Phospholipase D2:A Pivotal Player Modulating RBL-2H3 Mast Cell Structure

The current study examined the role of PLD2 in the maintenance of mast cell structure. Phospholipase D (PLD) catalyzes hydrolysis of phosphatidylcholine to produce choline and phosphatidic acid (PA). PLD has two isoforms, PLD1 and PLD2, which vary in expression and localization depending on the cell type. The mast cell line RBL-2H3 was transfected to overexpress catalytically active (PLD2CA) and inactive (PLD2CI) forms of PLD2. The results of this study show that PLD2CI cells have a distinct star-shaped morphology, whereas PLD2CA and RBL-2H3 cells are spindle shaped. In PLD2CI cells, the Golgi complex was also disorganized with dilated cisternae, and more Golgi-associated vesicles were present as compared with the PLD2CA and RBL-2H3 cells. Treatment with exogenous PA led to the restoration of the wild-type Golgi complex phenotype in PLD2CI cells. Conversely, treatment of RBL-2H3 and PLD2CA cells with 1% 1-Butanol led to a disruption of the Golgi complex. The distribution of acidic compartments, including secretory granules and lysosomes, was also modified in PLD2CI cells, where they concentrated in the perinuclear region. These results suggest that the PA produced by PLD2 plays an important role in regulating cell morphology in mast cells. (J Histochem Cytochem 60:386-396, 2012)

Characterization of suramin binding sites on the human group IIA secreted phospholipase A(2) by site-directed mutagenesis and molecular dynamics simulation

Suramin is a polysulphonated naphthylurea with inhibitory activity against the human secreted group IIA phospholipase A(2) (hsPLA2GIIA), and we have investigated suramin binding to recombinant hsPLA2GIIA using site-directed mutagenesis and molecular dynamics (MD) simulations. The changes in suramin binding affinity of 13 cationic residue mutants of the hsPLA2GIIA was strongly correlated with alterations in the inhibition of membrane damaging activity of the protein. Suramin binding to hsPLA2GIIA was also studied by MD simulations, which demonstrated that altered intermolecular potential energy of the suramin/mutant complexes was a reliable indicator of affinity change. Although residues in the C-terminal region play a major role in the stabilization of the hsPLA2GIIA/suramin complex, attractive and repulsive hydrophobic and electrostatic interactions with residues throughout the protein together with the adoption of a bent suramin conformation, all contribute to the stability of the complex. Analysis of the h5PLA2GIIA/suramin interactions allows the prediction of the properties of suramin analogues with improved binding and higher affinities which may be candidates for novel phospholipase A(2) inhibitors. (C) 2012 Elsevier Inc. All rights reserved.

The bactericidal effect of human secreted group IID phospholipase A(2) results from both hydrolytic and non-hydrolytic activities

The Human Secreted Group IID Phospholipase A(2) (hsPLA2GIID) may be involved in the human acute immune response. Here we have demonstrated that the hsPLA2GIID presents bactericidal and Ca2+-independent liposome membrane-damaging activities and we have compared these effects with the catalytic activity of active-site mutants of the protein. All mutants showed reduced hydrolytic activity against DOPC:DOPG liposome membranes, however bactericidal effects against Escherichia coli and Micrococcus luteus were less affected, with the D49K mutant retaining 30% killing of the Gram-negative bacteria at a concentration of 10 mu g/mL despite the absence of catalytic activity. The H48Q mutant maintained Ca2+-independent membrane-damaging activity whereas the G30S and D49K mutants were approximately 50% of the wild-type protein, demonstrating that phospholipid bilayer permeabilization by the hsPLA2GIID is independent of catalytic activity. We suggest that this Ca2+-independent damaging activity may play a role in the bactericidal function of the protein. (C) 2012 Elsevier Masson SAS. All rights reserved.

Hepatocyte nuclear factors 1α/4α and forkhead box A2 regulate the solute carrier 2A2 (Slc2a2) gene expression in the liver and kidney of diabetic rats

AIMS: Solute carrier 2a2 (Slc2a2) gene codifies the glucose transporter GLUT2, a key protein for glucose flux in hepatocytes and renal epithelial cells of proximal tubule. In diabetes mellitus, hepatic and tubular glucose output has been related to Slc2a2/GLUT2 overexpression; and controlling the expression of this gene may be an important adjuvant way to improve glycemic homeostasis. Thus, the present study investigated transcriptional mechanisms involved in the diabetes-induced overexpression of the Slc2a2 gene. MAIN METHODS: Hepatocyte nuclear factors 1α and 4α (HNF-1α and HNF-4α), forkhead box A2 (FOXA2), sterol regulatory element binding protein-1c (SREBP-1c) and the CCAAT-enhancer-binding protein (C/EBPβ) mRNA expression (RT-PCR) and binding activity into the Slc2a2 promoter (electrophoretic mobility assay) were analyzed in the liver and kidney of diabetic and 6-day insulin-treated diabetic rats. KEY FINDINGS: Slc2a2/GLUT2 expression increased by more than 50% (P<0.001) in the liver and kidney of diabetic rats, and 6-day insulin treatment restores these values to those observed in non-diabetic animals. Similarly, the mRNA expression and the binding activity of HNF-1α, HNF-4α and FOXA2 increased by 50 to 100% (P<0.05 to P<0.001), also returning to values of non-diabetic rats after insulin treatment. Neither the Srebf1 and Cebpb mRNA expression, nor the SREBP-1c and C/EBP-β binding activity was altered in diabetic rats. SIGNIFICANCE: HNF-1α, HNF-4α and FOXA2 transcriptional factors are involved in diabetes-induced overexpression of Slc2a2 gene in the liver and kidney. These data point out that these transcriptional factors are important targets to control GLUT2 expression in these tissues, which can contribute to glycemic homeostasis in diabetes.

The regulation of NHE₁ and NHE₃ activity by angiotensin II is mediated by the activation of the angiotensin II type I receptor/phospholipase C/calcium/calmodulin pathway in distal nephron cells

Angiotensin II (Ang II), acting via the AT1 receptor, induces an increase in intracellular calcium [Ca(2+)]i that then interacts with calmodulin (CaM). The Ca(2+)/CaM complex directly or indirectly activates sodium hydrogen exchanger 1 (NHE1) and phosphorylates calmodulin kinase II (CaMKII), which then regulates sodium hydrogen exchanger 3 (NHE3) activity. In this study, we investigated the cellular signaling pathways responsible for Ang II-mediated regulation of NHE1 and NHE3 in Madin-Darby canine kidney (MDCK) cells. The NHE1- and NHE3-dependent pHi recovery rates were evaluated by fluorescence microscopy using the fluorescent probe BCECF/AM, messenger RNA was evaluated with the reverse transcription polymerase chain reaction (RT-PCR), and protein expression was evaluated by immunoblot. We demonstrated that treatment with Ang II (1pM or 1 nM) for 30 min induced, via the AT1 but not the AT2 receptor, an equal increase in NHE1 and NHE3 activity that was reduced by the specific inhibitors HOE 694 and S3226, respectively. Ang II (1 nM) did not change the total expression of NHE1, NHE3 or calmodulin, but it induced CaMKII, cRaf-1, Erk1/2 and p90(RSK) phosphorylation. The stimulatory effects of Ang II (1 nM) on NHE1 or NHE3 activity or protein abundance was reduced by ophiobolin-A (CaM inhibitor), KN93 (CaMKII inhibitor) or PD98059 (Mek inhibitor). These results indicate that after 30 min, Ang II treatment may activate G protein-dependent pathways, including the AT1/PLC/Ca(2+)/CaM pathway, which induces CaMKII phosphorylation to stimulate NHE3 and induces cRaf-1/Mek/Erk1/2/p90(RSK) activity to stimulate NHE1

Evaluation of pyroptosis in macrophages using cytosolic delivery of purified flagellin

Pyroptosis is a molecularly controlled form of cell death that exhibits some features of apoptosis as well of necrosis. Pyroptosis is induced by inflammasome-activated caspase-1 or caspase-11 (caspase-4 in humans), as a result of distinct pathogenic or damage stimuli. Although pyroptosis displays some morphological and biochemical features of apoptosis, it has an inflammatory outcome due to the loss of plasma membrane integrity and the consequent release of intracellular contents, reminiscent to necrosis. Here, we use cytosolic delivery of purified flagellin as an experimental tool to trigger pyroptosis and describe potential methods to study this form of cell death. Finally, we discuss the advantages and limitations of these methods

Cytosolic flagellin-induced lysosomal pathway regulates inflammasome-dependent and -independent macrophage responses

NAIP5/NLRC4 (neuronal apoptosis inhibitory protein 5/nucleotide oligomerization domain-like receptor family, caspase activation recruitment domain domain-containing 4) inflammasome activation by cytosolic flagellin results in caspase-1-mediated processing and secretion of IL-1β/IL-18 and pyroptosis, an inflammatory cell death pathway. Here, we found that although NLRC4, ASC, and caspase-1 are required for IL-1β secretion in response to cytosolic flagellin, cell death, nevertheless, occurs in the absence of these molecules. Cytosolic flagellin-induced inflammasome-independent cell death is accompanied by IL-1α secretion and is temporally correlated with the restriction of Salmonella Typhimurium infection. Despite displaying some apoptotic features, this peculiar form of cell death do not require caspase activation but is regulated by a lysosomal pathway, in which cathepsin B and cathepsin D play redundant roles. Moreover, cathepsin B contributes to NAIP5/NLRC4 inflammasome-induced pyroptosis and IL-1α and IL-1β production in response to cytosolic flagellin. Together, our data describe a pathway induced by cytosolic flagellin that induces a peculiar form of cell death and regulates inflammasome-mediated effector mechanisms of macrophages

I valori di H1-A2 e H1-A3 come correlati della intensità "rivisitata": aspetti e problemi

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Genome sequence of Desulfovibrio sp. A2, a highly copper resistant, sulfate-reducing bacterium isolated from effluents of a zinc smelter at the Urals

Desulfovibrio sp. A2 is an anaerobic gram-negative sulfate-reducing bacterium with remarkable tolerance to copper. It was isolated from wastewater effluents of a zinc smelter at the Urals. Here, we report the 4.2-Mb draft genome sequence of Desulfovibrio sp. A2 and identify potential copper resistance mechanisms.

Aggretin, a heterodimeric C-type lectin from Calloselasma rhodostoma (malayan pit viper), stimulates platelets by binding to alpha 2beta 1 integrin and glycoprotein Ib, activating Syk and phospholipase Cgamma 2, but does not involve the glycoprotein VI/Fc receptor gamma chain collagen receptor

Aggretin, a potent platelet activator, was isolated from Calloselasma rhodostoma venom, and 30-amino acid N-terminal sequences of both subunits were determined. Aggretin belongs to the heterodimeric snake C-type lectin family and is thought to activate platelets by binding to platelet glycoprotein alpha(2)beta(1). We now show that binding to glycoprotein (GP) Ib is also required. Aggretin-induced platelet activation was inhibited by a monoclonal antibody to GPIb as well as by antibodies to alpha(2)beta(1). Binding of both of these platelet receptors to aggretin was confirmed by affinity chromatography. No binding of other major platelet membrane glycoproteins, in particular GPVI, to aggretin was detected. Aggretin also activates platelets from Fc receptor gamma chain (Fcgamma)-deficient mice to a greater extent than those from normal control mice, showing that it does not use the GPVI/Fcgamma pathway. Platelets from Fcgamma-deficient mice expressed fibrinogen receptors normally in response to collagen, although they did not aggregate, indicating that these platelets may partly compensate via other receptors including alpha(2)beta(1) or GPIb for the lack of the Fcgamma pathway. Signaling by aggretin involves a dose-dependent lag phase followed by rapid tyrosine phosphorylation of a number of proteins. Among these are p72(SYK), p125(FAK), and PLCgamma2, whereas, in comparison with collagen and convulxin, the Fcgamma subunit neither is phosphorylated nor coprecipitates with p72(SYK). This supports an independent, GPIb- and integrin-based pathway for activation of p72(SYK) not involving the Fcgamma receptor.

GPI-specific phospholipase D (GPI-PLD) is expressed during mouse development and is localized to the extracellular matrix of the developing mouse skeleton

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abundant in serum and has a well-characterized biochemistry; however, its physiological role is completely unknown. Previous investigations into GPI-PLD have focused on the adult animal or on in vitro systems and a putative role in development has been neither proposed nor investigated. We describe the first evidence of GPI-PLD expression during mouse embryonic ossification. GPI-PLD expression was detected predominantly at sites of skeletal development, increasing during the course of gestation. GPI-PLD was observed during both intramembraneous and endochondral ossification and localized predominantly to the extracellular matrix of chondrocytes and to primary trabeculae of the skeleton. In addition, the mouse chondrocyte cell line ATDC5 expressed GPI-PLD after experimental induction of differentiation. These results implicate GPI-PLD in the process of bone formation during mouse embryogenesis.