) elevated ALP and OCN mRNA levels and absorbance values of ARS content accompanied with increased Atg7 level and LC3BII/I ratio in combination with a decrease in the P62 level in OA osteoblasts. Furthermore, the promoting effect of PLC-γ1 inhibition on ALP and OCN mRNA levels and absorbance values of ARS content was reversed by endoplasmic reticulum (ER) stress activator HA15, as well as autophagic inhibitors CQ and 3MA. Injection with PLC-γ1 inhibitor U73122 from the surface of lateral epicondyle of femur reduced aberrant subchondral bone formation and attenuated articular cartilage degeneration in ACLT-rat., Conclusion: Aberrant changes of OA subchondral bone structure were concomitant with altered osteoblastic phenotype and mineralization. Impaired autophagy contributed to decreased osteoblastic mineralization in the early stage of OA. PLC-γ1 inhibition promoted osteoblastic mineralization through increasing autophagy in OA osteoblasts, which was partially attributed to suppression of ER stress. Targeting PLC-γ1 in subchondral bone osteoblasts could be more efficacious for OA therapy through treating the bone and cartilage at the same time. In summary, we hypothesize that suppressing PLCγ1 promotes mineralization of osteoarthritic subchondral bone osteoblasts via increasing autophagy, thereby ameliorating articular cartilage degeneration., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Integrins are a large family of cell surface receptors mediating the interaction of cells with their microenvironment and they play an important role in glioma biology. In the present work, we reported the anti-tumor effect of Sm-PLGV a phospholipase A 2 from Tunisian scorpion venom glands-as well as its recombinant forms expressed in Escherichia coli-through interference with integrin receptor function in malignant glioma cells U87. These phospholipases inhibited in a dose dependent manner the adhesion, migration and invasion onto fibrinogen and fibronectin without any cytotoxicity. We showed that Sm-PLGV and its recombinant constructs blocked U87 migration by reducing their velocity and directional persistence. The inhibitory effect was related to a blockage of the integrins αvβ3 and α5β1 function. Inactivation of the enzymatic activity of Sm-PLGV by chemical modification with p-bromophenacyl bromide did not affect its anti-tumor properties, suggesting the presence of 'pharmacological sites' distinct from the catalytic site in scorpion venom phospholipases A 2 ., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The antimicrobial and antiparasite activity of phospholipase A(2) (PLA(2)) from snakes and bees has been extensively explored. We studied the antiplasmodial effect of the whole venom of the snake Bothrops asper and of two fractions purified by ion-exchange chromatography: one containing catalytically-active phospholipases A(2) (PLA(2)) (fraction V) and another containing a PLA(2) homologue devoid of enzymatic activity (fraction VI). The antiplasmodial effect was assessed on in vitro cultures of Plasmodium falciparum. The whole venom of B. asper, as well as its fractions V and VI, were active against the parasite at 0.13 ± 0.01 µg/mL, 1.42 ± 0.56 µg/mL and 22.89 ± 1.22 µg/mL, respectively. Differences in the cytotoxic activity on peripheral blood mononuclear cells between the whole venom and fractions V and VI were observed, fraction V showing higher toxicity than total venom and fraction VI. Regarding toxicity in mice, the whole venom showed the highest lethal effect in comparison to fractions V and VI. These results suggest that B. asper PLA(2) and its homologue have antiplasmodial potential.

The apicomplexan parasite Cryptosporidium parvum is an important cause of diarrhea in humans and cattle, and it can persistently infect immunocompromised hosts. No consistently effective parasite-specific pharmaceuticals or immunotherapies for control of cryptosporidiosis are presently available. The innate immune system represents the first line of host defense against a range of infectious agents, including parasitic protozoa. Several types of antimicrobial peptides and proteins, collectively referred to herein as biocides, constitute a major effector component of this system. In the present study, we evaluated lactoferrin, lactoferrin hydrolysate, 5 cationic peptides (lactoferricin B, cathelicidin LL37, indolicidin, β-defensin 1, β-defensin 2), lysozyme, and 2 phospholipases (phospholipase A2, and phosphatidylinositol-specific phospholipase C) for anti-cryptosporidial activity. The biocides were evaluated either alone or in combination with 3E2, a monoclonal antibody (MAb) against C. parvum that inhibits sporozoite attachment and invasion. Sporozoite viability and infectivity were used as indices of anti-cryptosporidial activity in vitro. All biocides except lactoferrin had a significant effect on sporozoite viability and infectivity. Lactoferrin hydrolysate and each of the 5 cationic peptides were highly parasiticidal and strongly reduced sporozoite infectivity. While each phospholipase also had parasiticidal activity, it was significantly less than that of lactoferrin hydrolysate and each of the cationic peptides. However, each phospholipase reduced sporozoite infectivity comparably to that observed with lactoferrin hydrolysate and the cationic peptides. Moreover, when 3 of the cationic peptides (cathelicidin LL37, β-defensin 1, and β-defensin 2) were individually combined with MAb 3E2, a significantly greater reduction of sporozoite infectivity was observed over that by 3E2 alone. In contrast, reduction of sporozoite infectivity by a combination of either phospholipase with MAb 3E2 was no greater than that by 3E2 alone. These collective observations suggest that cationic peptides and phospholipases neutralize C. parvum by mechanisms that are predominantly either parasiticidal or non-parasiticidal, respectively.

Vipera ammodytes is the most venomous European snake, whose venom has been used as antigen for immunization of antivenom-producing animals. Same as venom of any other snake, it is a complex mixture of proteins, peptides and other compounds which biochemical and pharmacological variability has been demonstrated at interspecies and intraspecies level. In this work we demonstrated intraspecific variability between 8 venom production batches using both the conventional and the new methodology. Moreover, in contrast to the literature on different venoms' variability, for the first time we were able to select those biochemical differences that are related to and give information on the venom's toxicity and immunogenicity. We have shown that methods quantifying ammodytoxin (the most toxic compound identified so far in the Vipera ammodytes ammodytes venom) content of the venom clearly distinguish between high and low immunogenic venoms., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Venoms are complex mixtures of proteins, peptides and other compounds whose biochemical and biological variability has been clearly demonstrated. These molecules have been used as antigens for immunization of anti-venom-producing animals (horses or sheep). Ammodytoxins (Atx) are potently neurotoxic compounds, and the most toxic compounds isolated so far from the Vipera ammodytes ammodytes (Vaa) venom. Recently we have shown that the level of antibodies specific to Vaa venom's most toxic component, ammodytoxin A (AtxA), (anti-AtxA IgG) in Vaa venom immunized rabbit sera highly correlated to the venom toxicity-neutralization potential of these sera. Here we investigated whether Atx content of Vaa venom could influence the outcome of immunization procedure. The novel ELISA was developed for precise determination of Atx content and Atx was quantified in venom samples used for immunization of rabbits. We clearly showed that animals immunized with the venom containing lower amount of Atx produced sera with significantly lower venom toxicity-neutralizing power and, vice versa, animals immunized with venoms containing higher amount of Atx produced sera with higher venom toxicity-neutralizing ability. Thus, the content of Atx in Vaa venom is a relevant parameter of its suitability in the production of highly protective Vaa anti-venom., (Copyright 2010 Elsevier Inc. All rights reserved.)

A difficulty in explaining the mechanism whereby archaeal lipid membrane vesicles (archaeosomes) deliver entrapped protein antigens to the MHC class I cytosolic pathway from phagolysosomes of antigen-presenting cells has been the observation that they tend not to fuse. Here, we determine that archaeosomes, composed of archaeal isoprenoid mixtures of glyco and phospholipids, can be highly fusogenic when exposed to the pH and enzymes found in late phagolysosomes. Fusions were strictly dependent on acidic pH and the presence of alpha- or beta-glucosidase. Resonance energy transfer (RET) assays demonstrated that fusion conditions induced lipid mixing of archaeosome lipids with self-unlabeled archaeosomes. Because PC/PG/cholesterol liposomes by themselves did not fuse, it was possible to unequivocally show a fusion of rhodamine-labeled liposomes with archaeosomes by fluorescence microscopy and to demonstrate lipid mixing between labeled liposomes and archaeosomes by the RET assay. Radiotracer and (1)H NMR studies revealed that glycolipids in fused archaeosomes were not degraded significantly by glucosidase treatment during fusion. Rather, the glucosidases dramatically induced small archaeosomes to rapidly and visually aggregate at pH 4.8, but not 6.8, thus bringing membranes together appropriately as a first step in the fusion process. (1)H NMR was used to demonstrate that conditions causing aggregation correlated with binding of glucosidase to the archaeosomes. Binding at acidic pH occurred by the electrostatic interaction of positively charged glucosidase with the anionic phospholipids, although the interaction also occurred with the gentiobiosyl lipids. The data indicate a mechanism of membrane-membrane fusion for archaeal glycolipid membranes induced by glycosidase and illustrate the importance for inclusion of glycolipids in compositions of vesicles designed to deliver protein antigens to the cytosol for MHC class I presentation.

Cell surface glycosylation patterns are markers of cell type and status. However, the mechanisms regulating surface glycosylation patterns remain unknown. Using a panel of carbohydrate markers, we have shown that cell surface sialylation and fucosylation are upregulated in L1-transfected embryonic stem cells (L1-ESCs). Consistently, the mRNA levels of sialyltransferase ST6Gal1 and ST3Gal4, and fucosyltransferase FUT9 were significantly increased in L1-transfected ESCs. Activation of L1 signaling promoted cell survival and inhibited cell proliferation. ShRNAs knocking down FUT9, ST6Gal1 and ST3Gal4 blocked these effects. A phospholipase Cgamma (PLCgamma) inhibitor and shRNA reduced ST6Gal1, ST3Gal4 and FUT9 mRNA levels in the L1-ESCs. Thus, embryonic stem cell surface sialylation and fucosylation are regulated via PLCgamma by L1, with which they cooperate to modulate cell survival and proliferation.

Venoms of several animals have been used to study various physiopathologic processes, and also to offer opportunity to design and develop new therapeutic drugs. We briefly review certain wasp venom components and their biological effects, which may be potential sources of novel pharmacologically active compounds.

Two experiments were conducted to evaluate the effects of enzyme addition in chicken diets containing high oleic acid sunflower seeds (HOASS). In the first experiment (4 to 21 d of age), enzyme addition (lipase, phospholipase, and a combination of these) was used at the inclusion level of 1 g/kg in diets containing HOASS (250 g/kg) compared with a control corn-soybean diet. Weight gain, feed consumption, relative liver weight, fat digestibility, and amylase, lipase, serum lactate dehydrogenase (LDH), and creatine phosphokinase (CPK) activities were reduced, and feed conversion, relative duodenum, jejunum, ileum, and ceca lengths, plasma uric acid, cholesterol, and glucose concentrations were increased in the unsupplemented HOASS diet compared with the control diet. The addition of enzymes to the HOASS diet increased weight gain, feed consumption, relative pancreas and liver weights, fat digestibility, amylase and lipase activities, plasma uric acid, calcium, serum LDH and CPK, and total protein concentration and reduced feed conversion, relative spleen weight, relative duodenum, jejunum, ileum, and ceca lengths, plasma cholesterol, and glucose compared with the unsupplemented HOASS diet. In the second experiment (0 to 21 d of age), the same enzymes (0.5 g/kg each) were included in diets containing 150 g/kg of HOASS compared with a conventional sunflower meal diet (150 g/kg). The HOASS diet did not affect performance but reduced relative pancreas and abdominal fat weights and relative duodenum and ceca lengths, and increased crude fat, CP, and essential and nonessential amino acid digestibilities (except Ser, which was reduced) compared with the control diet. The addition of enzymes in the HOASS diet increased weight gain, feed consumption, and relative pancreas weight and reduced feed conversion, CP, and essential and nonessential amino acid digestibilities compared with the unsupplemented HOASS diet. In conclusion, the addition of 250 g of HOASS/kg in the diets caused a negative effect on performance, digestive organ sizes, fat and protein digestibilities, and pancreatic enzymes and modified blood parameters. However, the inclusion of HOASS at 150 g/kg improved some of these parameters and amino acid digestibilities. The enzyme addition counteracted some of these effects.

Although the flexor digitorum profundus (FDP) tendon's gliding resistance is low, the lubrication mechanism that enables this is unclear. The principal lubricants in joints, such as hyaluronic acid, phospholipids, and lubricin, a lubricating glycoprotein, are known to be present in the canine tendon. In this study, we assessed the effect of these lubricants in the tendon by measuring gliding resistance before and after their removal. Canine FDP tendons were treated with hyaluronidase, phospholipase, lipid solvent, and/or trypsin. The gliding resistance of FDP tendons significantly increased after all treatments (p < 0.05). The largest effect on gliding resistance was observed after trypsin digestion. Scanning electron microscopy and immunostaining for hyaluronic acid and lubricin were used to qualitatively assess the tendon surface after treatments. The trypsin digestion produced the most irregular surface, with many exposed collagen fibers. The results of this study suggest that phospholipids, hyaluronic acid, and protein components are all involved in maintaining the low gliding resistance of the FDP tendon., ((c) 2008 Orthopaedic Research Society)

The signal transduction cascade mediating muscarinic receptor modulation of N-type Ca2+ channel activity by the slow pathway has remained incompletely characterized despite focused investigation. Recently we confirmed a role for the G-protein Gq and identified phospholipase C (PLC), phospholipase A2 (PLA2), and arachidonic acid (AA) as additional molecules involved in N-current inhibition in superior cervical ganglion (SCG) neurons by the slow pathway. We have further characterized this signal transduction cascade by testing whether additional molecules downstream of phosphatidylinositol-4,5-bisphosphate (PIP2) are required. The L-channel antagonist nimodipine was bath-applied to block L-current. Pretreating cells with pertussis toxin (PTX) minimized M2/M4 muscarinic receptor inhibition of N-current by the membrane-delimited pathway. Consistent with our previous studies, pharmacologically antagonizing M1 muscarinic receptors (M1Rs), Gqalpha, PLC, PLA2, and AA minimized N-current inhibition by the muscarinic agonist oxotremorine-M (Oxo-M). When cells were left untreated with PTX, leaving the membrane-delimited pathway intact and the same antagonists retested, Oxo-M decreased whole cell currents. Moreover, inhibited currents displayed slowed activation kinetics, indicating intact N-current inhibition by the membrane-delimited pathway. These findings indicate that the antagonists used to block the slow pathway acted selectively. PLA2 cleaves AA from phospholipids, generating additional metabolites. We tested whether the metabolite lysophosphatidic acid (LPA) mimicked the inhibitory actions of Oxo-M. In contrast to AA, applying LPA did not inhibit whole cell currents. Taken together, these findings suggest that the slow pathway requires M1Rs, Gqalpha, PLC, PIP2, PLA2, and AA for N-current inhibition.

Antibacterial, antiparasitidal and antiviral properties have recently been attributed to members of the secreted phospholipases A(2) (sPLA(2)s) superfamily. Seven sPLA(2)s from groups IA, IB, IIA and III, were tested here in different culture conditions for inhibition of the in vitro intraerythrocytic development of Plasmodium falciparum, the causative agent of the most severe form of human malaria. In the presence of human serum, all sPLA(2)s were inhibitory, with three out of seven exhibiting IC(50)<0.1 nM. In all cases, inhibition could be induced by enzymatic pre-treatment of the serum. By contrast, no effect was observed when parasites were grown in a semi-defined medium (AlbuMAX II) devoid of lipoproteins and containing 10 times less phospholipids than the medium with human serum, strongly suggesting that hydrolysis of serum generating toxic lipid by-products, rather than a direct interaction of the sPLA(2) with the infected erythrocyte, is a general feature of the anti-Plasmodium properties of sPLA(2)s. Furthermore, in serum, six out of the seven sPLA(2)s were toxic against both trophozoite and schizont stages of the parasite development, contrasting with the trophozoite-selective bee venom enzyme's toxicity. Deciphering the molecular mechanisms at play in the phenotypic singularity of the bee venom enzyme toxicity might offer new prospects in antimalarial fight.

Propolis is a resinous substance collected by honeybees from plant sources. Its antimicrobial activity has been well documented but little is specifically known about its activity on virulence factors of Candida albicans. The aim of this work was therefore to evaluate in vitro the propolis effect on yeast-mycelial conversion (Y-M), extracellular phospholipase activity and fungal adhesion to epithelial cells. The two propolis samples used significantly inhibited the C. albicans strains tested, showing a rapid (between 30 seconds and 15 minutes), dose-dependent cytocidal activity and an inhibitory effect on Y-M conversion at a concentration of 0.22 mg/ml. Moreover, the hyphal length was reduced even at lower propolis concentration. Propolis also caused a dose- and time-dependent inhibition of phospholipase activity. No clear effect was shown on adherence to buccal epithelial cells and surface structure hydrophobicity, but damage to the plasma membrane structure was demonstrated with the Propidium Iodide test.

[3H]Spermidine (SPD) was accumulated in subcellular fractions enriched of the nucleus in a temperature-dependent manner with a saturable profile in murine brain. The accumulation reached a plateau within 30 min at 2 degrees C and 30 degrees C, while excess unlabeled SPD significantly inhibited the accumulation at 2 degrees C without markedly affecting that at 30 degrees C when added after equilibrium. The temperature-dependent accumulation of [3H]SPD was significantly inhibited by the triamine SPD and the tetraamine spermine, but not by the diamine putrescine. Phospholipases were invariably effective in significantly inhibiting the accumulation at 30 degrees C in a concentration-dependent manner. Amongst different discrete murine central structures examined, the temperature-dependent [3H]SPD accumulation was highest in neocortex with progressively lower activities in striatum, hypothalamus, spinal cord, medulla-pons, hippocampus, midbrain and cerebellum. These results suggest the possible presence of a hitherto unidentified nuclear transport system for particular polyamines in murine brain.

When finding the mechanism of the in vitro ethanol membrane action it was established that alcohol in moderate concentrations enhanced the inactivation of the rat brain cortex Na+, K(+)-ATPase after modification of the microsomal fraction by phospholipase A2 from cobra venom. It is supposed that ethanol sensitivity is determined by the structural state of the functional protein-lipid enzyme complexes in membrane. The decrease of the intrinsic tryptophan fluorescence of the membrane proteins and fluorescence of the probe 1-anilinonaphthalene-8-sulfonate (both without and under membrane surface charge shielding) in the presence of ethanol was revealed. It is concluded, that conformational changes of the membrane polar surface area coincide with the enzyme inactivation by ethanol.

Lactobacilli are members of the normal mucosal microflora of most animals. Many isolates of Lactobacillus spp. are adherent to epithelial cells. In this study, using Lactobacillus acidophilus and L. agilis, we detected adherence in a pattern that suggested that the bacteria were binding to extracellular matrix proteins. Fluorescent microscopy, by using anti-fibronectin antibody, demonstrated that the isolates localize in those areas where fibronectin was detected. In addition, fibronectin pretreatment of the bacterial cells decreased adherence to Intestinal 407 epithelial cell monolayers. Cellular binding to fibronectin was detected by enzyme-linked immunosorbent assay and affinity binding to radio-labeled fibronectin. Fibronectin may be one of the eukaryotic receptors mediating attachment of Lactobacillus to mucosal surfaces.

The adhesion molecule lymphocyte function-associated antigen 3 (LFA-3) (CD58) is an important regulator of immune cell function which occurs as both surface-associated and 'soluble' forms. This study has investigated the inter-relationship and the effects of cytokines on the expression of LFA-3 isoforms. The surface antigen was found to be relatively unaffected by cytokines, but the release of soluble LFA-3 (sLFA-3) was highly responsive to interleukin 1beta (IL-1beta), interferon gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha). This modulation was cell-specific, particularly with regard to IFN-gamma, which up-regulated sLFA-3 release by A431 cells but down-regulated the release of the soluble form from HEp2 and HepG2 cells. We further demonstrated that LFA-3 is also present in a cytoplasmic 'pool' in each of the cells and, moreover, that cleavage of LFA-3 from the cell surface by phospholipase C resulted in an increase in the levels of the intracellular LFA-3 and replacement of the membrane-associated antigen. These observations suggest that the expression of the surface, soluble and intracellular forms of LFA-3 may be linked by regulatory mechanisms which are likely to exert an important influence on inflammatory interactions.

Prior treatment with ferrous chloride led to marked inhibition of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne (MK-801) binding to an open ion channel associated with the N-methyl-D-aspartate (NMDA) receptor in a concentration-dependent manner at concentrations of higher than 1 microM in rat brain synaptic membranes. Both phospholipases A2 and C significantly prevented the inhibition when treated before the treatment with ferrous chloride, while neither superoxide dismutase nor alpha-tocopherol affected the inhibition even when treated simultaneously with ferrous chloride. Of various saturated and unsaturated free fatty acids, moreover, both oleic and arachidonic acids exclusively decreased the potency of ferrous chloride to inhibit binding when membranes were first treated with fatty acids, followed by the second treatment with ferrous chloride. These results suggest that membrane phospholipids may be at least in part responsible for interference by ferrous ions with opening processes of the native NMDA channel through molecular mechanisms associated with the liberation of unsaturated free fatty acids in rat brain.

Prior treatment with ferrous chloride led to the marked inhibition of [3H](+)-5-methyl-10, 11-dihydro-5H-dibenzo[a, d]cyclohepten-5, 10-imine (MK-801) binding to an open ion channel associated with the N-methyl-D-aspartate (NMDA) receptor in a concentration-dependent manner at concentrations of higher than 1 microM in rat brain synaptic membranes. Both phospholipases A2 and C significantly prevented the inhibition when treated before the treatment with ferrous chloride, while neither superoxide dismutase nor alpha-tocopherol affected the inhibition even when treated simultaneously with ferrous chloride. Of various saturated and unsaturated fatty acids, moreover, both oleic and arachidonic acids exclusively decreased the potency of ferrous chloride to inhibit binding when membranes were treated with fatty acids, followed by a second treatment with ferrous chloride. These results suggest that membrane phospholipids may be, at least in part, responsible for the interference by ferrous ions in the opening processes of the native NMDA channel through molecular mechanisms associated with the release of unsaturated fatty acids in rat brain.

Many cellular functions depend on rapid cytoskeletal rearrangements localized to specific cytoplasmic domains. Tight regulation of the submembranous microfilament network is accomplished in large part in erythrocytes and granulocytes by actin binding proteins that cap the fast-growing barbed filament ends. Study of this dynamic system is necessarily hampered by the confounding perturbations of cell lysis and dilution. In this paper, we characterize the functional properties of the membrane-associated spectrin-actin complex from human erythrocytes as it exists after hypotonic lysis. Purified spectrin-actin "seeds" extracted from erythrocyte membranes effectively nucleated actin elongation from their barbed ends. However, polymerization from spectrin-actin complexes associated with the membrane fraction prematurely slowed despite the presence of G-actin in great excess of the critical monomer concentration. The addition of cytochalasin B decreased (rather than augmented) the slowing of elongation attributable to the membrane fraction, indicating that capping of barbed filament ends (not monomer sequestration) was the major mechanism underlying this effect. The paradoxical implication of our findings is that, despite the presence of excess capper(s) in the membrane fraction, the membrane-associated spectrin-actin seeds were not capped until after dilution into physiological ionic strength buffer containing monomeric actin. Furthermore, by comparing the degrees of contamination of the extracted and membrane-associated spectrin-actin preparations, it appeared that recognized capping proteins (including gelsolin and capping protein beta2) were not the predominant cappers found in the membrane pellet after hypotonic lysis. We hypothesize that the barbed ends of membrane-associated spectrin-actin complexes, while not excluding actin monomers, may be selectively inaccessible to certain cappers (perhaps simply as the result of steric hindrance). Growth from such complexes in vivo could be limited by the availability of polymerization-competent G-actin.

Xenorhabdus spp. and Photorhabdus spp., entomopathogenic bacteria symbiotically associated with nematodes of the families Steinernematidae and Heterorhabditidae, respectively, were shown to produce different lipases when they were grown on suitable nutrient agar. Substrate specificity studies showed that Photorhabdus spp. exhibited a broad lipase activity, while most of the Xenorhabdus spp. secreted a specific lecithinase. Xenorhabdus spp. occur spontaneously in two variants, phase I and phase II. Only the phase I variants of Xenorhabdus nematophilus and Xenorhabdus bovienii strains produced lecithinase activity when the bacteria were grown on a solid lecithin medium (0.01% lecithin nutrient agar; 24 h of growth). Five enzymatic isomers responsible for this activity were separated from the supernatant of a X. nematophilus F1 culture in two chromatographic steps, cation-exchange chromatography and C18 reverse-phase chromatography. The substrate specificity of the X. nematophilus F1 lecithinase suggested that a phospholipase C preferentially active on phosphatidylcholine could be isolated. The entomotoxic properties of each isomer were tested by injection into the hemocoels of insect larvae. None of the isomers exhibited toxicity with the insects tested, Locusta migratoria, Galleria mellonella, Spodoptera littoralis, and Manduca sexta. The possible role of lecithinase as either a virulence factor or a symbiotic factor is discussed.

Heat treatment of normal human serum reveals otherwise masked anti-cardiolipin antibodies (aCL). We studied the mechanism of masking and the nature of the inhibitor of these aCL IgG. Other forms of treatment, besides heating for 30 min at 56 degrees C, can also unmask hidden aCL IgG. These include acid pH, hypermolar buffers and phospholipase digestion. When unmasked, these aCL recognize other anionic and zwitterionic phospholipids, but do not react with DNA, cell antigens or IgG. Using thin layer chromatography we demonstrate that the heat-labile inhibitor(s) of these aCL are phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine. These antibodies are not beta2-glycoprotein-I dependent and actually compete with this protein for phospholipid binding. The hidden antibodies are comprised of two populations of IgG autoantibodies: one reactive with cardiolipin, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine and sphingomyelin, and the other reactive almost exclusively with phosphatidylcholine and phosphorylcholine on enzyme-linked immunosorbent assay plates or when exposed by bromelain on the erythrocyte surface. Our data suggest that hidden aCL are natural oligoreactive IgG anti-phospholipid autoantibodies that circulate masked by their antigen.

Loss of amino acids into the coronary artery perfusate, which is exacerbated during anoxic stress, may have important implications for the ability of hearts subjected to ischemia or anoxia to recover function during reoxygenation. This work investigates the mechanisms underlying the amino acid efflux. Rat Langendorff heart preparations were used to study amino acid loss into coronary artery perfusates during anoxia or anoxia/reoxygenation sequences. Coronary flow rates, heart rates and intra-aortic pressures were recorded. Changes in myocardial amino acid concentrations were equated with amino acid levels in collected anoxic perfusate. With the exception of taurine, the differences in amino acid levels between normoxic and anoxic hearts were smaller than the amounts lost into the coronary perfusates, indicating ongoing replenishment of most amino acids during the anoxic episode. Fifteen-minute periods of exposure to low oxygen levels (P02 18-20 mmHg) resulted in large percentage increases in perfusate amino acid levels which returned slowly towards control levels upon reoxygenation. Anion channel blockers, anthracene-9-carboxylic acid, furosemide, and 4-acetamido-4-isothiocyanostilbene-2,2'-disulfonic acid (SITS), depressed anoxia-elicited increases in amino acid release. Phospholipase inhibition with quinacrine, 4-bromophenacyl bromide and 7,7-dimethyl-eicosadenoic acid (DEDA) depressed the anoxia-evoked release of amino acids. Combined applications of SITS and DEDA exhibited additive effects, virtually abolishing anoxia-evoked release of all the amino acids. The protein kinase C inhibitor, chelerythrine chloride, and the protein tyrosine kinase inhibitors, genistein and lavendustin A, inhibited anoxia-evoked amino acid release. Polyunsaturated fatty acids, arachidonic and linoleic, reduced anoxia-evoked amino acid release whereas monosaturated (oleic) and saturated (stearic) acids were ineffective. The glutamate transport inhibitor, dihydrokainate, depressed anoxia-evoked glutamate and aspartate release. These results suggest that at least three possible mechanisms for the anoxia-evoked amino acid efflux including (a) diffusional release through volume activated anion channels, (b) leakage across myocyte plasma membranes as a consequence of phospholipase activation and (c) reversal of Na+ dependent high-affinity transporters.

As previously reported, Listeria monocytogenes infection of P388D1 macrophages results in a rapid induction of NF-kappaB DNA-binding activity. Here we show that this induction of NF-kappaB activity occurs in a biphasic mode: first, a transient, IkappaBalpha degradation-dependent phase of activity, also induced by the nonvirulent species Listeria innocua, which is mediated by binding of the bacteria to the macrophage, or by adding Listeria-derived lipoteichoic acid to the macrophage; the second persistent phase of activation is only markedly induced when the bacteria enter the cytoplasm of the host cell and express the virulence genes plcA and plcB, encoding two phospholipases. We suggest that products of the enzymatic activity of phospholipases directly interfere with host cell signal transduction pathways, thus leading to persistent NF-kappaB activation via persistent IkappaBbeta degradation.

We reported earlier that isolated plasma membranes trigger a number of responses comprising contact inhibition of motility, including loss of oriented detyrosinated microtubules (Glu MTs) from the lamella of motile fibroblasts. In this study, we show that the membranes trigger this loss of Glu MTs, not by binding to cells, but by removing an essential component from the medium necessary to maintain oriented Glu MTs. Preincubation of membranes with medium containing serum followed by removal of the membranes by sedimentation rendered the membrane-treated medium capable of triggering the loss of oriented Glu MTs. Membrane activity was inhibited by high concentrations of serum and removal of serum from medium triggered the loss of oriented Glu MTs similar to that triggered by membranes. These results suggest that the membranes trigger the loss of Glu MTs by inactivating factors in serum that are required for the maintenance of oriented Glu MTs. By fractionating serum, we have identified lysophosphatidic acid (LPA) as the principal serum factor that is responsible for supporting oriented Glu MTs. The activity of LPA to maintain oriented Glu MTs upon serum withdrawal was half maximal at 100 nM and no activity was observed with structurally related phospholipids. Serum LPA levels were sufficient to account for the ability of serum to support oriented Glu MTs. Enzymatic degradation of serum LPA strongly reduced the ability of serum to support oriented Glu MTs. That membranes degrade LPA was shown by the ability of membranes to block LPA's ability to maintain oriented Glu MTs, and by direct measurement of the loss of radiolabeled LPA after incubation with membranes in vitro. These results show that isolated plasma membranes trigger the loss of Glu MTs from the lamella of motile cells by degrading serum LPA. Coupled with earlier results showing that membranes trigger a number of contact inhibition responses, our data suggest a new model for contact inhibition of motility in which local degradation of LPA and/or interference with LPA-stimulated signalling pathways initiates a contact inhibition response in colliding cells.

Guinea pig skin becomes more pigmented following exposure to UV rays. This melanization was accompanied by enhanced intensity of tyrosinase-staining and increased number of tyrosinase-positive melanocytes (MELty+), with resultant enhancement of melanin synthesis. To clarify the regulatory mechanism for melanization following UV irradiation, organ-cultured guinea pig skins have been used to examine their melanogenic responses to exogenous stimulation. This organ culture system responded well to UV irradiation, by increasing melanogenic activity. Also, in this system, phospholipases (PL), arachidonic acid, interleukin-1 alpha and melanocyte-stimulating hormone, but not endothelin-1 or phosphatidylinositol-specific PLC (PI-PLC), stimulated melanogenesis to various extents as indicated by the number of MELty+ and morphological changes. Among them, the PLA2 and PLD were found to have a potent stimulatory property for melanocytes. They might affect melanocytes directly or indirectly through an effect on keratinocytes. These results suggest that PLA2 and PLD play a key role in epidermal hyperpigmentation after UV irradiation or inflammation.

Recent studies indicate that sphingolipids mediate several cellular processes. We assessed roles of sphingolipids in the regulation of E-selectin expression in human umbilical vein endothelial cells. All exogenously-added sphingolipids (sphingosine, C2-ceramide, sphingosine 1-phosphate, and N,N-dimethylsphingosine) failed to induce E-selectin expression by themselves. C2-ceramide at 5 micron enhanced interleukin-1 beta (IL-1 beta)-induced E-selectin expression 2.7-fold, whereas other sphingolipids tested had no effects on this process. Sphingomyelinase, but not phospholipases A2, C, or D, mimicked the enhancing effect of C2-ceramide. Northern blot analyses revealed that C2-ceramide and sphingomyelinase increased interleukin-1 beta-induced E-selectin gene transcription levels. C2-ceramide and sphingomyelinase induced NF-kappaB activation by themselves and enhanced activation by IL-1 beta, which is essential for E-selectin expression. Immunological analyses with anti-NF-kappaB antibodies showed that subunit composition of NF-kappaB activated by IL-1 beta differs from that activated by C2-ceramide, suggesting that signaling pathways utilized by these stimuli may be different. Treatment with C2-ceramide or sphingomyelinase did not alter NF-ELAM1 specific binding activity. IL-1 beta induced sphingomyelin hydrolysis to ceramide; intracellular ceramide level increased to 182% of control value at 30 min. Taken together, these findings suggest that (i) sphingomyelin hydrolysis to ceramide does not trigger, but rather enhances cytokine-induced E-selectin expression, in part through NF-kappaB; (ii) sphingomyelin hydrolysis to ceramide does not mediate all the effects of IL-1 beta, although it may play important roles in IL-1 beta signal transduction in human umbilical vein endothelial cells.

In experiment 1, endometrial explants from 3 cyclic (Day 17) cows were incubated with arachidonic acid (AA), phospholipase A-2 (PLA-2) and calcium ionophore A23187 (CaI) or control. AA (0.2 mg), PLA-2 (1 U/ml) and CaI (4 micrograms/ml) increased PGF and PGE secretion. In experiment 2, endometrial explants from cyclic (n = 4) and pregnant (n = 3) cows were incubated +/- Ca++ and with either: control, AA, PLA-2, CaI, PLA-2 + CaI, or AA + CaI. PG secretion was higher in cultures with Ca++. In presence of Ca++, PGF secretion was lower for pregnant than cyclic endometrium. AA with Ca++ stimulated PGF and PGE secretion, indicating that AA availability may limit PG secretion. The stimulatory effect of PLA-2 on PGF and PGE secretion was greater in pregnant than cyclic endometrium. However, CaI inhibited the PLA-2 response of pregnant, but not cyclic endometrium. In experiment 3, endometrium (4 cyclic cows) failed to convert 3H-PGF2 alpha to PGE2 or 3H-PGE2 to PGF2 alpha. Responsiveness of PG secretion to PLA-2, and CaI is altered by reproductive status suggesting that these factors may be involved in the differential regulation of PG production during early pregnancy in cattle.

Ca2+ and Mg2+ caused a concentration-dependent activation of ATP hydrolysis by mitochondrial membranes of Hymenolepis diminuta, a rat intestinal cestode. Ca2+ was the more potent, but Mg2+ the more effective. The Lineweaver-Burk plot yielded Km and Vmax values of 1.15 nM and 217.4 nmol Pi min-1 mg-1 protein for Ca(2+)-dependent activity, and 1.86 mM and 333.3 nmol Pi min-1 mg-1 protein for Mg(2+)-dependent activity, respectively. Neither Na+ nor K+, nor a combination of the two cations, induced the hydrolysis of ATP. Ouabain, a specific inhibitor of Na+/K+ ATPase, did not affect the rate of ATP hydrolysis induced by Mg2+ alone or in combination with Na+ or K+. The membrane-bound enzyme was not affected by neuraminidase and concanavalin A. Ca2+ and Mg2+ also induced appreciable hydrolysis of other nucleoside triphosphates by the membranes. Some known anthelmintics, e.g. niclosamide, praziquantel and mebendazole, had no effect on ATPase activities. In addition to other compounds including respiratory inhibitors and uncouplers of phosphorylation, ruthenium red, which blocks Ca2+ influx into the cestode mitochondria, had no influence on the rate of ATP hydrolysis induced by the cations. Triton X-100 was found most suitable for solubilization of both activities. The differences between cestode ATPase and its mammalian counterpart have been discussed.

In vitro addition of Ca2+ ions was effective in almost doubling binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne (MK-801) before equilibrium to an ion channel associated with an N-methyl-D-aspartate (NMDA)-sensitive subclass of brain excitatory amino acid receptors. The addition of inhibitors for phospholipase (PLase) A2 markedly reduced binding in the absence of added Ca2+ ions, while Ca2+ ions restored the reduction to the level found in the absence of the inhibitors. Pretreatment with PLases A2 and C but not D was effective in potentiating [3H]MK-801 binding in a biphasic manner at a concentration range of 5 mU/ml-10 U/ml. Moreover, PLases A2 and C at low concentrations not only suppressed the abilities of 3 different agonists to potentiate [3H]MK-801 binding before equilibrium, but also reduced that of Ca2+ ions. These results suggest that Ca2+ ions may potentiate [3H]MK-801 binding to the NMDA channel highly permeable to Ca2+ ions through a mechanism common to that underlying potentiation by exogenous PLase A2 and/or C.

Ferricyanide reductase activity of plasma membranes isolated from Ehrlich ascites tumour cells was very sensitive to trypsin treatment. The decreases of activity observed after treatment with different glycosidases suggests that ferricyanide reductase is a glycoprotein. The opposite effects of phospholipase A2 and phospholipase C on the redox activity indicate that the phospholipidic environment plays an important role in the function of ferricyanide reductase. Sodium ions at millimolar concentrations, and some divalent cations at micromolar concentrations (Ca2+, Mg2+, Sr2+, and Mn2+) behaved as stimulators of ferricyanide reductase activity.

Tumor necrosis factor (TNF) is a potent inducer of human immunodeficiency virus (HIV) proviral transcription and subsequent mature virus production. Recent investigations have shown that TNF can use a signal transduction pathway in HL-60 cells involving sphingomyelin hydrolysis to ceramide with subsequent stimulation of a ceramide-linked kinase. When sphingomyelinase was added exogenously to activate this cascade in HIV-1-infected HL-60 cells, it mimicked TNF-induced HIV production. Phospholipases A2, C, or D, which do not generate ceramide, had no effect; however, a synthetic ceramide analog added exogenously potently induced HIV production. In addition, anti-TNF antibodies blocked much of the effect of both sphingomyelinase and the synthetic ceramide analog on virus expression, suggesting that, although signaling is initiated through the sphingomyelin pathway, it is sustained by autocrine TNF synthesis. Thus, direct activation of the sphingomyelin pathway recapitulated the effect of TNF on both HIV and TNF production. These studies indicate that the sphingomyelin pathway is involved in TNF signaling for HIV production in chronically infected myeloid cells.

We have investigated the ability of APC Class II molecules to bind and release exogenous peptides, two phenomena that are still poorly understood. In order to investigate the half-life of the complex of an exogenous peptide with DR molecules we have evaluated the uptake and release of the radiolabeled peptide 17-29-Tyr of influenza virus matrix protein (MA 17-29-Y) by a B-EBV cell line at different times and under different conditions. We have found that the kinetics of both binding and release of the peptide are very fast in living cells; using glutaraldehyde-fixed cells, the kinetics of the two phenomena are slow, closely resembling those observed with the same peptide and purified, immobilized DR molecules. As confirmed by the study of a specific T-cell clone activation, the Class II-MA 17-29-Y complexes are short-living ones, with an average half-life of 55 min, and the DR molecules that bind exogenous peptides continuously undergo peptidic exchange. These data, taken together, suggest that the APC are endowed with cellular mechanisms that increase the efficiency of both the loading and the unloading of Class II HLA with exogenous peptides. These mechanisms do not appear to require ATP or to involve newly synthesized Class II molecules, intracellular acidic compartments, or the microtubule-microfilament system. On the other hand, an undamaged cell membrane appears to be crucial for an efficient binding.

Vasoactive intestinal peptide (VIP) receptors were investigated in rat peritoneal macrophage membranes (RPMM) using [125I]VIP as ligand. The receptor binding was rapid, reversible, saturable, specific, and dependent on time, temperature, and membrane concentration. The Scatchard analysis of binding data was consistent with the existence of two classes of VIP binding sites with Kd values of 0.60 +/- 0.08 and 275 +/- 39 nM and binding capacities of 580 +/- 71 and 72,500 +/- 810 fmol VIP/mg protein, respectively. The interaction showed a high degree of specificity, as suggested by competitive displacement experiments with several peptides structurally or not structurally related to VIP. These pharmacological studies showed the following order of potency: VIP (IC50 = 1 nM) > rGRF (IC50 = 13 nM) > PHI (IC50 = 421 nM) >> secretin. Glucagon, somatostatin, insulin octapeptide of cholecystokinin [CCK(26-33)], and pancreastatin were ineffective at concentrations up to 1 microM. Binding of [125I]VIP to membranes is markedly reduced by increasing the ionic strength of incubation medium. Treatment of membranes with dithiothreitol, trypsin, and phospholipases A2 and C resulted in a loss of the ability of these membranes to bind VIP. However, treatment with phospholipase D did not affect binding of VIP by membranes. The molecular characterization of VIP receptors in RPMM was performed after [125I]VIP cross-linking to membranes using the cross-linker dithiobis (succinimidyl propionate). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins revealed specific [125I]VIP-protein complexes of M(r) 55,000 +/- 1700, 35,000 +/- 900, and 22,000 +/- 500.

The ability to produce a cytolytic toxin contributes to the success of many organisms in a particular niche by such diverse means as lysis of a phagolysosomal membrane of the macrophage by hemolysin from the intracellular parasite Trypanosoma cruzi, disruption of leukocyte activity by the Escherichia coli hemolysin, and destruction of invading bacteria by hemolysin from the annelid Glycera dibranchiata. The relative contribution of erythrocyte lysis to survival of the cytolysin producer is still under investigation. Nevertheless, the hemolytic phenotype is both a powerful tool for identifying novel cytolysins and a convenient marker for studying cytolytic activity in established toxins.

Pretreatment of 1321N1 human astrocytoma cells with serum induces a pronounced increase in subsequent stimulation by forskolin and other agents of intracellular cyclic AMP accumulation, a phenomenon referred to as sensitization (Mol. Pharmacol. 39, 399-406, 1991). Pretreatment of these cells with lysophosphatidic acid induced sensitization to a similar extent as that with serum (approximately fivefold for forskolin stimulation and twofold for isoproterenol and prostaglandin E1 stimulation), with half-maximal effects at approximately 30 nM lysophosphatidic acid. Phosphatidic acid was effective but less potent whereas other lipids were ineffective. Sensitization by serum and by lysophosphatidic acid were almost completely inhibited by pertussis toxin pretreatment and partially inhibited by prolonged phorbol ester exposure to induce protein kinase C down-regulation. Among nine cell lines tested, those that exhibited sensitization with serum showed comparable sensitization with lysophosphatidic acid. The effects of both lysophosphatidic acid and serum were markedly inhibited by treatment with phospholipase B but only minimally altered with phospholipases A2, D, and C. Exposure of cells to phospholipase C alone induced approximately threefold sensitization, but both serum and lysophosphatidic acid were able to induce further three- to fourfold sensitization above that induced by phospholipase C alone. In contrast, the effects of serum and lysophosphatidic acid were not additive with each other. Together these results suggest that lysophosphatidic acid or a closely related compound present in serum is the factor responsible for sensitization of the cyclic AMP pathway.