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Background: High burden of drug-resistant (DR) tuberculosis (TB) is a significant threat to national TB control programs all over the world and in the Russian Federation. Different Mycobacterium tuberculosis (MTB) genotypes are hypothesized to have specific characteristics affecting TB control programs. For example, Beijing strains are supposed to have higher mutation rates compared to strains of other genotypes and subsequently higher capability to develop drug-resistance., Results: Clinical MTB isolates from HIV- and HIV+ patients from four regions of Russia were analyzed for genotypes and mutations conferring resistance to Isoniazid, Rifampicin, Ethambutol, aminoglycosides, and fluoroquinolones. Analysis of genotypes and polymorphism of genomic loci according to the HIV status of the patients - sources of MTB isolates were performed. Studied MTB isolates from HIV- TB patients belonged to 15 genotypes and from HIV + TB patients - to 6 genotypes. Beijing clinical isolates dominated in HIV- (64,7%) and HIV+ (74,4%) groups. Other isolates were of LAM (including LAM1 and LAM9), Ural, and 4 minor groups of genotypes (including 5 subclones T). The spectrum of genotypes in the HIV- group was broader than in the HIV+ group. PR of B0/W148 Beijing was significantly lower than of other Beijing genotypes in susceptible and MDR-XDR isolates. Rates of isolates belonging to non-Beijing genotypes were higher than Beijing in susceptible isolates from HIV- patients., Conclusions: Beijing genotype isolates prevailed in clinical isolates of all drug susceptibility profiles both from HIV- and HIV+ patients, although B0/W148 Beijing genotype did not dominate in this study. Genome loci and mutations polymorphisms were more pronounced in clinical isolates from HIV- patients, than from HIV+., (© 2022. The Author(s).)

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease with no effective medical therapies. Recent research has focused on identifying the biological processes essential to the development and progression of fibrosis, and on the mediators driving these processes. Lysophosphatidic acid (LPA), a biologically active lysophospholipid, is one such mediator. LPA has been found to be elevated in bronchoalveolar lavage (BAL) fluid of IPF patients, and through interaction with its cell surface receptors, it has been shown to drive multiple biological processes implicated in the development of IPF. Accordingly, the first clinical trial of an LPA receptor antagonist in IPF has recently been initiated. In addition to being a therapeutic target, LPA also has potential to be a biomarker for IPF. There is increasing interest in exhaled breath condensate (EBC) analysis as a non-invasive method for biomarker detection in lung diseases, but to what extent LPA is present in EBC is not known., Methods: In this study, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to assess for the presence of LPA in the EBC and plasma from 11 IPF subjects and 11 controls., Results: A total of 9 different LPA species were detectable in EBC. Of these, docosatetraenoyl (22:4) LPA was significantly elevated in the EBC of IPF subjects when compared to controls (9.18 pM vs. 0.34 pM; p = 0.001). A total of 13 different LPA species were detectable in the plasma, but in contrast to the EBC, there were no statistically significant differences in plasma LPA species between IPF subjects and controls., Conclusions: These results demonstrate that multiple LPA species are detectable in EBC, and that 22:4 LPA levels are elevated in the EBC of IPF patients. Further research is needed to determine the significance of this elevation of 22:4 LPA in IPF EBC, as well as its potential to serve as a biomarker for disease severity and/or progression.

Rationale: Bioactive lipid mediators, derived from membrane lipid precursors, are released into the airway and airspace where they bind high-affinity cognate receptors and may mediate asthma pathogenesis. Lysophosphatidic acid (LPA), a bioactive lipid mediator generated by the enzymatic activity of extracellular autotaxin (ATX), binds LPA receptors, resulting in an array of biological actions on cell proliferation, migration, survival, differentiation, and motility, and therefore could mediate asthma pathogenesis., Objectives: To define a role for the ATX-LPA pathway in human asthma pathogenesis and a murine model of allergic lung inflammation., Methods: We investigated the profiles of LPA molecular species and the level of ATX exoenzyme in bronchoalveolar lavage fluids of human patients with asthma subjected to subsegmental bronchoprovocation with allergen. We interrogated the role of the ATX-LPA pathway in allergic lung inflammation using a murine allergic asthma model in ATX-LPA pathway-specific genetically modified mice., Measurements and Main Results: Subsegmental bronchoprovocation with allergen in patients with mild asthma resulted in a remarkable increase in bronchoalveolar lavage fluid levels of LPA enriched in polyunsaturated 22:5 and 22:6 fatty acids in association with increased concentrations of ATX protein. Using a triple-allergen mouse asthma model, we showed that ATX-overexpressing transgenic mice had a more severe asthmatic phenotype, whereas blocking ATX activity and knockdown of the LPA2 receptor in mice produced a marked attenuation of Th2 cytokines and allergic lung inflammation., Conclusions: The ATX-LPA pathway plays a critical role in the pathogenesis of asthma. These preclinical data indicate that targeting the ATX-LPA pathway could be an effective antiasthma treatment strategy.

Aims: To test the role of sphingosine-1-phosphate (S1P) signaling system in the in vivo setting of resuscitation and survival after cardiac arrest., Main Methods: A mouse model of potassium-induced cardiac arrest and resuscitation was used to test the importance of S1P homeostasis in resuscitation and survival. C57BL/6 and sphingosine kinase-1 knockout (SphK1-KO) female mice were arrested for 8 min then subjected to 5 minute CPR with epinephrine bolus given at 90s after the beginning of CPR. Animal survival was monitored for 4h post-resuscitation. Upregulation of tissue and circulatory S1P levels were achieved via inhibition of S1P lyase by 2-acetyl-5-tetrahydroxybutyl imidazole (THI). Plasma and heart tissue S1P and ceramide levels were quantified by targeted ESI-LC/MS/MS., Key Findings: Lack of SphK1 and low tissue/circulatory S1P levels in SphK1-KO mice led to poor animal resuscitation after cardiac arrest and to impaired survival post-resuscitation. Inhibition of S1P lyase in SphK1-KO mice drastically improved animal resuscitation and survival. Improved resuscitation and survival of THI-treated SphK1-KO mice were better correlated with cardiac dihydro-S1P (DHS1P) than S1P levels. The lack of SphK1 and the inhibition of S1P lyase by THI were accompanied by modulation in cardiac S1PR1 and S1PR2 expression and by selective changes in plasma N-palmitoyl- and N-behenoyl-ceramide levels., Significance: Our data provide evidence for the crucial role for SphK1 and S1P signaling system in resuscitation and survival after cardiac arrest, which may form the basis for development of novel therapeutic strategy to support resuscitation and long-term survival of cardiac arrest patients., (© 2013.)

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease, wherein transforming growth factor β (TGF-β) and sphingosine-1-phosphate (S1P) contribute to the pathogenesis of fibrosis. However, the in vivo contribution of sphingosine kinase (SphK) in fibrotic processes has not been documented. Microarray analysis of blood mononuclear cells from patients with IPF and SphK1- or SphK2-knockdown mice and SphK inhibitor were used to assess the role of SphKs in fibrogenesis. The expression of SphK1/2 negatively correlated with lung function and survival in patients with IPF. Also, the expression of SphK1 was increased in lung tissues from patients with IPF and bleomycin-challenged mice. Knockdown of SphK1, but not SphK2, increased survival and resistance to pulmonary fibrosis in bleomycin-challenged mice. Administration of SphK inhibitor reduced bleomycin-induced mortality and pulmonary fibrosis in mice. Knockdown of SphK1 or treatment with SphK inhibitor attenuated S1P generation and TGF-β secretion in a bleomycin-induced lung fibrosis mouse model that was accompanied by reduced phosphorylation of Smad2 and MAPKs in lung tissue. In vitro, bleomycin-induced expression of SphK1 in lung fibroblast was found to be TGF-β dependent. Taken together, these data indicate that SphK1 plays a critical role in the pathology of lung fibrosis and is a novel therapeutic target.

Sphingoid base-1-phosphates represent a very low portion of the sphingolipid pool but are potent bioactive lipids in mammals. This study was undertaken to determine whether these lipids are produced in palmitate-treated pancreatic β cells and what role they play in palmitate-induced β cell apoptosis. Our lipidomic analysis revealed that palmitate at low and high glucose supplementation increased (dihydro)sphingosine-1-phosphate levels in INS-1 β cells. This increase was associated with an increase in sphingosine kinase 1 (SphK1) mRNA and protein levels. Over-expression of SphK1 in INS-1 cells potentiated palmitate-induced accumulation of dihydrosphingosine-1-phosphate. N,N-dimethyl-sphingosine, a potent inhibitor of SphK, potentiated β-cell apoptosis induced by palmitate whereas over-expression of SphK1 significantly reduced apoptosis induced by palmitate with high glucose. Endoplasmic reticulum (ER)-targeted SphK1 also partially inhibited apoptosis induced by palmitate. Inhibition of INS-1 apoptosis by over-expressed SphK1 was independent of sphingosine-1-phosphate receptors but was associated with a decreased formation of pro-apoptotic ceramides induced by gluco-lipotoxicity. Moreover, over-expression of SphK1 counteracted the defect in the ER-to-Golgi transport of proteins that contribute to the ceramide-dependent ER stress observed during gluco-lipotoxicity. In conclusion, our results suggest that activation of palmitate-induced SphK1-mediated sphingoid base-1-phosphate formation in the ER of β cells plays a protective role against palmitate-induced ceramide-dependent apoptotic β cell death., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We recently demonstrated that hyperoxia (HO) activates lung endothelial cell NADPH oxidase and generates reactive oxygen species (ROS)/superoxide via Src-dependent tyrosine phosphorylation of p47(phox) and cortactin. Here, we demonstrate that the non-muscle ~214-kDa myosin light chain (MLC) kinase (nmMLCK) modulates the interaction between cortactin and p47(phox) that plays a role in the assembly and activation of endothelial NADPH oxidase. Overexpression of FLAG-tagged wild type MLCK in human pulmonary artery endothelial cells enhanced interaction and co-localization between cortactin and p47(phox) at the cell periphery and ROS production, whereas abrogation of MLCK using specific siRNA significantly inhibited the above. Furthermore, HO stimulated phosphorylation of MLC and recruitment of phosphorylated and non-phosphorylated cortactin, MLC, Src, and p47(phox) to caveolin-enriched microdomains (CEM), whereas silencing nmMLCK with siRNA blocked recruitment of these components to CEM and ROS generation. Exposure of nmMLCK(-/-) null mice to HO (72 h) reduced ROS production, lung inflammation, and pulmonary leak compared with control mice. These results suggest a novel role for nmMLCK in hyperoxia-induced recruitment of cytoskeletal proteins and NADPH oxidase components to CEM, ROS production, and lung injury.

A defining feature of acute lung injury (ALI) is the increased lung vascular permeability and alveolar flooding, which leads to associated morbidity and mortality. Specific therapies to alleviate the unremitting vascular leak in ALI are not currently clinically available; however, our prior studies indicate a protective role for sphingosine-1-phosphate (S1P) in animal models of ALI with reductions in lung edema. As S1P levels are tightly regulated by synthesis and degradation, we tested the hypothesis that inhibition of S1P lyase (S1PL), the enzyme that irreversibly degrades S1P via cleavage, could ameliorate ALI. Intratracheal instillation of LPS to mice enhanced S1PL expression, decreased S1P levels in lung tissue, and induced lung inflammation and injury. LPS challenge of wild-type mice receiving 2-acetyl-4(5)-[1(R),2(S),3(R),4-tetrahydroxybutyl]-imidazole to inhibit S1PL or S1PL(+/-) mice resulted in increased S1P levels in lung tissue and bronchoalveolar lavage fluids and reduced lung injury and inflammation. Moreover, down-regulation of S1PL expression by short interfering RNA (siRNA) in primary human lung microvascular endothelial cells increased S1P levels, and attenuated LPS-mediated phosphorylation of p38 mitogen-activated protein kinase and I-κB, IL-6 secretion, and endothelial barrier disruption via Rac1 activation. These results identify a novel role for intracellularly generated S1P in protection against ALI and suggest S1PL as a potential therapeutic target.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that mediates cellular functions by ligation via G protein-coupled S1P receptors. In addition to its extracellular action, S1P also has intracellular effects; however, the signaling pathways modulated by intracellular S1P remain poorly defined. We have previously demonstrated a novel pathway of intracellular S1P generation in human lung endothelial cells (ECs). In the present study, we examined the role of intracellular S1P generated by photolysis of caged S1P on EC barrier regulation and signal transduction. Intracellular S1P released from caged S1P caused mobilization of intracellular calcium, induced activation of MAPKs, redistributed cortactin, vascular endothelial cadherin, and β-catenin to cell periphery, and tightened endothelial barrier in human pulmonary artery ECs. Treatment of cells with pertussis toxin (PTx) had no effect on caged S1P-mediated effects on Ca(2+) mobilization, reorganization of cytoskeleton, cell adherens junction proteins, and barrier enhancement; however, extracellular S1P effects were significantly attenuated by PTx. Additionally, intracellular S1P also activated small GTPase Rac1 and its effector Ras GTPase-activating-like protein IQGAP1, suggesting involvement of these proteins in the S1P-mediated changes in cell-to-cell adhesion contacts. Downregulation of sphingosine kinase 1 (SphK1), but not SphK2, with siRNA or inhibition of SphK activity with an inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (CII) attenuated exogenously administrated S1P-induced EC permeability. Furthermore, S1P1 receptor inhibitor SB649164 abolished exogenous S1P-induced transendothelial resistance changes but had no effect on intracellular S1P generated by photolysis of caged S1P. These results provide evidence that intracellular S1P modulates signal transduction in lung ECs via signaling pathway(s) independent of S1P receptors.

Reactive oxygen species (ROS) generation, particularly by the endothelial NADPH oxidase family of proteins, plays a major role in the pathophysiology associated with lung inflammation, ischemia/reperfusion injury, sepsis, hyperoxia, and ventilator-associated lung injury. We examined potential regulators of ROS production and discovered that hyperoxia treatment of human pulmonary artery endothelial cells induced recruitment of the vesicular regulator, dynamin 2, the non-receptor tyrosine kinase, c-Abl, and the NADPH oxidase subunit, p47(phox), to caveolin-enriched microdomains (CEMs). Silencing caveolin-1 (which blocks CEM formation) and/or c-Abl expression with small interference RNA inhibited hyperoxia-mediated tyrosine phosphorylation and association of dynamin 2 with p47(phox) and ROS production. In addition, treatment of human pulmonary artery endothelial cells with dynamin 2 small interfering RNA or the dynamin GTPase inhibitor, Dynasore, attenuated hyperoxia-mediated ROS production and p47(phox) recruitment to CEMs. Using purified recombinant proteins, we observed that c-Abl tyrosine-phosphorylated dynamin 2, and this phosphorylation increased p47(phox)/dynamin 2 association (change in the dissociation constant (K(d)) from 85.8 to 6.9 nm). Furthermore, exposure of mice to hyperoxia increased ROS production, c-Abl activation, dynamin 2 association with p47(phox), and pulmonary leak, events that were attenuated in the caveolin-1 knock-out mouse confirming a role for CEMs in ROS generation. These results suggest that hyperoxia induces c-Abl-mediated dynamin 2 phosphorylation required for recruitment of p47(phox) to CEMs and subsequent ROS production in lung endothelium.

Phosphatidic acid generated by the activation of phospholipase D (PLD) functions as a second messenger and plays a vital role in cell signaling. Here we demonstrate that PLD-dependent generation of phosphatidic acid is critical for Rac1/IQGAP1 signal transduction, translocation of p47(phox) to cell periphery, and ROS production. Exposure of [(32)P]orthophosphate-labeled human pulmonary artery endothelial cells (HPAECs) to hyperoxia (95% O(2) and 5% CO(2)) in the presence of 0.05% 1-butanol, but not tertiary-butanol, stimulated PLD as evidenced by accumulation of [(32)P]phosphatidylbutanol. Infection of HPAECs with adenoviral constructs of PLD1 and PLD2 wild-type potentiated hyperoxia-induced PLD activation and accumulation of O(2)(.)/reactive oxygen species (ROS). Conversely, overexpression of catalytically inactive mutants of PLD (hPLD1-K898R or mPLD2-K758R) or down-regulation of expression of PLD with PLD1 or PLD2 siRNA did not augment hyperoxia-induced [(32)P]phosphatidylbutanol accumulation and ROS generation. Hyperoxia caused rapid activation and redistribution of Rac1, and IQGAP1 to cell periphery, and down-regulation of Rac1, and IQGAP1 attenuated hyperoxia-induced tyrosine phosphorylation of Src and cortactin and ROS generation. Further, hyperoxia-mediated redistribution of Rac1, and IQGAP1 to membrane ruffles, was attenuated by PLD1 or PLD2 small interference RNA, suggesting that PLD is upstream of the Rac1/IQGAP1 signaling cascade. Finally, small interference RNA for PLD1 or PLD2 attenuated hyperoxia-induced cortactin tyrosine phosphorylation and abolished Src, cortactin, and p47(phox) redistribution to cell periphery. These results demonstrate a role of PLD in hyperoxia-mediated IQGAP1 activation through Rac1 in tyrosine phosphorylation of Src and cortactin, as well as in p47(phox) translocation and ROS formation in human lung endothelial cells.

In vascular endothelium, the major research focus has been on reactive oxygen species (ROS) derived from Nox2. The role of Nox4 in endothelial signal transduction, ROS production, and cytoskeletal reorganization is not well defined. In this study, we show that human pulmonary artery endothelial cells (HPAECs) and human lung microvascular endothelial cells (HLMVECs) express higher levels of Nox4 and p22(phox) compared to Nox1, Nox2, Nox3, or Nox5. Immunofluorescence microscopy and Western blot analysis revealed that Nox4 and p22(phox), but not Nox2 or p47(phox), are localized in nuclei of HPAECs. Further, knockdown of Nox4 with siRNA decreased Nox4 nuclear expression significantly. Exposure of HPAECs to hyperoxia (3-24 h) enhanced mRNA and protein expression of Nox4, and Nox4 siRNA decreased hyperoxia-induced ROS production. Interestingly, Nox4 or Nox2 knockdown with siRNA upregulated the mRNA and protein expression of the other, suggesting activation of compensatory mechanisms. A similar upregulation of Nox4 mRNA was observed in Nox2 2(-/-) ko mice. Downregulation of Nox4, or pretreatment with N-acetylcysteine, attenuated hyperoxia-induced cell migration and capillary tube formation, suggesting that ROS generated by Nox4 regulate endothelial cell motility. These results indicate that Nox4 and Nox2 play a physiological role in hyperoxia-induced ROS production and migration of ECs.

The generation of reactive oxygen species (ROS) in the vasculature plays a major role in the genesis of endothelial cell (EC) activation and barrier function. Of the several potential sources of ROS in the vasculature, the endothelial NADPH oxidase family of proteins is a major contributor of ROS associated with lung inflammation, ischemia/reperfusion injury, sepsis, hyperoxia, and ventilator-associated lung injury. The NADPH oxidase in lung ECs has most of the components found in phagocytic oxidase, and recent studies show the expression of several homologues of Nox proteins in vascular cells. Activation of NADPH oxidase of nonphagocytic vascular cells is complex and involves assembly of the cytosolic (p47(phox), p67(phox), and Rac1) and membrane-associated components (Noxes and p22(phox)). Signaling pathways leading to NADPH oxidase activation are not completely defined; however, they do appear to involve the cytoskeleton and posttranslation modification of the components regulated by protein kinases, protein phosphatases, and phospholipases. Furthermore, several key components regulating NADPH oxidase recruitment, assembly, and activation are enriched in lipid microdomains to form a functional signaling platform. Future studies on temporal and spatial localization of Nox isoforms will provide new insights into the role of NADPH oxidase-derived ROS in the pathobiology of lung diseases.

Particulate matter (PM) in ambient air is a risk factor for human respiratory and cardiovascular diseases. The delivery of PM to airway epithelial cells has been linked to release of proinflammatory cytokines; however, the mechanisms of PM-induced inflammatory responses are not well-characterized. This study demonstrates that PM induces cyclooxygenase (COX)-2 expression and IL-6 release through both a reactive oxygen species (ROS)-dependent NF-kappaB pathway and an ROS-independent C/EBPbeta pathway in human bronchial epithelial cells (HBEpCs) in culture. Treatment of HBEpCs with Baltimore PM induced ROS production, COX-2 expression, and IL-6 release. Pretreatment with N-acetylcysteine (NAC) or EUK-134, in a dose-dependent manner, attenuated PM-induced ROS production, COX-2 expression, and IL-6 release. The PM-induced ROS was significantly of mitochondrial origin, as evidenced by increased oxidation of the mitochondrially targeted hydroethidine to hydroxyethidium by reaction with superoxide. Exposure of HBEpCs to PM stimulated phosphorylation of NF-kappaB and C/EBPbeta, while the NF-kappaB inhibitor, Bay11-7082, or C/EBPbeta siRNA attenuated PM-induced COX-2 expression and IL-6 release. Furthermore, NAC or EUK-134 attenuated PM-induced activation of NF-kappaB; however, NAC or EUK-134 had no effect on phosphorylation of C/EBPbeta. In addition, inhibition of COX-2 partly attenuated PM-induced Prostaglandin E2 and IL-6 release.

We have demonstrated that LPA (lysophosphatidic acid)-induced IL (interleukin)-8 secretion was partly mediated via transactivation of EGFR [EGF (epidermal growth factor) receptor] in HBEpCs (human bronchial epithelial primary cells). The present study provides evidence that LPA-induced transactivation of EGFR regulates COX (cyclo-oxygenase)-2 expression and PGE(2) [PG (prostaglandin) E(2)] release through the transcriptional factor, C/EBPbeta (CCAAT/enhancer-binding protein beta), in HBEpCs. Treatment with LPA (1 microM) stimulated COX-2 mRNA and protein expression and PGE(2) release via G(alphai)-coupled LPARs (LPA receptors). Pretreatment with inhibitors of NF-kappaB (nuclear factor-kappaB), JNK (Jun N-terminal kinase), or down-regulation of c-Jun or C/EBPbeta with specific siRNA (small interference RNA) attenuated LPA-induced COX-2 expression. Downregulation of EGFR by siRNA or pretreatment with the EGFR tyrosine kinase inhibitor, AG1478, partly attenuated LPA-induced COX-2 expression and phosphorylation of C/EBPbeta; however, neither of these factors had an effect on the NF-kappaB and JNK pathways. Furthermore, LPA-induced EGFR transactivation, phosphorylation of C/EBPbeta and COX-2 expression were attenuated by overexpression of a catalytically inactive mutant of PLD2 [PLD (phospholipase D) 2], PLD2-K758R, or by addition of myristoylated PKCzeta [PKC (protein kinase C) zeta] peptide pseudosubstrate. Overexpression of the PLD2-K758R mutant also attenuated LPA-induced phosphorylation and activation of PKCzeta. These results demonstrate that LPA induces COX-2 expression and PGE(2) production through EGFR transactivation-independent activation of transcriptional factors NF-kappaB and c-Jun, and EGFR transactivation-dependent activation of C/EBPbeta in HBEpCs. Since COX-2 and PGE(2) have been shown to be anti-inflammatory in airway inflammation, the present data suggest a modulating and protective role of LPA in regulating innate immunity and remodelling of the airways.

Background: Lysophosphatidic acid (LPA) is a biologically active lysophospholipid and a component of normal plasma. LPA binds to receptors expressed on circulating and structural lung cells and affects cell growth and activation. Whether LPA is present in the lung has not been previously reported., Objective: To develop an assay to measure LPA in bronchoalveolar lavage (BAL) fluids, and to study the association between LPA and allergic airway inflammation., Methods: Seventeen allergic subjects underwent bronchoscopy and segmental allergen challenge, followed 18 h later by BAL. Supernatants were analysed for LPA content using liquid chromatography and mass spectroscopy. Expression of LPA receptors on primary bronchial epithelial cells was analysed by immunolabelling, and the effects of LPA on epithelial cell barrier function was investigated by measuring transepithelial resistance., Results: LPA was detectable in BAL from control lung segments, and significantly increased 18 h after allergen challenge. Polyunsaturated species of LPA were especially increased following segmental allergen challenge. LPA levels did not strongly correlate with the number or percentages of eosinophils, neutrophils of lymphocytes, whereas MIP-3alpha (CCL20) levels correlated significantly with the allergen-driven influx of lymphocytes. The levels of LPA from control sites correlated inversely with BAL protein content, suggesting that LPA promoted epithelial barrier integrity at baseline. Experiments using primary human bronchial epithelial cells confirmed that LPA tightened the epithelial cell barrier., Conclusion: Lysophosphatidic acid is detectable in human BAL fluids at baseline and its expression increases during allergic inflammation. LPA does not appear to be a dominant chemoattractant for eosinophils or lymphocytes during allergic airway inflammation. In the absence of ongoing inflammation, LPA may promote epithelial barrier integrity.

Sphingosine kinase 1 (SK1) is one of the two known kinases, which generates sphingosine-1-phosphate (S1P), a potent endogenous lipid mediator involved in cell survival, proliferation, and cell-cell interactions. Activation of SK1 and intracellular generation of S1P were suggested to be part of the growth and survival factor-induced signaling, and overexpression of SK1 provoked cell tumorigenic transformation. Using a highly selective and sensitive LC-MS/MS approach, here we show that SK1 overexpression, but not SK2, in different primary cells and cultured cell lines results in predominant upregulation of the synthesis of dihydrosphingosine-1-phosphate (DHS1P) compared to S1P. Stable isotope pulse-labeling experiments in conjunction with LC-MS/MS quantitation of different sphingolipids demonstrated strong interference of overexpressed SK1 with the de novo sphingolipid biosynthesis by deviating metabolic flow of newly formed sphingoid bases from ceramide formation toward the synthesis of DHS1P. On the contrary, S1P biosynthesis was not directly linked to the de novo sphingoid bases transformations and was dependent on catabolic generation of sphingosine from complex sphingolipids. As a result of SK1 overexpression, migration and Ca2+-response of human pulmonary artery endothelial cells (HPAEC) to stimulation with external S1P, but not thrombin, was strongly impaired. In contrast, selective increase in intracellular content of DHS1P or S1P through the uptake and phosphorylation of corresponding sphingoid bases had no effect on S1P-induced signaling or facilitation of wound healing. Furthermore, infection of human bronchial epithelial cells (HBEpC) with RSV A-2 virus increased SK1-mediated synthesis of DHS1P and S1P, whereas TNF-alpha enhanced only S1P production in HPAEC. These findings uncover a new functional role for SK1, which can control survival/death (DHS1P-S1P/ceramides) balance by targeting sphingolipid de novo biosynthesis and selectively generating DHS1P at a metabolic step preceding ceramide formation.

Sphingosine-1-phosphate (S1P) and dihydrosphingosine-1-phosphate (DHS1P) are important signaling sphingolipids. The presence of nanomolar levels of S1P and DHS1P in tissues, cells, and biological fluids requires a highly sensitive and selective assay method for their reliable detection and quantitation. Preliminary findings employing positive ion electrospray ionization (ESI) liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis indicated significant sample carryover from previous injections of authentic standards of S1P and DHS1P. This article details a negative ion ESI LC-MS/MS technique following modification of the zwitterionic nature of S1P and DHS1P via derivatization. A highly selective and sensitive LC-MS/MS technique capable of reliable detection of less than 50 fmol of the derivatives of S1P and DHS1P without significant sample carryover was developed. Standard curves for S1P and DHS1P are linear over wide ranges (0-300 pmol) of analyte concentrations with correlation coefficients (r2) greater than 0.995. The levels of S1P and DHS1P in human platelet poor plasma were 590.8+/-42.1 and 130.7+/-20.7 pmol/ml, respectively. The levels of S1P and DHS1P in fetal bovine serum were 141.7+/-4.6 and 0.6+/-0.2 pmol/ml, respectively. The addition of sphingosine (1 microM) to human pulmonary artery endothelial cells in culture resulted in a more than 20-fold increase in the cellular level of S1P, whereas the level of DHS1P was unchanged.

Purpose: Polycarpine from ascidian Polycarpa aurata was previously found to be active against different human tumor cells. In this study, we investigated the antitumor mechanisms of polycarpine and its synthetic derivative, desmethoxyethoxy-polycarpine (dimethylpolycarpine), through the induction of apoptosis. This new knowledge regarding the proapoptotic action of polycarpine and dimethylpolycarpine should lead to a better understanding of their effects and development of a new class of anticancer drugs., Methods: Apoptosis was clearly observed by flow cytometry and Western blotting using an antibody against cleaved caspase-3 as an apoptotic marker., Results: Polycarpines differentially activated p38 kinase, JNKs, and ERKs in JB6 Cl 41 cells. The polycarpines-induced apoptosis was decreased in cells expressing a dominant-negative mutant of JNK. Both compounds stimulated p53-dependent transcriptional activity and phosphorylation. Induction of p53-phosphorylation at serine 15 was suppressed in JNKI and JNK2 knockout cells. Furthermore, polycarpines were unable to induce apoptosis in p53-deficient MEFs in contrast to a strong induction of apoptosis in wild type MEFs, suggesting that p53 is involved in apoptosis induced by polycarpines. The p53 phosphorylation in turn was mediated by activated JNKs., Conclusions: These results indicate that all three MAPK signaling pathways are involved in the response of JB6 cells to treatment with polycarpines. Evidence also supports a proapoptotic role of the JNKs signaling pathway in vivo and clearly indicates that JNKs are required for phosphorylation of c-Jun, activation of p53, and subsequent apoptosis induced by polycarpines.

The complex therapy in patients with primary open-angle glaucoma, with normalized intraocular pressure but with declined visual functions included the use of the Russian neurometabolic preparation pantogam. The improvement of the visual function appeared as enlargement of the borders of visual fields, decreased area and sensitivity deficit (according to computer perimetric data). Doppler ultrasonography has demonstrated an increase in the mean blood velocity in the orbital vessels and a decrease in eyeball blood supply defects after pantogam therapy which is a favourable sign of a glaucomatous process. The authors consider the use of pantogam to be rational in the complex therapy of patients with glaucoma.

A new fatty acid, (5Z,9Z)-22-methyl-5,9-tetracosadienoic acid (1a), and a rare fatty acid, (5Z,9Z)-23-methyl-5,9-tetracosadienoic acid (2a), the predominant constituents of the free fatty acid fraction from the lipids of the sponge Geodinella robusta, were isolated and partly separated by reversed phase high-performance liquid chromatography, followed by multifold crystallization from MeOH to give 1a and 2a in 70% and 60% purity, respectively. These fatty acids were identified as (5Z,9Z)-22- and (5Z,9Z)-23-methyl-5,9-tetracosadienoic acids by nuclear magnetic resonance techniques, including distortionless enhancement by polarization transfer, heteronuclear multiple quantum connectivity, and correlation spectroscopy experiments, as well as from mass-spectrometric data for their methyl esters, the methyl esters of their perhydro derivatives, and their pyrrolidides. Mixtures of 1a and 2a showed cytotoxic activity against mouse Ehrlich carcinoma cells and a hemolytic effect on mouse erythrocytes. The sterol fraction from the same sponge was analyzed by gas-liquid chromatography-mass spectrometry, and 24-methylenecholesterol was identified as a main constituent of this fraction. The implications of the co-occurrence of membranolytic long-chain fatty acids and 24-methylenecholesterol as a main membrane sterol are discussed in terms of the phenomenon of biochemical coordination.

The paper presents a complex psychophysiological analysis of the effect of a combined administration of pantogam and potassium orotate (kalii orotas) on the dynamics of cognitive function in patients with neurotic disorders. The investigation was conducted in an 8-stage consecutive cycle and employed computer-aided diagnostic system. It was established that the combined use of pantogam and potassium orotate produces a positive effect upon the dynamics of restoration of the attention and memory mechanisms in neurotic patients.

Halenaquinol inhibited the partial reactions of ATP hydrolysis by rat brain cortex Na(+),K(+)-ATPase, such as [3H]ATP binding to the enzyme, Na(+)-dependent front-door phosphorylation from [gamma-(33)P]ATP, and also Na(+)- and K(+)-dependent E(1)<-->E(2) conformational transitions of the enzyme. Halenaquinol abolished the positive cooperativity between the Na(+)- and K(+)-binding sites on the enzyme. ATP and sulfhydryl-containing reagents (cysteine and dithiothreitol) protected the Na(+),K(+)-ATPase against inhibition. Halenaquinol can react with additional vital groups in the enzyme after blockage of certain sulfhydryl groups with 5,5'-dithio-bis-nitrobenzoic acid. Halenaquinol inhibited [3H]ouabain binding to Na(+),K(+)-ATPase under phosphorylating and non-phosphorylating conditions. Binding of fluorescein 5'-isothiocyanate to Na(+),K(+)-ATPase and intensity of fluorescence of enzyme tryptophanyl residues were decreased by halenaquinol. We suggest that interaction of halenaquinol with the essential sulfhydryls in/or near the ATP-binding site of Na(+),K(+)-ATPase resulted in a change of protein conformation and subsequent alteration of overall and partial enzymatic reactions.

The distribution of free sterols, polyhydroxysteroids and steroid glycosides in different body components of the Far-eastern starfish Patiria (=Asterina) pectinifera has been studied. It was shown that free sterol fractions from aboral and oral body walls, gonads, stomach and pyloric ceca contained Delta(7) sterols with a preponderance of 5alpha-cholest-7-en-3beta-ol. All these body components had also toxic steroid oligoglycosides. However, polyhydroxysteroids and related low molecular weight steroid glycosides were found in stomach and pyloric ceca only. In pyloric ceca, the sulfated monoside 'asterosaponin' P(1) was identified as a main polar steroid, whereas 6-sodium sulfate of cholestane-3beta,4beta,6alpha,7alpha,8,15beta,16beta,26-octaol predominated in the stomach. Probable biological functions of polar steroids and free sterols in this starfish were discussed. It was suggested that some polyhydroxysteroids and related monoglycosides play the same biological role as bile alcohols and bile acids do in vertebrates.

Halenaquinol, a natural cardioactive pentacyclic hydroquinone from the sponge Petrosia seriata, was found to be a powerful inhibitor of the rat brainstem and of the rat brain cortex Na+, K(+)-ATPases and the rabbit muscle sarcoplasmic reticulum Ca(2+)-ATPase with I50 values of 7.0 x 10(-7), 1.3 x 10(-6) and 2.5 x 10(-6) M, respectively. Halenaquinol also inhibited K(+)-phosphatase activity of the rat brain cortex Na+, K(+)-ATPase with an I50 value of 3 x 10(-6) M. Ouabain-insensitive Mg(2+)-ATPase activity of the microsomal fraction of the rat brain cortex was weakly inhibited by halenaquinol. Inhibition was irreversible, dose- and time-dependent. Naphthohydroquinone fragment in structures of halenaquinol, related natural and model compounds was very important for an inhibiting effect.

Effects of two triterpene glycosides, isolated from the holothurian Psolus fabricii, on rat brain Na+, K(+)-ATPase (Na, K-pump; EC 3.6.1.3) were investigated. Psolusosides A and B (PsA and PsB) inhibited rat brain Na+, K(+)-ATPase with I50 values of 1 x 10(-4) M and 3 x 10(-4) M, respectively. PsA significantly stimulated [3H]ATP binding to Na+, K(+)-ATPase, weakly increased [3H]ouabain binding to the enzyme, and inhibited K(+)-phosphatase activity to a smaller degree than the total reaction of ATP hydrolysis. In contrast, PsB decreased [3H]ATP binding to Na+, K(+)-ATPase, and had no effect on [3H]ouabain binding to the enzyme. K(+)-Phosphatase activity was inhibited by PsB in parallel with Na+, K(+)-ATPase activity. The fluorescence intensity of tryptophanyl residues of Na+, K(+)-ATPase was increased by PsA and decreased by PsB in a dose-dependent manner. The excimer formation of pyrene, a hydrophobic fluorescent probe, was decreased by PsA only. The different characteristics of inhibition mode for these substances were explained by peculiarities of their chemical structures and distinctive affinity to membrane cholesterol.

Sapogenins from the starfish Asterias amurensis and Lethasterias nanimensis chelifera, 5 alpha-pregn-9(11)-ene-3 beta,6 alpha-diol-20-one, 5 alpha-cholest-9(11)-ene-3 beta,6 alpha-diol-23-one, 5 alpha-cholesta-9(11),24(25)-diene-3 beta,6 alpha-diol-23-one, (20E)-5 alpha-cholesta-9(11),20(22)-diene-3 beta,6 alpha-diol-23-one and 24 zeta-methyl-5 alpha-cholesta-9(11),20(22)-diene-3 beta,6 alpha-diol-23-one, stimulated the contractile force of the heart of the mollusk Spisula sachalinensis at concentration of 5 x 10(-5) M. Ouabain, a specific inhibitor of Na+,K(+)-ATPase, at concentration of 5 x 10(-5) M had no effect on this physiological model. Starfish sapogenins of the cholestane series moderately inhibited rat brain cortex Na+,K(+)-ATPase and decreased Ca2+ influx into Ehrlich carcinoma cells. In contrast, pregnane asterogenin asterone did not inhibit Na+,K(+)-ATPase and increased the influx of Ca2+ into cells. These effects were not the result of cell membrane damage, because none of the compounds tested have hemolytic activity.

The effect of triterpene glycosides from holothurians on Na+-K+-ATPase of rat brain was investigated. The marine glycosides are irreversible inhibitors of the enzyme with an average I50 value of 10(-4) M. ATP had a low protective effect against inhibition. The inhibitory effect was increased by preincubation with MgCl2. There was alteration of the activation curve of Na+-K+-ATPase by NaCl and KCl in the presence of glycosides. Triterpene glycosides inhibited the K+-phosphatase activity, but to a smaller degree than the ATPase activity. Na+-K+-ATPase of pig kidney was less sensitive to the marine triterpene glycosides than the brain enzyme. The marine glycosides did not alter the specific binding of [3H]-ouabain to the Na+-K+-ATPase.

High-angle X-ray diffraction spectra showed that triterpene glycosides form crystalline complexes with membrane cholesterol. Electron microscopy demonstrated a decreased vesicle size, of the membrane preparation from rat brain which is enriched in Na+-K+-ATPase, by the triterpene glycosides. The Arrhenius plot was linear in the presence of triterpene glycosides. The half-width of the phosphatidylcholine N-methyl proton line in proton NMR spectra was not altered in the presence of marine glycosides. The excimer formation of pyrene, a hydrophobic fluorescent probe, was significantly decreased by triterpene glycosides. The increase of tryptophanyl residue fluorescence demonstrated a change of the Na+-K+-ATPase conformation after treatment with cytotoxic glycosides.

3,5-Dibromo-1-acetoxy-4-oxo-2,5-cyclohexadien-1-acetonitrile (dienone A) inhibited Na+ - K+-ATPase with a half-maximal inhibition concentration (I50) equal to 2.9 X 10(-6)M. Inhibition was time- and pH-dependent and complete after 20-30 min preincubation within a range of pH from 7.0 to 9.0. Kinetic evaluation of the cationic substrate activation of Na+ - K+-ATPase indicated mixed type inhibition with regard to Na+ and K+ and competitive inhibition with regard to ATP activation of the enzyme. The presence of Mg2+ caused an increased inhibition. Also, K+-p-nitrophenyl phosphatase activity was altered by dienone A and mixed type inhibition with regard to p-nitrophenyl phosphate and K+ was demonstrated. Inhibition was partially restored by repeated washing. Preincubation with sulfhydryl reagents protected the enzyme from inhibition. A significant linear correlation between reactive enzyme sulfhydryl contents [SH] and Na+ - K+-ATPase activity in the presence of varying concentrations of dienone A was observed. One of the factors causing cytotoxic activity of this compound might be its interaction with some thiol groups of the membrane-bound Na+ - K+-ATPase.

Marine glycosides from the sea cucumbers Actinopyga agassizi, Holothuria atra, Bohadschia argus, Cucumaria fraudatrix, Astichopus multifidus and Thelenota ananas inhibit both Na+-K+ ATPase and Mg2+-ATPase of rat brain in vitro. The glycoside-cholesterol complex of these compounds does not influence ATPase activity. Asterosaponins from starfishes Linckia guildingi and Linckia laevigata possess a slight inhibiting effect. The triterpene glycosides from sea cucumbers are more powerful inhibitors than steroidal glycosides from starfishes.

The bromine-containing compounds from sponges of the Aplysinidae family inhibit, in vitro, the Na+ -K+ -ATPase activity of the rat brain microsomal fraction. The extent of inhibition is dependent on concentration and chemical structure of the compounds. The substances containing the dienone fragment, such as 3,5-dibromo-1-hydroxy-4-oxo-2,5-cyclohexadien-1-acetamide (IV), 3,5-dibromo-1-acetoxy-4-oxo-2,5-cyclohexadien-1-acetonitrile (V) and 3,5-dibromo-1-hydroxy-4-oxo-2,5-cyclohexadien-1-ethylacetate (VI), are powerful inhibitors of Na+ -K+ -ATPase.

A simultaneous determination of 3 gonadotropic hormones of the hypophysis (follitropin, lutronin and prolactin) using a radioimmunoassay in 33 patients with trophoblastic uterine tumors and 22 women on the 1st-8th day after normal delivery, has shown that there are not only significant differences in the nature of the secretion of hypophyseal gonadotropic hormones in pathology as compared to the normal postnatal period but also differences in different forms of trophoblastic tumors. These differences were characterized by a progressive, according to the severity of disease, independence between trophoblastic tissue function and gonadotropic function of the hypophysis. A marked suppression of follicle-stimulating function of the hypophysis after normal delivery decreased hydatidiform mole and trophoblastic tumors and was not observed in patients with chorioepithelioma of the uterus.