Resistance to Gemcitabine in Pancreatic Cancer Is Connected to Methylglyoxal Stress and Heat Shock Response.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with poor prognosis. Gemcitabine is the first-line therapy for PDAC, but gemcitabine resistance is a major impediment to achieving satisfactory clinical outcomes. This study investigated whether methylglyoxal (MG), an oncometabolite spontaneously formed as a by-product of glycolysis, notably favors PDAC resistance to gemcitabine.

Biochemical analysis of the NAD+-dependent malate dehydrogenase, a substrate of several serine/threonine protein kinases of Mycobacterium tuberculosis.

PknD is one of the eleven eukaryotic-like serine/threonine protein kinases (STPKs) of Mycobacterium tuberculosis (Mtb). In vitro phosphorylation assays with the active recombinant PknD showed that the intracellular protein NAD+-dependent malate dehydrogenase (MDH) is a substrate of this kinase. MDH, an energy-supplying enzyme, catalyzes the interconversion of malate and oxaloacetate and plays crucial roles in several metabolic pathways including the citric acid cycle.

ATP antagonizes thrombin-induced signal transduction through 12(S)-HETE and cAMP.

In this study we have investigated the role of extracellular ATP on thrombin induced-platelet aggregation (TIPA) in washed human platelets. ATP inhibited TIPA in a dose-dependent manner and this inhibition was abolished by apyrase but not by adenosine deaminase (ADA) and it was reversed by extracellular magnesium. Antagonists of P2Y1 and P2Y12 receptors had no effect on this inhibition suggesting that a P2X receptor controlled ATP-mediated TIPA inhibition.

[P2X4 or P2X7: which of these two receptors is the best target to promote salivation?].

P2X purinergic receptors are receptors which, after ATP binding, form a channel permeant to monovalent and divalent cations. Acinar and ductal cells from salivary glands express P2X4 and P2X7 receptors. The P2X4 receptor has a high affinity for ATP, rapidly desensitizes and is mostly located on the basal membrane of acinar cells.

Inhibition by EGTA of the formation of a biofilm by clinical strains of Staphylococcus aureus.

The effect of EGTA on the adhesion and on the formation of a biofilm by two reference and eight clinical strains of Staphylococcus aureus was studied. All the clinical strains were isolated from patients from Kinshasa. Spa typing confirmed that these clinical strains were distinct.

Contribution of the production of quormones to some phenotypic characteristics of Pseudomonas aeruginosa clinical strains.

The contribution of quorum sensing in some phenotypic and pathogenic characteristics of Pseudomonas aeruginosa was studied. The production of acylhomoserine lactones (AHL) by planktonic cultures of eight clinical and reference strains of P. aeruginosa was evaluated using two biosensors.

Study of the effect of antimicrobial peptide mimic, CSA-13, on an established biofilm formed by Pseudomonas aeruginosa.

The formation of a Pseudomonas aeruginosa biofilm, a complex structure enclosing bacterial cells in an extracellular polymeric matrix, is responsible for persistent infections in cystic fibrosis patients leading to a high rate of morbidity and mortality. The protective environment created by the tridimensional structure reduces the susceptibility of the bacteria to conventional antibiotherapy. Cationic steroid antibiotics (CSA)-13, a nonpeptide mimic of antimicrobial peptides with antibacterial activity on planktonic cultures, was evaluated for its ability to interact with sessile cells.

Activation of calcium-insensitive phospholipase A(2) (iPLA(2)) by P2X(7) receptors in murine peritoneal macrophages.

Free fatty acid releases are triggered by PLA2 activation and are substrates for many enzymes such as cyclooxygenases. These reactions are responsible for the production of many prostaglandins implicated in the inflammation yet many purinergic receptors have been implicated in diseases characterised by chronic inflammation. The role of P2X receptors was evaluated in LPS-primed murine peritoneal macrophages which were labelled with either [(3)H]-oleic acid or [(3)H]-arachidonic acid.

Identification of peptides derived from the human antimicrobial peptide LL-37 active against biofilms formed by Pseudomonas aeruginosa using a library of truncated fragments.

Persistent Pseudomonas aeruginosa infections are a major cause of morbidity and mortality in cystic fibrosis (CF) patients and are linked to the formation of a biofilm. The development of new biofilm inhibition strategies is thus a major challenge. LL-37 is the only human antimicrobial peptide derived from cathelicidin.

Effect of pluronic acid F-127 on the toxicity towards eukaryotic cells of CSA-13, a cationic steroid analogue of antimicrobial peptides.

Aims: CSA-13 is an antimicrobial cationic steroid with some toxicity against eukaryotic cells. The purpose of this work was to test whether pluronic acid F-127 could interfere with the toxicity of CSA-13 on human umbilical vein endothelial (HUVEC) without modifying its bactericidal activity against Pseudomonas aeruginosa.

Methods And Results: The addition of pluronic acid F-127 slightly decreased the number of dead cells after exposure to CSA-13.

Study of the formation of a biofilm by clinical strains of Staphylococcus aureus.

A study on biofilm formation was carried out using five methicillin-sensitive [MSSA] and five methicillin-resistant [MRSA] strains of S. aureus. In each group, there were four strains isolated from patients from Kinshasa (Democratic Republic of Congo, DRC) and one reference strain.

Secretion of IL-1β triggered by dynasore in murine peritoneal macrophages.

The interaction of lipopolysaccharide-primed murine peritoneal macrophages with ivermectin, an antiparasite drug which potentiates P2X(4) receptors and dynasore which inhibits the GTPase activity of dynamin, a protein contributing to the internalization of plasma membrane proteins, was tested. Murine peritoneal macrophages express P2X(4) receptors which are mostly intracellular. In cells from P2X(7)-knockout mice (KO mice), 10 µm adenosine triphosphate (ATP) provoked a transient increase of the intracellular concentration of calcium.

Effect of a low concentration of a cationic steroid antibiotic (CSA-13) on the formation of a biofilm by Pseudomonas aeruginosa.

Aims: Cationic steroids like CSA-13 have been designed by analogy with antimicrobial cationic peptides and have bactericidal properties. The purpose of this work was to evaluate the effect of a low concentration (1 mg l(-1)) of CSA-13 on the formation of a biofilm by eight strains of Pseudomonas aeruginosa (four mucoid and four nonmucoid strains) on an inert surface.

Method And Results: The biofilm formation was measured with the Crystal Violet method.

Ivermectin-dependent release of IL-1beta in response to ATP by peritoneal macrophages from P2X(7)-KO mice.

The response to ATP of peritoneal macrophages from wild-type (WT) and P2X(7)-invalidated (KO) mice was tested. Low concentrations (1-100 μM) of ATP transiently increased the intracellular concentration of calcium ([Ca(2+)](i)) in cells from both mice. The inhibition of the polyphosphoinositide-specific phospholipase C with U73122 inhibited this response especially in WT mice suggesting that the responses coupled to P2Y receptors were potentiated by the expression of P2X(7) receptors.

Efficacy of the combination of tobramycin and a macrolide in an in vitro Pseudomonas aeruginosa mature biofilm model.

Respiratory disease is the main cause of morbidity and mortality in patients with cystic fibrosis (CF). In particular, patients suffer from chronic infection due to biofilm formation by opportunistic Pseudomonas aeruginosa (32). Therefore, there is an urgent need to develop alternative ways to treat biofilm-associated clinical infections.

Distinct regulation by lipopolysaccharides of the expression of interleukin-1β by murine macrophages and salivary glands.

The regulation of interleukin (IL)-1 expression and secretion by salivary glands and macrophages in response to lipopolysaccharides (LPS) was compared. In wild-type mice, injection of LPS significantly decreased the volume of saliva stimulated by pilocarpine and increased its protein and amylase concentration. It did not modify the salivary concentration of IL-1β.

Stimulation by P2X₇ receptors of calcium-dependent production of reactive oxygen species (ROS) in rat submandibular glands.

Background: Agonists of P2X₇ receptors increase the production of reactive oxygen species (ROS) in immunocytes. In this work we tested this response and its effect on mitochondrial inner membrane potential (Deltapsi(m)) in exocrine glands.

Methods: The production of ROS by rat submandibular glands was investigated by measuring the oxidation of dichlorodihydrofluorescein (DCFH), a fluorescent probe.

Interaction between tobramycin and CSA-13 on clinical isolates of Pseudomonas aeruginosa in a model of young and mature biofilms.

The bactericidal activity of a cholic acid antimicrobial derivative, CSA-13, was tested against eight strains of Pseudomonas aeruginosa (both reference and clinical strains) and compared with the response to tobramycin. In planktonic cultures, the minimal inhibitory and minimal bactericidal concentrations of CSA-13 and tobramycin were in the 1-25 mg/L range except for one mucoid clinical strain which was much less sensitive to tobramycin (minimal bactericidal concentration, 65-125 mg/L). In young (24 h) biofilms, the sensitivity to CSA-13 was reduced (half-maximal concentration CSA-13 averaged 88 mg/L) and varied among the eight strains.

Study of the initial phase of biofilm formation using a biofomic approach.

Aims: The purpose of this work was to study the initial steps of formation of a biofilm using the BioFilm Ring Test and the Crystal violet staining technique.

Methods And Results: Eight strains of Pseudomonas aeruginosa were studied. The two methods revealed that four strains formed a rapid biofilm.

Regulation by CRAMP of the responses of murine peritoneal macrophages to extracellular ATP.

Peritoneal macrophages were isolated from wild type (WT) mice and from mice invalidated for the P2X(7) receptor (KO) which had been pretreated with thioglycolate. In cells from WT mice, 1 mM ATP increased the intracellular concentration of calcium ([Ca(2+)](i)), the uptake of ethidium bromide, the production of reactive oxygen species (ROS), the secretion of IL-1beta, the release of oleic acid and of lactate dehydrogenase; it decreased the intracellular concentration of potassium ([K(+)](i)). In KO mice, ATP transiently increased the [Ca(2+)](i) confirming that the P2X(7) receptor is a major receptor of peritoneal macrophages.

Membrane compartments and purinergic signalling: the role of plasma membrane microdomains in the modulation of P2XR-mediated signalling.

Purinergic signalling is implicated in virtually any cellular and physiological function. These functions are mediated through the activation of different receptor subfamilies, among which P2X receptors (P2XRs) are ligand-gated ion channels that respond mostly to ATP. In addition to forming a nonselective cation channel, these receptors engage with a complex network of signalling pathways, including protein kinase cascades, lipid signal mediators and proteases.

Pharmacological evidence for the stimulation of NADPH oxidase by P2X(7) receptors in mouse submandibular glands.

ATP in the 100 muM-1 mM concentration range provoked a calcium-independent increase of the oxidation of dichlorodihydrofluorescein (DCFH) to dichlorofluorescein (DCF) by mouse submandibular cells. 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (BzATP), a P2X(7) agonist, but not a muscarinic or an adrenergic agonist, reproduced the effect of ATP. The inhibition of phospholipase C by U73122 or the potentiation of P2X(4) receptor activation with ivermectin did not modify the response to ATP.

Contribution of two ionotropic purinergic receptors to ATP responses in submandibular gland ductal cells.

The effect of extracellular ATP on salivary gland function was compared in wild-type (WT) and P2X(7) knockout (KO) mice. The increase in the intracellular concentration of calcium ([Ca(2+)](i)) in response to carbachol was similar in submandibular ductal cells of WT and KO mice. ATP and its analog, benzoyl-ATP, induced a sustained increase in the [Ca(2+)](i) in WT animals.

Characterization and comparison of raft-like membranes isolated by two different methods from rat submandibular gland cells.

Lipid rafts are defined as cholesterol and sphingolipid enriched domains in biological membranes. Their role in signalling and other cellular processes is widely accepted but the methodology used for their biochemical isolation and characterization remains controversial. Raft-like membranes from rat submandibular glands were isolated by two different protocols commonly described in the literature; one protocol was based on selective solubilization by Triton X-100 at low temperature and the other protocol consisted in extensive sonication.

P2X7 and phospholipid signalling: the search of the "missing link" in epithelial cells.

The purinergic receptor P2X(7) is widely expressed in epithelial cells. This receptor shares in common with the other P2X receptors the ability to form a non-selective cation channel. On the other hand, the COOH terminus of P2X(7) seems to allow this receptor to couple to a spectrum of downstream effectors responsible for the regulation of cell death and pore formation among other functions.