RESUMO
BACKGROUND The aim of this study was to determine if components of the endocannabinoid system are modulated in uterine leiomyomas (fibroids). Components studied included cannabinoid receptors 1 (CB1) and 2 (CB2); the G protein-coupled receptor GPR55; transient potential vanilloid receptor 1 (TRPV1) and the endocannabinoid modulating enzymes N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH), and their N-acylethanolamine (NAE) ligands: N-arachidonylethanolamine (AEA), N-oleoylethanolamine (OEA), and N-palmityolethanaolamine (PEA). MATERIAL AND METHODS Transcript levels of CB1, CB2, TRPV1, GPR55, NAPE-PLD, and FAAH were measured using RT-PCR and correlated with the tissue levels of the 3 NAEs in myometrial tissues. The tissues studied were: 1) fibroids, 2) myometrium adjacent/juxtaposed to the fibroid lesions, and 3) normal myometrium. Thirty-seven samples were processed for NAE measurements and 28 samples were used for RT-PCR analyses. RESULTS FAAH expression was significantly lower in fibroids, resulting in a NAPE-PLD: FAAH ratio that favors higher AEA levels in pre-menopausal tissues, whilst PEA levels were significantly lower, particularly in post-menopausal women, suggesting PEA protects against fibroid pathogenesis. The CB1: CB2 ratio was lower in fibroids, suggesting that loss of CB1 expression affects the fibroid cell phenotype. Significant correlations between reduced FAAH, CB1, and GPR55 expression and PEA in fibroids indicate that the loss of these endocannabinoid system components are biomarkers of leiomyomata. CONCLUSIONS Loss of expression of CB1, FAAH, GPR55, and PEA production are linked to the pathogenesis of uterine fibroids and further understanding of this might eventually lead to better disease indicators or the development of therapeutic potentials that might eventually be used in the management of uterine fibroids.
Assuntos
Endocanabinoides/metabolismo , Leiomioma/metabolismo , Leiomioma/fisiopatologia , Adulto , Idoso , Amidoidrolases/análise , Biópsia , Etanolaminas/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Pessoa de Meia-Idade , Ácidos Oleicos/metabolismo , Fosfolipase D/análise , Receptor CB1 de Canabinoide/análise , Receptor CB2 de Canabinoide/análise , Receptores de Canabinoides , Receptores Acoplados a Proteínas G/análise , Canais de Cátion TRPV/análise , Útero/fisiopatologiaRESUMO
Diphtheria by Corynebacterium ulcerans is increasingly occurring in children, adolescents and adults. In addition to diphtheria toxin (DT), phospholipase D (PLD) is considered a virulence factor of C. ulcerans. In the present study, a first case of concurrent diphtheria by a PLD-negative C. ulcerans and infectious mononucleosis (IM) was verified. Clinical and microbiological profiles and binding properties to human Fibrinogen (Fbg), Fibronectin (Fn) and type I collagen (col I) biotinylated proteins and virulence to Caenorhabditis elegans were investigated for C. ulcerans strain 2590 (clinical isolate) and two control strains, including PLD-positive BR-AD22 wild type and PLD-negative ELHA-1 PLD mutant strains. MALDI-TOF assays and a multiplex PCR of genes coding for potentially toxigenic corynebacteria identified strain 2590 as non-DT producing. Interestingly, strain 2590 did not express PLD activity in the CAMP test although the presence of the pld gene was verified. PLD-negative 2590 and a PLD-positive 210932 strains showed similar affinity to Fbg, Fn and type I collagen. C. elegans were able to escape from C. ulcerans strains, independent of PLD and DT production. Higher mortality of nematodes was verified for PLD-negative strains. Additional studies concerning multifactorial virulence potential of C. ulcerans, including environmental conditions remain necessary.
Assuntos
Infecções por Corynebacterium/microbiologia , Corynebacterium/isolamento & purificação , Difteria/microbiologia , Mononucleose Infecciosa/microbiologia , Adolescente , Animais , Antibacterianos/farmacologia , Caenorhabditis elegans , Corynebacterium/classificação , Corynebacterium/efeitos dos fármacos , Corynebacterium/genética , Humanos , Masculino , Fosfolipase D/análise , Fosfolipase D/metabolismo , Fatores de Virulência/análise , Fatores de Virulência/metabolismoRESUMO
Phosphatidylcholine (PC)-specific phospholipase D (PLD) hydrolyzes the phosphodiester bond of the PC to generate phosphatidic acid (PA) and regulates several subcellular functions. Mammalian genomes contain two genes encoding distinct isoforms of PLD in contrast with invertebrate genomes that include a single PLD gene. However, the significance of two genes within a genome encoding the same biochemical activity remains unclear. Recently, loss of function in the only PLD gene in Drosophila was reported to result in reduced PA levels and a PA-dependent collapse of the photoreceptor plasma membrane due to defects in vesicular transport. Phylogenetic analysis reveals that human PLD1 (hPLD1) is evolutionarily closer to dPLD than human PLD2 (hPLD2). In the present study, we expressed hPLD1 and hPLD2 in Drosophila and found that while reconstitution of hPLD1 is able to completely rescue retinal degeneration in a loss of function dPLD mutant, hPLD2 was less effective in its ability to mediate a rescue. Using a newly developed analytical method, we determined the acyl chain composition of PA species produced by each enzyme. While dPLD was able to restore the levels of most PA species in dPLD3.1 cells, hPLD1 and hPLD2 each were unable to restore the levels of a subset of unique species of PA. Finally, we found that in contrast with hPLD2, dPLD and hPLD1 are uniquely distributed to the subplasma membrane region in photoreceptors. In summary, hPLD1 likely represents the ancestral PLD in mammalian genomes while hPLD2 represents neofunctionalization to generate PA at distinct subcellular membranes.
Assuntos
Mutação com Perda de Função , Fosfolipase D/genética , Degeneração Retiniana/genética , Animais , Linhagem Celular , Drosophila , Expressão Gênica , Humanos , Fosfolipase D/análise , Fosfolipase D/metabolismo , Filogenia , Degeneração Retiniana/metabolismo , Espectrometria de Massas em Tandem , TransgenesRESUMO
Extensive rodent literature suggests that the endocannabinoid (eCB) system present in the nucleus accumbens (NAc) modulates dopamine (DA) release in this area. However, expression patterns of the cannabinoid receptor type 1 (CB1R), the synthesizing enzyme N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD), and the degradation enzyme fatty acid amide hydrolase (FAAH) in the NAc have not yet been described in non-human primates. The goal of this study is therefore to characterize the expression and localization of the eCB system within the NAc of vervet monkeys (Chlorocebus sabaeus) using Western blots and immunohistochemistry. Results show that CB1R, NAPE-PLD, and FAAH are expressed across the NAc rostrocaudal axis, both in the core and shell. CB1R, NAPE-PLD, and FAAH are localized in medium spiny neurons (MSNs) and fast-spiking GABAergic interneurons (FSIs). Dopaminergic projections and astrocytes did not express CB1R, NAPE-PLD, or FAAH. These data show that the eCB system is present in the vervet monkey NAc and supports its role in the primate brain reward circuit.
Assuntos
Amidoidrolases/análise , Chlorocebus aethiops/anatomia & histologia , Núcleo Accumbens/química , Fosfolipase D/análise , Receptor CB1 de Canabinoide/análise , Animais , Feminino , Imuno-Histoquímica , Masculino , Microscopia Confocal , Núcleo Accumbens/anatomia & histologia , Núcleo Accumbens/ultraestruturaRESUMO
As one of the emerging inorganic graphene analogues, two-dimensional titanium carbide (Ti3C2) nanosheets have attracted extensive attention in recent years because of their remarkable structural and electronic properties. Herein, a sensitive and selective nanoprobe to fluorescently probe phospholipase D activity was developed on the basis of an ultrathin Ti3C2 nanosheets-mediated fluorescence quenching effect. Ultrathin Ti3C2 nanosheets with â¼1.3 nm in thickness were synthesized from bulk Ti3AlC2 powder by a two-step exfoliation procedure and further modified by a natural phospholipid that is doped with rhodamine B-labeled phospholipid (RhB-PL-Ti3C2). The close proximity between RhB and Ti3C2 leads to efficient fluorescence quenching (>95%) of RhB by energy transfer. Phospholipase D-catalyzed lipolysis of the phosphodiester bond in RhB-PL results in RhB moving away from the surface of Ti3C2 nanosheets and subsequent fluorescence recovery of RhB, providing a fluorescent "switch-on" assay for the phospholipase D activity. The proposed nanoprobe was successfully applied to quantitatively determine phospholipase D activity with a low limit of detection (0.10 U L-1) and to measure its inhibition. Moreover, in situ monitoring and imaging the activity of phospholipase D in living cells were achieved using this biocompatible nanoprobe. These results reveal that Ti3C2 nanosheets-based probes exhibit great potential in fluorometric assay and clinical diagnostic applications.
Assuntos
Corantes Fluorescentes/química , Nanopartículas/química , Imagem Óptica , Fosfolipase D/análise , Fosfolipídeos/química , Titânio/química , Linhagem Celular Tumoral , Corantes Fluorescentes/síntese química , Humanos , Células MCF-7RESUMO
Here we developed a simple, sensitive and accurate PLD detection method based on a target-controlled gating liposome (TCGL) "off-on" cascade amplified strategy and personal glucose meters (PGMs). It showed excellent sensitivity with a detection limit of 0.005 U L(-1) and well performed PLD activity analysis in breast cancer cells and inhibitor drug screening.
Assuntos
Automonitorização da Glicemia/instrumentação , Neoplasias da Mama/enzimologia , Ensaios Enzimáticos/instrumentação , Fosfolipase D/análise , Amilose/metabolismo , Técnicas Biossensoriais/instrumentação , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos/instrumentação , Feminino , Glucana 1,4-alfa-Glucosidase/metabolismo , Glucose/análise , Glucose/metabolismo , Humanos , Limite de Detecção , Lipossomos/metabolismo , Lipossomos/ultraestrutura , Fosfolipase D/antagonistas & inibidores , Fosfolipase D/metabolismoRESUMO
Through its production of phosphatidic acid (PA), phospholipase D (PLD) is strongly involved in vesicular trafficking and cell signaling, making this enzyme an important therapeutic target. However, most PLD assays developed so far are either discontinuous or based on the indirect determination of choline released during PLD-catalyzed phosphatidylcholine hydrolysis, making its kinetic characterization difficult. We present here the development of a direct, specific, and continuous PLD assay that is based on the chelation-enhanced fluorescence property of 8-hydroxyquinoline (8HQ) following Ca(2+) complexation with PLD-generated PA. The real-time fluorescence intensity from 8HQ/Ca(2+)/PA complexes can be converted to concentrations of product using a calibration curve, with a detection limit of 1.2 µM of PA on a microplate scale, thus allowing measurement of the PLD-catalyzed reaction rate parameters. Hence, this assay is well adapted for studying the substrate specificity of PLD, together with its kinetic parameters, using natural phospholipids with various headgroups. In addition, the assay was found to be effective in monitoring the competitive inhibition of PA formation in the production of phosphatidylalcohols following the addition of primary alcohols, such as ethanol, propan-1-ol, or butan-1-ol. Finally, this assay was validated using the purified recombinant Vigna unguiculata PLD, as well as the PLD from Streptomyces chromofuscus, cabbage, or peanuts, and no PA production could be detected using phospholipase A1, phospholipase A2, or phospholipase C, allowing for a reliable determination of PLD activity in crude protein extract samples. This easy to handle PLD assay constitutes, to our knowledge, the first direct and continuous PA determination method on a microplate scale.
Assuntos
Fluorescência , Oxiquinolina/química , Fosfolipase D/análise , Álcoois/farmacologia , Arachis/enzimologia , Brassica/enzimologia , Fabaceae/enzimologia , Estrutura Molecular , Fosfolipase D/antagonistas & inibidores , Fosfolipase D/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo , Streptomyces/enzimologiaRESUMO
Phospholipase D (PLD) is a critical component of intracellular signal transduction and has been implicated in many important biological processes. It has been observed that there are abnormalities in PLD expression in many human cancers, and PLD is thus recognized as a potential diagnostic biomarker as well as a target for drug discovery. We report for the first time a phospholipid-modified nanoprobe for ratiometric upconversion fluorescence (UCF) sensing and bioimaging of PLD activity. The nanoprobe can be synthesized by a facile one-step self-assembly of a phospholipid monolayer composed of poly(ethylene glycol) (PEG)ylated phospholipid and rhodamine B-labeled phospholipid on the surface of upconversion nanoparticles (UCNPs) NaYF4: 20%Yb, 2%Er. The fluorescence resonance energy transfer (FRET) process from the UCF emission at 540 nm of the UCNPs to the absorbance of the rhodamine B occurs in the nanoprobe. The PLD-mediated hydrolysis of the phosphodiester bond makes rhodamine B apart from the UCNP surface, leading to the inhibition of FRET. Using the unaffected UCF emission at 655 nm as an internal standard, the nanoprobe can be used for ratiometric UCF detection of PLD activity with high sensitivity and selectivity. The PLD activity in cell lysates is also determined by the nanoprobe, confirming that PLD activity in a breast cancer cell is at least 7-fold higher than in normal cell. Moreover, the nanoprobe has been successfully applied to monitoring PLD activity in living cells by UCF bioimaging. The results reveal that the nanoprobe provides a simple, sensitive, and robust platform for point-of-care diagnostics and drug screening in biomedical applications.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Imagem Molecular/métodos , Nanopartículas/química , Fosfolipase D/análise , Fosfolipídeos/química , Linhagem Celular/enzimologia , Linhagem Celular Tumoral/enzimologia , Fluorescência , Humanos , Hidrólise , Imagem Molecular/instrumentação , Polietilenoglicóis/química , Rodaminas/química , Soluções , Itérbio/química , Ítrio/químicaRESUMO
The hypothalamus and the endocannabinoid system are important players in the regulation of energy homeostasis. In a previous study, we described the ultrastructural distribution of CB1 receptors in GABAergic and glutamatergic synaptic terminals of the dorsomedial region of the ventromedial nucleus of the hypothalamus (VMH). However, the specific localization of the enzymes responsible for the synthesis of the two main endocannabinoids in the hypothalamus is not known. The objective of this study was to investigate the precise subcellular distribution of N-arachidonoylphospatidylethanolamine phospholipase D (NAPE-PLD) and diacylglycerol lipase α (DAGL-α) in the dorsomedial VMH of wild-type mice by a high resolution immunogold electron microscopy technique. Knock-out mice for each enzyme were used to validate the specificity of the antibodies. NAPE-PLD was localized presynaptically and postsynaptically but showed a preferential distribution in dendrites. DAGL-α was mostly postsynaptic in dendrites and dendritic spines. These anatomical results contribute to a better understanding of the endocannabinoid modulation in the VMH nucleus. Furthermore, they support the idea that the dorsomedial VMH displays the necessary machinery for the endocannabinoid-mediated modulation of synaptic transmission of brain circuitries that regulate important hypothalamic functions such as feeding behaviors.
Assuntos
Imuno-Histoquímica , Lipase Lipoproteica/análise , Fosfolipase D/análise , Núcleo Hipotalâmico Ventromedial/enzimologia , Animais , Feminino , Lipase Lipoproteica/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Fosfolipase D/metabolismo , Inclusão do Tecido , Núcleo Hipotalâmico Ventromedial/ultraestruturaRESUMO
Tyrosyl-DNA phosphodiesterase I (Tdp1) is a member of the phospholipase D superfamily that hydrolyzes 3'-phospho-DNA adducts via two conserved catalytic histidines-one acting as the lead nucleophile and the second acting as a general acid/base. Substitution of the second histidine specifically to arginine contributes to the neurodegenerative disease spinocerebellar ataxia with axonal neuropathy (SCAN1). We investigated the catalytic role of this histidine in the yeast protein (His432) using a combination of X-ray crystallography, biochemistry, yeast genetics, and theoretical chemistry. The structures of wild-type Tdp1 and His432Arg both show a phosphorylated form of the nucleophilic histidine that is not observed in the structure of His432Asn. The phosphohistidine is stabilized in the His432Arg structure by the guanidinium group that also restricts the access of nucleophilic water molecule to the Tdp1-DNA intermediate. Biochemical analyses confirm that His432Arg forms an observable and unique Tdp1-DNA adduct during catalysis. Substitution of His432 by Lys does not affect catalytic activity or yeast phenotype, but substitutions with Asn, Gln, Leu, Ala, Ser, and Thr all result in severely compromised enzymes and DNA topoisomerase I-camptothecin dependent lethality. Surprisingly, His432Asn did not show a stable covalent Tdp1-DNA intermediate that suggests another catalytic defect. Theoretical calculations revealed that the defect resides in the nucleophilic histidine and that the pK(a) of this histidine is crucially dependent on the second histidine and on the incoming phosphate of the substrate. This represents a unique example of substrate-activated catalysis that applies to the entire phospholipase D superfamily.
Assuntos
Fosfolipase D/química , Diester Fosfórico Hidrolases/química , Domínio Catalítico/genética , Cristalografia por Raios X , Histidina/química , Histidina/genética , Histidina/metabolismo , Humanos , Cinética , Modelos Biológicos , Modelos Moleculares , Simulação de Dinâmica Molecular , Família Multigênica/genética , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Organismos Geneticamente Modificados , Fosfolipase D/análise , Fosfolipase D/genética , Diester Fosfórico Hidrolases/análise , Diester Fosfórico Hidrolases/genética , Leveduras/genética , Leveduras/metabolismoRESUMO
Phospholipase D (PLD, EC 3.1.4.4) is a key enzyme involved in phospholipid catabolism, initiating a lipolytic cascade in membrane deterioration during senescence and stress, which was cloned from Jatropha curcas L., an important plant species as its seed is the raw material for biodiesels. The cDNA was 2,886 bp in length with a complete open reading frame of 2,427 bp which encoded a polypeptide of 808 amino acids including a putative signal peptide of 53 amino acid residues and a mature protein of 755 amino acids with a predicted molecular mass of 86 kD and a pI of 5.44, having two highly conserved HKD' motifs. Phylogenetic analysis indicated the J. curcas PLD alpha (JcPLDalpha) showed a high similarity to other PLD alpha from plants. Semi-quantitative RT-PCR analysis revealed that it was especially abundant in root, stem, leaf, endosperm and flower, weakly in seed. And the JcPLDalpha was increasedly expressed in leaf undergoing environmental stress such as salt (300 mM NaCl), drought (30% PEG), cold (4degreeC) and heat (50degreeC). The JcPLDalpha protein was successfully expressed in Escherichia coli and showed high enzymatic activities. Maximal activity was at pH 8 and 60degreeC.
Assuntos
Jatropha/genética , Fosfolipase D/genética , Clonagem Molecular , DNA Complementar/análise , DNA Complementar/isolamento & purificação , Genes de Plantas , Concentração de Íons de Hidrogênio , Jatropha/enzimologia , Jatropha/metabolismo , Fosfolipase D/análise , Fosfolipase D/isolamento & purificação , Fosfolipase D/metabolismo , Filogenia , Análise de Sequência de DNA , TemperaturaRESUMO
The ability of negatively charged phosphatidates to form complexes with Fe(3+) ions was used to design a simple spectrophotometric assay for the quantitative determination of phosphatidic acid (PA). In the reaction with the purple iron(III)-salicylate, PA extracts Fe(3+) ions and decreases the absorbance at 490 nm. Lower competition with salicylate for Fe(3+) ions was observed with single negatively charged phosphatidates such as phosphatidylglycerol (PG), whereas neutral phosphatidates such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE) showed no influence on the absorbance of the iron(III) complex. The detection limit of the method on a microplate scale was 10 microM PA. Based on these results, an assay for determining the activity of phospholipase D (PLD) toward natural phospholipids such as PC, PE, and PG was developed. In contrast to other spectroscopic PLD assays, this method is able to determine PLD activity toward different lipids or even lipid mixtures.
Assuntos
Ferro/química , Ácidos Fosfatídicos/análise , Fosfolipase D/análise , Espectrofotometria/métodos , Brassica/enzimologia , Ácidos Fosfatídicos/química , Ácidos Fosfatídicos/metabolismo , Fosfolipase D/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Salicilatos/química , Glycine maxRESUMO
Hematopoietic stem cells (HSCs) are generally defined by their dual properties of pluripotency and extensive self-renewal capacity. However, a lack of experimental clarity as to what constitutes extensive self-renewal capacity coupled with an absence of methods to prospectively isolate long-term repopulating cells with defined self-renewal activities has made it difficult to identify the essential components of the self-renewal machinery and investigate their regulation. We now show that cells capable of repopulating irradiated congenic hosts for 4 months and producing clones of cells that can be serially transplanted are selectively and highly enriched in the CD150(+) subset of the EPCR(+)CD48(-)CD45(+) fraction of mouse fetal liver and adult bone marrow cells. In contrast, cells that repopulate primary hosts for the same period but show more limited self-renewal activity are enriched in the CD150(-) subset. Comparative transcriptome analyses of these 2 subsets with each other and with HSCs whose self-renewal activity has been rapidly extinguished in vitro revealed 3 new genes (VWF, Rhob, Pld3) whose elevated expression is a consistent and selective feature of the long-term repopulating cells with durable self-renewal capacity. These findings establish the identity of a phenotypically and molecularly distinct class of pluripotent hematopoietic cells with lifelong self-renewal capacity.
Assuntos
Separação Celular/métodos , Citometria de Fluxo/métodos , Células-Tronco Hematopoéticas/citologia , Animais , Animais Congênicos , Antígenos CD/análise , Antígenos de Diferenciação/análise , Células da Medula Óssea/citologia , Divisão Celular , Células Cultivadas/transplante , Perfilação da Expressão Gênica , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Imunofenotipagem , Antígenos Comuns de Leucócito/análise , Fígado/citologia , Fígado/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Fosfolipase D/análise , Quimera por Radiação , Receptores de Superfície Celular/análise , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Proteína rhoB de Ligação ao GTP/análise , Proteína rhoB de Ligação ao GTP/genética , Fator de von Willebrand/análise , Fator de von Willebrand/genéticaAssuntos
Ácidos Araquidônicos/metabolismo , Transtorno do Deficit de Atenção com Hiperatividade/enzimologia , Encéfalo/enzimologia , Moduladores de Receptores de Canabinoides/metabolismo , Dopamina/metabolismo , Alcamidas Poli-Insaturadas/metabolismo , Adolescente , Amidoidrolases/análise , Amidoidrolases/sangue , Ácidos Araquidônicos/análise , Transtorno do Deficit de Atenção com Hiperatividade/sangue , Transtorno do Deficit de Atenção com Hiperatividade/fisiopatologia , Encéfalo/fisiopatologia , Moduladores de Receptores de Canabinoides/análise , Criança , Estudos de Coortes , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Regulação para Baixo/fisiologia , Endocanabinoides , Humanos , Linfócitos/metabolismo , Masculino , Vias Neurais/metabolismo , Vias Neurais/fisiopatologia , Fosfolipase D/análise , Fosfolipase D/sangue , Alcamidas Poli-Insaturadas/análise , Valor Preditivo dos TestesRESUMO
Phospholipase D (PLD) is an enzyme producing phosphatidic acid and choline through hydrolysis of phosphatidylcholine. The enzyme has been identified as a member of a variety of signal transduction cascades and as a key regulator of numerous intracellular vesicle trafficking processes. A role for PLD in regulating glucose homeostasis is emerging as the enzyme has recently been identified in events regulating exocytosis of insulin from pancreatic beta-cells and also in insulin-stimulated glucose uptake through controlling GLUT4 vesicle exocytosis in muscle and adipose tissue. We present methodologies for assessing cellular PLD activity in secretagogue-stimulated insulin-secreting pancreatic beta-cells and also insulin-stimulated adipocyte and muscle cells, two of the principal insulin-responsive cell types controlling blood glucose levels.
Assuntos
Adipócitos/enzimologia , Células Secretoras de Insulina/enzimologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Insulina/farmacologia , Células Musculares/enzimologia , Fosfolipase D/análise , Fosfolipase D/metabolismo , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Secreção de Insulina , Camundongos , Células Musculares/citologia , Células Musculares/efeitos dos fármacos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Fosfolipídeos/análise , Fosfolipídeos/isolamento & purificação , Fosfolipídeos/metabolismo , Coloração e RotulagemRESUMO
In the mammalian pineal gland, the rhythm in melatonin biosynthesis depends on the norepinephrine (NE)-driven regulation of arylalkylamine N-acetyltransferase (AANAT), the penultimate enzyme of melatonin biosynthesis. A recent study showed that phytocannabinoids like tetrahydrocannabinol reduce AANAT activity and attenuate NE-induced melatonin biosynthesis in rat pineal glands, raising the possibility that an endocannabinoid system is present in the pineal gland. To test this hypothesis, we analyzed cannabinoid (CB) receptors and specific enzymes for endocannabinoid biosynthesis or catabolism in rat pineal glands and cultured pinealocytes. Immunohistochemical and immunoblot analyses revealed the presence of CB1 and CB2 receptor proteins, of N-acyl phosphatidyl ethanolamine hydrolyzing phospholipase D (NAPE-PLD), an enzyme catalyzing endocannabinoid biosynthesis and of fatty acid amide hydrolase (FAAH), an endocannabinoid catabolizing enzyme, in pinealocytes, and in pineal sympathetic nerve fibers identified by double immunofluorescence with an antibody against tyrosine hydroxylase. The immunosignals for the CB2 receptor, NAPE-PLD, and FAAH found in pinealocytes did not vary under a 12 hr light:12 hr dark cycle. The CB1 receptor immunoreaction in pinealocytes was significantly reduced at the end of the light phase [zeitgeber time (ZT) 12]. The immunosignal for NAPE-PLD found in pineal sympathetic nerve fibers was reduced in the middle of the dark phase (ZT 18). Stimulation of cultured pinealocytes with NE affected neither the subcellular distribution nor the intensity of the immunosignals for the investigated CB receptors and enzymes. In summary, the pineal gland comprises indispensable compounds of the endocannabinoid system indicating that endocannabinoids may be involved in the control of pineal physiology.
Assuntos
Fibras Adrenérgicas/química , Moduladores de Receptores de Canabinoides/análise , Endocanabinoides , Glândula Pineal/química , Receptor CB1 de Canabinoide/análise , Receptor CB2 de Canabinoide/análise , Amidoidrolases/análise , Análise de Variância , Animais , Células Cultivadas , Immunoblotting , Imuno-Histoquímica , Luz , Masculino , Microscopia Confocal , Norepinefrina/farmacologia , Fosfolipase D/análise , Fotoperíodo , Ratos , Ratos Wistar , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Recent studies suggested that glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) correlated with tumor malignancy and prognosis of certain tumors. As hematopoietic stem/progenitor cells (HS/PC) homing was similar to tumor invasion and metastasis in some mechanisms, which arose our interests in whether GPI-PLD contribution to the homing of HS/PC. In this study, CD34(+) cells from umbilical cord blood (UCB), mobilized peripheral blood (MPB), and bone marrow (BM) were assayed for their differences in adhesion, migration, respectively. The expression of GPI-anchored proteins (CD48, CD90) on the cells were analyzed by flow cytometry. Semi-quantitive RT-PCR was used to detect GPI-PLD expression in the three different CD34(+) cells. The results showed that GPI-PLD had no effect on the adhesion of CD34(+) cells. While, spontaneous and SDF-1 induced migration of UCB and MPB, but not BM CD34(+) cells were decreased after 1,10-phenanthroline (an inhibitor of GPI-PLD) pretreatment. Furthermore, we found little difference in GPI-anchored adhesion molecules (CD48, CD90) expression between untreated and pretreated CD34(+) cells. GPI-PLD mRNA was low expressed in MPB and undetected in UCB and BM CD34(+) cells. Our results suggested that GPI-PLD probably had no contribution to HS/PC homing, which may due to its low or no expression in UCB, BM and MPB CD34(+) cells.
Assuntos
Antígenos CD34 , Movimento Celular , Células-Tronco Hematopoéticas/fisiologia , Fosfolipase D/fisiologia , Antígenos CD/análise , Células da Medula Óssea , Antígeno CD48 , Sangue Fetal , Humanos , Fosfolipase D/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Antígenos Thy-1/análiseRESUMO
Phospholipid metabolizing enzymes are important participants in neutrophil signal transduction pathways. The methods discussed herein describe assays for assessing the activities of phospholipase (PL)A2, PLC, PLD, and phosphoinositide 3-OH-kinase (PI3-K) in intact neutrophils. PLA2 activity is measured as the release of radiolabed arachidonic acid. PLC activity is measured as the accumulation of inositol 1,4,5-trisphosphate (IP3), a water-soluble product, using a commercially available radioreceptor assay kit. PLD activity is measured as the appearance of its radiolabeled products, phosphatidic acid and phosphatidylethanol. PI3-K activity is measured as the appearance of its radiolabeled product, phosphatidylinositol-3,4,5-trisphosphate.
Assuntos
Técnicas de Laboratório Clínico , Neutrófilos/metabolismo , Fosfolipídeos/análise , Fosfolipídeos/metabolismo , Animais , Cromatografia em Camada Fina/métodos , Humanos , Modelos Biológicos , Neutrófilos/enzimologia , Fosfatidilinositol 3-Quinases/análise , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipase D/análise , Fosfolipase D/metabolismo , Fosfolipases A2/análise , Fosfolipases A2/metabolismo , Fosfolipases Tipo C/análise , Fosfolipases Tipo C/metabolismoRESUMO
Tumor necrosis factor alpha (TNFalpha), a pleiotropic cytokine, activates both apoptotic and pro-survival signals depending on the cell model. Using ECV304 cells, which can be made TNFalpha-sensitive by cycloheximide (CHX) co-treatment, we evaluated the potential roles of ceramide and phospholipase D (PLD) in TNFalpha-induced apoptosis. TNFalpha/CHX induced a robust increase in ceramide levels after 16 h of treatment when cell death was maximal. PLD activity was increased at early time point (1h) whereas both PLD activity and PLD1 protein were strongly decreased after 24h. TNFalpha/CHX-induced cell death was significantly lowered by exogenous bacterial PLD and phoshatidic acid, and in cells overexpressing PLD1. Conversely, cells depleted in PLD proteins by small interference RNA (siRNA) treatment exhibited higher susceptibility to apoptosis. These results show that PLD exerts a protective role against TNFalpha-induced cell death.
Assuntos
Apoptose/efeitos dos fármacos , Endotélio Vascular/citologia , Fosfolipase D/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Linhagem Celular , Ceramidas/genética , Cicloeximida/farmacologia , Humanos , Fosfolipase D/análise , Fosfolipase D/genética , Substâncias Protetoras , RNA Interferente Pequeno/farmacologia , TransfecçãoRESUMO
Signaling by the mammalian target of rapamycin (mTOR) has been reported to be necessary for mechanical load-induced growth of skeletal muscle. The mechanisms involved in the mechanical activation of mTOR signaling are not known, but several studies indicate that a unique [phosphotidylinositol-3-kinase (PI3K)- and nutrient-independent] mechanism is involved. In this study, we have demonstrated that a regulatory pathway for mTOR signaling that involves phospholipase D (PLD) and the lipid second messenger phosphatidic acid (PA) plays a critical role in the mechanical activation of mTOR signaling. First, an elevation in PA concentration was sufficient for the activation of mTOR signaling. Second, the isozymes of PLD (PLD1 and PLD2) are localized to the z-band in skeletal muscle (a critical site of mechanical force transmission). Third, mechanical stimulation of skeletal muscle with intermittent passive stretch ex vivo induced PLD activation, PA accumulation, and mTOR signaling. Finally, pharmacological inhibition of PLD blocked the mechanically induced increase in PA and the activation of mTOR signaling. Combined, these results indicate that mechanical stimuli activate mTOR signaling through a PLD-dependent increase in PA. Furthermore, we showed that mTOR signaling was partially resistant to rapamycin in muscles subjected to mechanical stimulation. Because rapamycin and PA compete for binding to the FRB domain on mTOR, these results suggest that mechanical stimuli activate mTOR signaling through an enhanced binding of PA to the FRB domain on mTOR.