Phosphatidylcholine-specific Phospholipase C Research Articles

Exploring the Genomic Landscape of Bacillus paranthracis PUMB_17 as a Proficient Phosphatidylcholine-Specific Phospholipase C Producer.

Phospholipases find versatile applications across industries, including detergent production, food modification, pharmaceuticals (especially in drug delivery systems), and cell signaling research. In this study, we present a strain of Bacillus paranthracis for the first time, demonstrating significant potential in the production of phosphatidylcholine-specific phospholipase C (PC-PLC). The investigation thoroughly examines the B. paranthracis PUMB_17 strain, focusing on the activity of PC-PLC and its purification process. Notably, the PUMB_17 strain displays extracellular PC-PLC production with high specific activity during the late exponential growth phase. To unravel the genetic makeup of PUMB_17, we employed nanopore-based whole-genome sequencing and subsequently conducted a detailed genome annotation. The genome comprises a solitary circular chromosome spanning 5,250,970 bp, featuring a guanine-cytosine ratio of 35.49. Additionally, two plasmids of sizes 64,250 bp and 5845 bp were identified. The annotation analysis reveals the presence of 5328 genes, encompassing 5186 protein-coding sequences, and 142 RNA genes, including 39 rRNAs, 103 tRNAs, and 5 ncRNAs. The aim of this study was to make a comprehensive genomic exploration that promises to enhance our understanding of the previously understudied and recently documented capabilities of Bacillus paranthracis and to shed light on a potential use of the strain in the industrial production of PC-PLC.

Unravelling the signaling pathway downstream of the neurokinin receptor in atrium: discovering novel anti-arrhythmic targets for treatment of atrial fibrillation

Abstract Background Stimulation of neurokinin receptor (NKR) 3 is anti-fibrillatory by prolonging the action potential duration (APD) of atrial cardiomyocytes via inhibition of a background potassium current (1). The NKR is a Gq/11 protein-coupled receptor, but the downstream intracellular pathway and the end-effector potassium (K+)-channel in atrium are unknown. We aimed at unravelling the components of this pathway in atrial cardiomyocytes. Methods Rabbit (New Zealand White) atrial cardiomyocytes were isolated from Langendorff-perfused hearts by enzymatic dissociation. Human immortalized atrial myocytes (HiAMs) were developed, and kindly provided by our collaborators (2). Adult human atrial cardiomyocytes were obtained by enzymatic dissociation from left atrial appendages (LAA) routinely removed from atrial fibrillation (AF) patients undergoing minimally invasive surgical pulmonary vein isolation in the clinics. Action potentials (APs) and K+ currents were recorded at 36.5°C with the amphotericin-B-perforated patch clamp technique. RNA was isolated from rabbit atrial tissue and sequenced on an Illumina HiSeq4000 platform. Results In rabbit atrial cardiomyocytes, stimulation of NKR3 prolonged APD by ∼50% and inhibited an background outward K+-current by ∼45%. Inhibition of phosphokinase C (PKC) did not prevent APD prolongation by NKR3 stimulation, but application of a diacylglycerol (DAG) analog inhibited an outward K+-current by ∼40% indicating that DAG, but not PKC, is downstream of NKR3. Inhibition of phosphatidylcholine-specific phospholipase C (PC-PLC), but not of phosphoinositide-specific PLC (PI-PLC), reduced APD increase by NKR3 stimulation by five-fold, indicating PC-PLC as the intracellular signaling molecule. In HiAMs, direct PLC activation prolonged APD by 34%. RNA sequencing analysis revealed potential end-targets of the NKR3 pathway in rabbit. Inhibition of small conductance potassium (KCNN2/KCNN3) or TASK-1 (KCNK3) channels (prolonging APD by ∼15%) did not abolish the APD prolongation effect by subsequent NKR3 stimulation (∼50%), indicating that these channels are not the end-effector K+-channels inhibited by NKR3 stimulation. Inhibition of constitutive KACh channels had little effect on APD at baseline. Nevertheless, APD shortening by agonist-induced KACh channel activity could be completely reversed by NKR3 stimulation, indicating that increased outward KACh current can be fully counterbalanced by the reduction in K+ background current. In human atrial cardiomyocytes from AF patients, stimulation of NKR3 prolonged APD by 25%, Conclusions PC-PLC and DAG, but not PI-PLC and PKC, are intracellularly downstream of NKR3 in atrial rabbit cardiomyocytes. Stimulation of the NKR pathway may have an anti-arrhythmic potential in patients with AF.

Phosphatidylcholine-Specific Phospholipase C as a Promising Drug Target.

Phosphatidylcholine-specific phospholipase C (PC-PLC) is an enzyme that catalyzes the formation of the important secondary messengers phosphocholine and diacylglycerol (DAG) from phosphatidylcholine. Although PC-PLC has been linked to the progression of many pathological conditions, including cancer, atherosclerosis, inflammation and neuronal cell death, studies of PC-PLC on the protein level have been somewhat neglected with relatively scarce data. To date, the human gene expressing PC-PLC has not yet been found, and the only protein structure of PC-PLC that has been solved was from Bacillus cereus (PC-PLCBc). Nonetheless, there is evidence for PC-PLC activity as a human functional equivalent of its prokaryotic counterpart. Additionally, inhibitors of PC-PLCBc have been developed as potential therapeutic agents. The most notable classes include 2-aminohydroxamic acids, xanthates, N,N'-hydroxyureas, phospholipid analogues, 1,4-oxazepines, pyrido[3,4-b]indoles, morpholinobenzoic acids and univalent ions. However, many medicinal chemistry studies lack evidence for their cellular and in vivo effects, which hampers the progression of the inhibitors towards the clinic. This review outlines the pathological implications of PC-PLC and highlights current progress and future challenges in the development of PC-PLC inhibitors from the literature.

QPCR detection of viable Bacillus cereus group cells in cosmetic products

Reference methods for microbiological safety assessments of cosmetics rely on culture methods that reveal colonies of live microorganisms on growth media. Rapid molecular technologies, such as qPCR, detects the presence of target DNA in samples from dead and viable cells. DNA intercalating dyes, such as propidium monoazide (PMAxx), are capable of restricting PCR amplification to viable microbial cells. Here we developed singleplex and multiplex real time (qPCR) assays for the detection of Bacillus cereus (B. cereus) using 16S rRNA and phosphatidylcholine-specific phospholipase C (PLC) gene specific sequences coupled with PMAxx. The limit of detection was determined to be ~ 1 log CFU/ml for 16S rRNA and 3 log CFU/ml for PLC detection in pure culture using an eye shadow isolate, B. cereus 3A. We assessed the inclusivity and exclusivity of our qPCR assays using 212 strains, including 143 members of B. cereus, 38 non- B. cereus. and 31 non-Bacillus species; inclusivity was 100% for the 16S rRNA and 97.9% for the PLC targets; the exclusivity was 100% for 16S rRNA and 98.6% for PLC targets. These qPCR assays were then used to assess samples of commercial cosmetics: one set of liquid face toners (N = 3), artificially contaminated with B. cereus 3A, and one set of powdered cosmetics (N = 8), previously determined to be contaminated with B. cereus. For some samples, test portions were analyzed by qPCR in parallel, with and without PMAxx treatment. All test portions were simultaneously streaked on BACARA plates to confirm viable cells of B. cereus, according to the culture method. We found no difference in sensitivity between the singleplex and the multiplex qPCR assays (P > 0.05). Inoculated samples that did not recover B. cereus on plates still showed amplification of the DNA targets. However, that amplification was significantly delayed in PMAxx –treated samples (P < 0.0001) with CT value differences of 7.82 for 16S rRNA and 7.22 for PLC. Likewise, amplification delay was significant (P < 0.0001) with inoculated samples that recovered B. cereus on plates with CT value differences of 2.96 and 2.36 for 16S rRNA and PLC, respectively, demonstrating the presence of dead cells in the samples. All our qPCR results correlated with detection on BACARA plates (kappa, k = 0.99), independently of the presence of PMAxx in the PCR assays. Nevertheless, the amplification threshold with PMAxx dyes was significantly higher than the non-PMAxx dyes. Our findings confirm qPCR can be used for more rapid detection of microorganisms in cosmetics, including B. cereus, and selective detection of viable cells can be improved using PMAxx dyes.

Tricyclodecan-9-yl-Xanthogenate (D609): Mechanism of Action and Pharmacological Applications

Tricyclodecan-9-yl xanthogenate (D609) is a synthetic tricyclic compound possessing a xanthate group. This xanthogenate compound is known for its diverse pharmacological properties. Over the last three decades, many studies have reported the biological activities of D609, including antioxidant, antiapoptotic, anticholinergic, anti-tumor, anti-inflammatory, anti-viral, anti-proliferative, and neuroprotective activities. Its mechanism of action is extensively attributed to its ability to cause the competitive inhibition of phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) and sphingomyelin synthase (SMS). The inhibition of PCPLC or SMS affects secondary messengers with a lipidic nature, i.e., 1,2-diacylglycerol (DAG) and ceramide. Various in vitro/in vivo studies suggest that PCPLC and SMS inhibition regulate the cell cycle, block cellular proliferation, and induce differentiation. D609 acts as a pro-inflammatory cytokine antagonist and diminishes Aβ-stimulated toxicity. PCPLC enzymatic activity essentially requires Zn2+, and D609 might act as a potential chelator of Zn2+, thereby blocking PCPLC enzymatic activity. D609 also demonstrates promising results in reducing atherosclerotic plaque formation, post-stroke cerebral infarction, and cancer progression. The present compilation provides a comprehensive mechanistic insight into D609, including its chemistry, mechanism of action, and regulation of various pharmacological activities.

D609 inhibition of phosphatidylcholine-specific phospholipase C attenuates prolonged insulin stimulation-mediated GLUT4 downregulation in 3T3-L1 adipocytes

Glucose uptake is stimulated by insulin via stimulation of glucose transporter 4 (GLUT4) translocation to the plasma membrane from intracellular compartments in adipose tissue and muscles. Insulin stimulation for prolonged periods depletes GLUT4 protein, particularly in highly insulin-responsive GLUT4 storage vesicles. This depletion mainly occurs via H2O2-mediated retromer inhibition. However, the post-receptor mechanism of insulin activation of oxidative stress remains unknown. Here, we show that phosphatidylcholine-specific phospholipase C (PC-PLC) plays an important role in insulin-mediated downregulation of GLUT4. In the study, 3T3-L1 adipocytes were exposed to a PC-PLC inhibitor, tricyclodecan-9-yl-xanthogenate (D609), for 30 min prior to the stimulation with 500 nM insulin for 4 h, weakening the depletion of GLUT4. D609 also prevents insulin-driven H2O2 generation in 3T3-L1 adipocytes. Exogenous PC-PLC and its product, phosphocholine (PCho), also caused GLUT4 depletion and promoted H2O2 generation in 3T3-L1 adipocytes. Furthermore, insulin-mediated the increase in the cellular membrane PC-PLC activity was observed in Amplex Red assays. These results suggested that PC-PLC plays an important role in insulin-mediated downregulation of GLUT4 and that PCho may serve as a signaling molecule.

Incorporation of a Nitric Oxide Donating Motif into Novel PC-PLC Inhibitors Provides Enhanced Anti-Proliferative Activity.

Inhibition of phosphatidylcholine-specific phospholipase C (PC-PLC) has previously been shown to be a potential target for novel cancer therapeutics. One downstream consequence of PC-PLC activity is the activation of NF-κB, a nuclear transcription factor responsible for transcribing genes related to oncogenic traits, such as proliferation, angiogenesis, metastasis, and cancer cell survival. Another biological pathway linked to NF-κB is the exogenous delivery of nitric oxide (NO), which decreases NF-κB activity through an apparent negative-feedback loop. In this study, we designed and synthesised 13 novel NO-releasing derivatives of our previously reported class of PC-PLC inhibitors, 2-morpholinobenzoic acids. These molecules contained a secondary benzylamine group, which was readily nitrosylated and subsequently confirmed to release NO in vitro using a DAF-FM fluorescence-based assay. It was then discovered that these NO-releasing derivatives possessed significantly improved anti-proliferative activity in both MDA-MB-231 and HCT116 cancer cell lines compared to their non-nitrosylated parent compounds. These results confirmed that the inclusion of an exogenous NO-releasing functional group onto a known PC-PLC inhibitor enhances anti-proliferative activity and that this relationship can be exploited in order to further improve the anti-proliferative activity of current/future PC-PLC inhibitors.

Development of 2-Morpholino-N-hydroxybenzamides as anti-proliferative PC-PLC inhibitors

Phosphatidylcholine-specific phospholipase C (PC-PLC) is a key enzyme involved in the metabolism of the mammalian phospholipid phosphatidylcholine into secondary messengers diacylglycerol (DAG) and phosphocholine. DAG and phosphocholine have been identified to amplify various cellular processes involved in oncogenesis such as proliferation, cell-cycle activation, differentiation and motility, therefore making PC-PLC a potential target for novel anti-cancer treatments. The current literature standard for PC-PLC inhibition, tricyclodecan-9-yl-potassium xanthate (D609), has been shown to arrest proliferation in multiple cancer cell lines, however, it is not drug-like resulting in low aqueous stability, making it a poor drug candidate. 2-Morpholinobenzoic acids have been shown to have improved PC-PLC inhibitory activity compared to D609, with molecular modelling identifying chelation of the carboxylic acid to catalytic Zn2+ ions in the PC-PLC active site being a key interaction. In this study, the carboxylic acid motif was replaced with a hydroxamic acid to strengthen the Zn2+ interaction. It was found that the hydroxamic acid derivatives displayed PC-PLC inhibitory activity similar, or better, than D609. Furthermore, these novel inhibitors had potent anti-proliferative activity in MDA-MB-231 and HCT-116 cancer cell lines, far greater than D609 and previous 2-morpholinobenzoic acids.

NIR Fluorescent Imaging and Photodynamic Therapy with a Novel Theranostic Phospholipid Probe for Triple-Negative Breast Cancer Cells.

New exogenous probes are needed for both imaging diagnostics and therapeutics. Here, we introduce a novel nanocomposite near-infrared (NIR) fluorescent imaging probe and test its potency as a photosensitizing agent for photodynamic therapy (PDT) against triple-negative breast cancer cells. The active component in the nanocomposite is a small molecule, pyropheophorbide a-phosphatidylethanolamine-QSY21 (Pyro-PtdEtn-QSY), which is imbedded into lipid nanoparticles for transport in the body. The probe targets abnormal choline metabolism in cancer cells; specifically, the overexpression of phosphatidylcholine-specific phospholipase C (PC-PLC) in breast, prostate, and ovarian cancers. Pyro-PtdEtn-QSY consists of a NIR fluorophore and a quencher, attached to a PtdEtn moiety. It is selectively activated by PC-PLC resulting in enhanced fluorescence in cancer cells compared to normal cells. In our in vitro investigation, four breast cancer cell lines showed higher probe activation levels than noncancerous control cells, immortalized human mammary gland cells, and normal human T cells. Moreover, the ability of this nanocomposite to function as a sensitizer in PDT experiments on MDA-MB-231 cells suggests that the probe is promising as a theranostic agent.

Functional role of phosphatidylcholine-specific phospholipase C in regulating leukotriene synthesis and degranulation in human eosinophils

Phosphatidylinositol-specific phospholipase C (PI-PLC) and cytosolic phospholipase A2 (cPLA2) regulate both eosinophil degranulation and leukotriene (LT) synthesis via PI-PLC-mediated calcium influx and cPLA2 activation. Phosphatidylcholine-specific phospholipase C (PC-PLC) likely plays a key role in cellular signaling, including the eosinophilic allergic inflammatory response. This study examined the role of PC-PLC in eosinophil LT synthesis and degranulation using tricyclodecan-9-yl-xanthogenate (D609), a PC-specific PLC inhibitor. D609 inhibited N-formyl-met-leu-phe + cytochalasin B (fMLP/B)-induced arachidonic acid (AA) release and leukotriene C4 (LTC4) secretion. However, at concentrations that blocked both AA release and LTC4 secretion, D609 had no significant inhibitory effect on stimulated cPLA2 activity. D609 also partially blocked fMLP/B-induced calcium influx, indicating that inhibition of AA release and LTC4 secretion by D609 is due to inhibition of calcium-mediated cPLA2 translocation to intracellular membranes, not inhibition of cPLA2 activity. In addition, D609 inhibited fMLP/B-stimulated eosinophil peroxidase release, indicating that PC-PLC regulates fMLP/B-induced eosinophil degranulation by increasing the intracellular calcium concentration ([Ca2+]i). Overall, our results showed that PC-PLC is critical for fMLP/B-stimulated eosinophil LT synthesis and degranulation. In addition, degranulation requires calcium influx, while PC-PLC regulates LTC4 synthesis through calcium-mediated cPLA2 activation.

Identification and Pathogenic Potential of Bacillus cereus Strains Isolated from a Dairy Processing Plant Producing PDO Taleggio Cheese.

Low levels of contamination by Bacillus cereus at the cheese farm is essential for reducing any opportunity for growth prior consumption. In this study, B. cereus distribution in a plant producing Protected Designation of Origin Taleggio cheese was investigated and the virulence potential of the isolates was evaluated. Seventy-four samples were collected from Food and Non Food Contact Surfaces (FCS, NFCS), saline curd, and Taleggio. The eleven isolates were identified, typified, and clustered. Strains were tested for the production of hemolysins, hemolysin BL (HBL), phosphatidylcholine-specific phospholipase C (PC-PLC), proteases, and biofilm, and for the presence of chromosomal toxin-encoding genes (sph, plcA, cytK, entFM, bcet, entS, nheA, nheB, nheC). B. cereus was detected on NFCS, FCS, and curd, but not in Taleggio. The isolates were grouped into six clusters, and all produced PC-PLC, hemolysins, and proteases, and most of them HBL (66.7%). All the clusters harbored the nheA, sph, plcA, entFM, and cytK genes, and some also nheB (83.3%), nheC (66.7%), bcet (50.0%), and entS (66.7%). All strains showed biofilm-forming ability. Our data reveal possible contamination of production plants and cheese curd by potentially virulent B. cereus, but bacterial absence in Taleggio highlights the efficacy of a proper management of the production phases in assuring consumer’s protection.

Development, synthesis and biological investigation of a novel class of potent PC-PLC inhibitors

Phospholipases are enzymes that are involved in the hydrolysis of acyl and phosphate esters of phospholipids, generating secondary messengers that have implications in various cellular processes including proliferation, differentiation and motility. As such inhibitors of phospholipases have been widely studied for their use as anti-cancer therapeutics. Phosphatidylcholine-specific phospholipase C (PC-PLC) is implicated in the progression of a number of cancer cell lines including aggressing triple-negative breast cancers. Most current studies on PC-PLC have utilised D609 as the standard inhibitor however it is known to have multiple failings, including poor stability in aqueous media. 2-Morpholinobenzoic acids were recently identified using vHTS as a potential class of lead compounds, with improvements over D609. In this work 129 analogues in this class were prepared and their PC-PLC inhibitory activity was assessed. It was found that the majority of these novel compounds had improved activity when compared to D609 with the most potent inhibitors completely inhibiting enzyme activity. It was determined that the best compound/s contained a morpholino and 2-substituted N-benzyl moieties with these findings explained using molecular modelling. The compounds reported here will allow for improved study of PC-PLC activity.

Phosphocholine Antagonizes Listeriolysin O-Induced Host Cell Responses of Listeria monocytogenes.

Bacterial toxins disrupt plasma membrane integrity with multitudinous effects on host cells. The secreted pore-forming toxin listeriolysin O (LLO) of the intracellular pathogen Listeria monocytogenes promotes egress of the bacteria from vacuolar compartments into the host cytosol often without overt destruction of the infected cell. Intracellular LLO activity is tightly controlled by host factors including compartmental pH, redox, proteolytic, and proteostatic factors, and inhibited by cholesterol. Combining infection studies of L. monocytogenes wild type and isogenic mutants together with biochemical studies with purified phospholipases, we investigate the effect of their enzymatic activities on LLO. Here, we show that phosphocholine (ChoP), a reaction product of the phosphatidylcholine-specific phospholipase C (PC-PLC) of L. monocytogenes, is a potent inhibitor of intra- and extracellular LLO activities. Binding of ChoP to LLO is redox-independent and leads to the inhibition of LLO-dependent induction of calcium flux, mitochondrial damage, and apoptosis. ChoP also inhibits the hemolytic activities of the related cholesterol-dependent cytolysins (CDC), pneumolysin and streptolysin. Our study uncovers a strategy used by L. monocytogenes to modulate cytotoxic LLO activity through the enzymatic activity of its PC-PLC. This mechanism appears to be widespread and also used by other CDC pore-forming toxin-producing bacteria.

Effect of imidazoline-1 receptor agonists on renal dysfunction in rats associated with chronic, sequential fructose and ethanol administration.

Insulin resistance and chronic alcoholism are risk factors for renal dysfunction. This study investigated the therapeutic effects of two imidazoline-1 receptor (I1R) agonists on renal dysfunction in rats after chronic, sequential fructose and ethanol administration. Daily drinking water was supplemented with fructose (10%, w/v) for 12weeks and then with ethanol (20%, v/v) for another 8weeks. Rats were treated with rilmenidine and clonidine in the last two weeks of the study. Blood glucose and serum insulin (sIns) levels, lipid profiles, kidney function and renal histopathology were evaluated at the end of the experiment. Additionally, renal gene expression of nischarin, phosphatidylcholine-specific phospholipase C (PC-PLC) and prostaglandin E2 (PGE2) were measured. Renal levels of superoxide dismutase (SOD), malondialdehyde (MDA), myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS) and total NO (tNO) were detected, and we determined the relative renal gene expression levels of alpha smooth muscle actin (α-SMA), hydroxyproline, interleukin 10 (IL-10), tumour necrosis factor alpha (TNF-α) and caspase-3. The results showed significant deterioration of blood glucose, sIns, lipid profiles, kidney function and renal histopathology in fructose/ethanol-fed rats. Additionally, markers of inflammation, fibrosis, apoptosis and oxidative stress were upregulated. The administration of rilmenidine or clonidine significantly improved blood glucose and sIns levels and reduced renal dysfunction. Our work showed that chronic, sequential fructose and ethanol administration induced fasting hyperglycaemia and renal impairment, and these effects were ameliorated by I1R agonists.

Discovery of novel PC-PLC activity inhibitors.

Phosphatidylcholine-specific phospholipase C (PC-PLC) is one of the important members of phospholipase family which is capable of specifically hydrolyzing the third phosphate linker of glycerophospholipid molecules, releasing phosphocholine and diacylglycerols (DAG). It is a crucial virulence factor of bacteria contributed to cell-to-cell spread and leading multiple diseases in mammals. Moreover, PC-PLC has a wide range of biological functions and involves in various cell signaling pathway, including apoptosis, proliferation, differentiation, and metastasis. In this study, we have synthesized 2 chiral compounds ((R)-7-amino-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-ol, called R-7ABO, and (S)-7-amino-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-ol, called S-7ABO) and discovered their inhibitory effect on PC-PLC activity which derived from Bacillus cereus (B.cereus) and human umbilical vein endothelial cells (HUVEC). Therefore, as two novel efficient PC-PLC inhibitors, R-7ABO and S-7ABO might become favorable tools of antibacterial therapy in B.cereus infection diseases and researching the function of PC-PLC in HUVECs.

Discovery of novel phosphatidylcholine-specific phospholipase C drug-like inhibitors as potential anticancer agents

Phosphatidylcholine–specific phospholipase C (PC-PLC) is a promising target for new anticancer treatment. Herein, we report our work in the discovery of novel drug-like PC-PLC inhibitors. Virtual screening led to the identification of promising hits from four different structural series that contain the molecular scaffold of benzenesulphonamides (10), pyrido[3,4-b]indoles (22), morpholinobenzoic acid (84) and benzamidobenzoic acid (80). 164 structural analogues were tested to investigate the chemical space around the hit series and to generate preliminary structurally activity relationships (SAR). Two of the pyrido[3,4-b]indoles (22_10 and 22_15) had comparable or better potency as D609, an established but non-drug-like PC-PLC inhibitor. Furthermore, three morpholinobenzoic acids (84, 84_4 and 84_5) had superior potency than D609. Therefore, this study paves the way towards the development of drug-like PL-PLC inhibitors as potential anticancer agents.

Mechanisms of U46619-induced contraction in mouse intrarenal artery.

Thromboxane A2 (TXA2 ) has been implicated in the pathogenesis of vascular complications, but the underlying mechanism remains unclear. The contraction of renal arterial rings in mice was measured by a Multi Myograph System. The intracellular calcium concentration ([Ca2+ ]i ) in vascular smooth muscle cells (VSMCs) was obtained by using a fluo-4/AM dye and a confocal laser scanning microscopy. The results show that the U46619-induced vasoconstriction of renal artery was completely blocked by a TXA2 receptor antagonist GR32191, significantly inhibited by a selective phospholipase C (PI-PLC) inhibitor U73122 at 10μmol/L and partially inhibited by a Phosphatidylcholine - specific phospholipase C (PC-PLC) inhibitor D609 at 50μmol/L. Moreover, the U46619-induced vasoconstriction was inhibited by a general protein kinase C (PKC) inhibitor chelerythrine at 10μmol/L, and a selective PKCδ inhibitor rottlerin at 10μmol/L. In addition, the PKC-induced vasoconstriction was partially inhibited by a Rho-kinase inhibitor Y-27632 at 10μmol/L and was further completely inhibited together with a putative IP3 receptor antagonist and store-operated Ca2+ (SOC) entry inhibitor 2-APB at 100μmol/L. On the other hand, U46619-induced vasoconstriction was partially inhibited by L-type calcium channel (Cav1.2) inhibitor nifedipine at 1μmol/L and 2-APB at 50 and 100μmol/L. Last, U46619-induced vasoconstriction was partially inhibited by a cell membrane Ca2+ activated C1- channel blocker 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) at 50 and 100μmol/L. Our results suggest that the U46619-induced contraction of mouse intrarenal arteries is mediated by Cav1.2 and SOC channel, through the activation of thromboxane-prostanoid receptors and its downstream signaling pathway.

Doxorubicin-induced elevated oxidative stress and neurochemical alterations in brain and cognitive decline: protection by MESNA and insights into mechanisms of chemotherapy-induced cognitive impairment ("chemobrain").

Chemotherapy-induced cognitive impairment (CICI) is now widely recognized as a real and too common complication of cancer chemotherapy experienced by an ever-growing number of cancer survivors. Previously, we reported that doxorubicin (Dox), a prototypical reactive oxygen species (ROS)-producing anti-cancer drug, results in oxidation of plasma proteins, including apolipoprotein A-I (ApoA-I) leading to tumor necrosis factor-alpha (TNF-α)-mediated oxidative stress in plasma and brain. We also reported that co-administration of the antioxidant drug, 2-mercaptoethane sulfonate sodium (MESNA), prevents Dox-induced protein oxidation and subsequent TNF-α elevation in plasma. In this study, we measured oxidative stress in both brain and plasma of Dox-treated mice both with and without MESNA. MESNA ameliorated Dox-induced oxidative protein damage in plasma, confirming our prior studies, and in a new finding led to decreased oxidative stress in brain. This study also provides further functional and biochemical evidence of the mechanisms of CICI. Using novel object recognition (NOR), we demonstrated the Dox administration resulted in memory deficits, an effect that was rescued by MESNA. Using hydrogen magnetic resonance imaging spectroscopy (H1-MRS) techniques, we demonstrated that Dox administration led to a dramatic decrease in choline-containing compounds assessed by (Cho)/creatine ratios in the hippocampus in mice. To better elucidate a potential mechanism for this MRS observation, we tested the activities of the phospholipase enzymes known to act on phosphatidylcholine (PtdCho), a key component of phospholipid membranes and a source of choline for the neurotransmitter, acetylcholine (ACh). The activities of both phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D were severely diminished following Dox administration. The activity of PC-PLC was preserved when MESNA was co-administered with Dox; however, PLD activity was not protected. This study is the first to demonstrate the protective effects of MESNA on Dox-related protein oxidation, cognitive decline, phosphocholine (PCho) levels, and PC-PLC activity in brain and suggests novel potential therapeutic targets and strategies to mitigate CICI.

Nonprotecting Group Synthesis of a Phospholipase C Activatable Probe with an Azo-Free Quencher.

The near-infrared fluorescent activatable smart probe Pyro-phosphatidylethanolamine (PtdEtn)-QSY was synthesized and observed to selectively fluoresce in the presence of phosphatidylcholine-specific phospholipase C (PC-PLC). PC-PLC is an important biological target as it is known to be upregulated in a variety of cancers, including triple negative breast cancer. Pyro-PtdEtn-QSY features a QSY21 quenching moiety instead of the Black Hole Quencher-3 (BHQ-3) used previously because the latter contains an azo bond, which could lead to biological instability.

Protein corona and phospholipase activity drive selective accumulation of nanomicelles in atherosclerotic plaques

ApoB-100 and Phosphatidylcholine-specific phospholipase C (PC-PLC) are important contributors to atherosclerosis development. ApoB-100 is the main structural protein of LDL, being directly associated with atherosclerosis plaque generation. PC-PLC is highly expressed in atherosclerosis lesions and contributes to their progression. We show how phosphatidylcholine-coated nanomicelles can be used for specific characterisation of atherosclerosis plaque. Results show that ApoB-100 in the protein corona of the nanomicelle targets the particles to atherosclerotic areas in apolipoprotein E−/− mice. Furthermore, PC-PLC selectively removes the polar heads from the phospholipid coating of the nanomicelles leading to their accumulation. To fully characterise the behaviour of the nanomicelles, we developed multimodal probes using a nanoemulsion step. Hybrid imaging revealed plaque accumulation of the nanomicelles and colocalisation with PC-PLC expression and ApoB-100 in the plaque. This study shows how protein corona composition and enzyme-driven nanomaterial accumulation can be used for detection of atherosclerosis.