Phosphatidylinositol-specific Phospholipase Research Articles

Membrane-associated carbonic anhydrase in the respiratory system of the Pacific hagfish (Eptatretus stouti)

Like other agnathans, the Pacific hagfish (Eptatretus stouti) lacks red blood cell (RBC) Cl(-)/HCO(3)(-) exchange. Despite this absence of anion exchange, the majority (86.7+/-1.4%) of the total CO(2) carried in the blood is found within the plasma as HCO(3)(-), and thus presumably is inaccessible to RBC carbonic anhydrase (CA). As such, a branchial plasma-accessible CA isozyme in hagfish would be beneficial for mobilizing the considerable plasma HCO(3)(-) stores for CO(2) excretion and blood acid-base balance. The current study used a combination of molecular and biochemical methods to identify two membrane-associated CA isozymes in the respiratory system of E. stouti. Using homology cloning methods, CA IV and XV-like isozymes were identified in the gill and RBC, respectively. Real-time PCR analysis of relative mRNA expression revealed that CA IV was specific to the gill, while CA XV was found in several tissues including the RBC, gill, liver, heart and muscle. Isolation of subcellular fractions of gill and RBC verified the presence of membrane-associated CA activity in each tissue that persisted after standard washing protocols. Unlike CA activity associated with the cytoplasmic fractions, the activity in gill membranes was not inhibited by sodium dodecyl sulphate, while RBC membrane activity was inhibited to a lesser degree than the cytoplasmic fraction. Additionally, incubation of gill membrane fractions with phosphatidylinositol-specific phospholipase C released significant CA activity into the supernatant indicating the presence of a glycophosphatidyl inositol-linkage to the membrane, as found with other CA IV and XV isozymes. These results demonstrate that Pacific hagfish possess gill and RBC plasma-accessible membrane-associated CA that may play important roles in respiratory gas exchange and acid-base balance.

RECK Forms Cowbell-shaped Dimers and Inhibits Matrix Metalloproteinase-catalyzed Cleavage of Fibronectin

The membrane-anchored protease regulator RECK plays important roles in mammalian development and tumor suppression. The biochemical bases of these bioactivities, however, remain poorly understood. Here we report on the properties of a recombinant RECK protein expressed in mouse fibroblasts and purified to near homogeneity. Multiple lines of evidence indicate that RECK forms dimers. Single particle reconstruction using transmission electron microscopy revealed a unique cowbell-like shaped RECK dimer. RECK is cleaved by MMP-2 and MMP-7 and competitively inhibits MMP-7-catalyzed cleavage of fibronectin. Forced RECK expression in HT1080 cells, whose endogenous RECK expression is minimal, leads to an increase in the amount of fibronectin associated with the cell. Our data demonstrate the ability of RECK to protect fibronectin from MMP-mediated degradation.

FCS of Mutated Phosphatidylinositol-specific Phospholipase C Enzymes Monitors the Interplay of Substrate and Activator Lipid Binding

Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes are activated on the surface of vesicles by nonsubstrate phospholipids with the extent of activation tuned by the lipid composition. Bacillus thuringiensis PI-PLC can be activated by a small amount of phosphatidylcholine (PC) towards its substrate PI. Fluorescence correlation spectroscopy (FCS) has been used to study fluorescently labeled PI-PLC and a series of mutants (P42G, K44A, K44E, Y88A, and Y246/247/248S) binding to small vesicles of a substrate analogue (phosphatidylglycerol) and PC as a function of the vesicle composition. For PI-PLC and most of the mutant proteins there is a synergistic effect of the two types of phospholipids in anchoring the enzyme to a vesicle. If a mutation alters the affinity of substrate at the active site, binding should vary with XPC and be enhanced in PC-rich region; on the other hand, if a mutation alters the affinity of activator, Kd will increase with increased XPC. In this way, careful determination of the apparent Kd helps to sort out effects of enzyme mutations on activator and substrate sites. FCS is particularly useful in exploring the contribution of Kd to PI cleavage by mutant PI-PLC enzymes. For a fixed XPC, if the bulk concentration of vesicles is above Kd, enzymatic activities should be similar, as long as vesicle binding is the critical step. However, it should drop dramatically when the bulk concentration is below Kd measured by FCS (a long as the substrate analogue is a good mimic for substrate). The results provide a direct analysis of vesicle binding and catalytic activity and shed light into how occupation of the activator site enhances activity. Currently, we are extending the analysis of this system with tethered small unilamellar vesicles and single-molecule methods.

Role of Rim Tyr/Trp Residues in Interfacial Activation of Phospholipase C Enzymes

The Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (btPI-PLC) as a model system and mammalian PI-PLCδ1 without an intact PH domain have been examined for the contribution of rim aromatic groups to protein binding to vesicles and the correlation of this with catalytic activity. In the bacterial enzyme, two tryptophan residues (Trp47 in the two-turn helix B and Trp242 in a disordered loop) are critical for binding to interfaces; of the many several Tyr residues mutated, replacement with alanine at several sites (close to helix B as well as the active site) weakens membrane binding. For many of these residues the loss in binding affinity approximates what is predicted for an Ala for Trp (or Tyr) substitution. If a single Tyr is modified there is little effect on the catalytic activity measured with mM substrate (although for Y88A an increase in specific activity is seen). In the mammalian enzyme, one Trp is in the hydrophobic rim ridge and could be analogous to Trp47 in bacterial PI-PLC. Replacement of the rim Trp has little effect on binding of the protein to non-substrate containing vesicles (measured by RET of the protein to labeled PE incorporated into the vesicles). However, the activity is significantly reduced. These results (and analyses of other surface variants) are discussed in terms of the multi-domain structure of the mammalian PLC contributing to binding but with X-Y domain exhibiting similar conformational changes to the bacterial enzyme.

Role of Ca2+ mobilization and Ca2+ sensitization in 8‐iso‐PGF2α‐induced contraction in airway smooth muscle

Isoprostanes are prostaglandin (PG)-like compounds synthesized by oxidative stress, not by cyclooxygenase, and increase in bronchoalveolar lavage fluid of patients with asthma. The airway inflammation implicated in this disease may be amplified by oxidants. Although isoprostanes are useful biomarkers for oxidative stress, the action of these agents on airways has not been fully elucidated. This study was designed to determine the intracellular mechanisms underlying the effects of oxidative stress on airway smooth muscle, focused on Ca(2+) signalling pathways involved in the effect of 8-iso-PGF(2 alpha). Using simultaneous recording of isometric tension and F(340)/F(380) (an indicator of intracellular concentrations of Ca(2+), [Ca(2+)]i, we examined the correlation between tension and [Ca(2+)]i in response to 8-iso-PGF(2 alpha) in the fura-2 loaded tracheal smooth muscle. Augmented tension and F(340)/F(380) by 8-iso-PGF(2 alpha) were attenuated by ICI-192605, an antagonist of thromboxane A(2) receptors (TP receptors). Moreover, D609, an antagonist of phosphatidylcholine-specific phospholipase C, markedly reduced both the tension and F(340)/F(380) induced by 8-iso-PGF(2 alpha), whereas U73122, an antagonist of phosphatidylinositol-specific phospholipase C, modestly inhibited them by 8-iso-PGF(2 alpha). SKF96365, a non-selective antagonist of Ca(2+) channels, markedly reduced both tension and F(340)/F(380) by 8-iso-PGF(2 alpha). However, diltiazem and verapamil, voltage-dependent Ca(2+) channel inhibitors, modestly attenuated tension although their reduction of F(340)/F(380) was not different from that by SKF96365. Y-27632, an inhibitor of Rho-kinase, significantly attenuated contraction induced by 8-iso-PGF(2 alpha) without reducing F(340)/F(380), whereas GF109203X and Go6983, protein kinase C inhibitors, did not markedly antagonize them although reducing F(340)/F(380) with a potency similar to Y-27632. 8-iso-PGF(2 alpha) causes airway smooth muscle contraction via activation of TP receptors. Ca(2+) mobilization by SKF96365- and D609-sensitive Ca(2+) influx and Ca(2+) sensitization by Rho-kinase contribute to the intracellular mechanisms underlying the action of 8-iso-PGF(2 alpha). Rho-kinase may be a therapeutic target for the physiologic abnormalities induced by oxidative stress in airways.

Helicobacter pylori VacA-induced Inhibition of GSK3 through the PI3K/Akt Signaling Pathway

Helicobacter pylori VacA toxin contributes to the pathogenesis and severity of gastric injury. We found that incubation of AZ-521 cells with VacA resulted in phosphorylation of protein kinase B (Akt) and glycogen synthase kinase-3beta (GSK3beta) through a PI3K-dependent pathway. Following phosphorylation and inhibition of GSK3beta,beta-catenin was released from a GSK3beta/beta-catenin complex, with subsequent nuclear translocation. Methyl-beta-cyclodextrin (MCD) and phosphatidylinositol-specific phospholipase C (PI-PLC), but not 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and bafilomycin A1, inhibited VacA-induced phosphorylation of Akt, indicating that it does not require VacA internalization and is independent of vacuolation. VacA treatment of AZ-521 cells transfected with TOPtkLuciferase reporter plasmid or control FOPtkLucifease reporter plasmid resulted in activation of TOPtkLuciferase, but not FOPtkLucifease. In addition, VacA transactivated the beta-catenin-dependent cyclin D1 promoter in a luciferase reporter assay. Infection of AZ-521 cells by a vacA mutant strain of H. pylori failed to induce phosphorylation of Akt and GSK3beta, or release of beta-catenin from a GSK3beta/beta-catenin complex. Taken together, these results support the conclusion that VacA activates the PI3K/Akt signaling pathway, resulting in phosphorylation and inhibition of GSK3beta, and subsequent translocation ofbeta-catenin to the nucleus, consistent with effects of VacA on beta-catenin-regulated transcriptional activity. These data introduce the possibility that Wnt-dependent signaling might play a role in the pathogenesis of H. pylori infection, including the development of gastric cancer.

Prostaglandin E2 Increases the Expression of B-Type Natriuretic Peptide Receptor through EP1 Receptor, Ca2+ Mobilization and Protein Kinase C Signaling Pathway in Rat Calvarial Osteoblasts

The C-type natriuretic peptide stimulates osteoblastic functions through the B-type natriuretic receptor (NPR-B). In this study, we examined the signaling pathway behind the regulation of NPR-B expression through the prostaglandin E2 (PGE2) receptor, EP1 subtype using rat calvarial osteoblasts. A23187 as a Ca2+ ionophore increased NPR-B expression dose-dependently. PGE2 or 17-phenyl-ω-trinor PGE2 (EP1A), an EP1 agonist, increased NPR-B expression, and the potentiating effects were blocked by treating with BAPTA-AM as an intracellular Ca2+ chelator. Activators of protein kinase C (PKC), 1-oleoyl-2-acetyl-sn-glycerol, a membrane-permeable diacylglycerol, and 12-o-tetradecanoyl-phorbol-13-acetate, also increased NPR-B expression, and the potentiating effects were blocked by treating with BAPTA-AM. The treatment of cells with GF109203X, a PKC inhibitor, blocked the PGE2- and EP1A-induced increase in NPR-B expression. From these results, we concluded that EP1-mediated increase in the expression of NPR-B requires not only Ca2+ mobilization but also PKC activation through the activation of phosphatidylinositol-specific phospholipase C.

Identification and Role of the Homodimerization Interface of the Glycosylphosphatidylinositol-anchored Membrane Type 6 Matrix Metalloproteinase (MMP25)

The membrane type (MT) 6 matrix metalloproteinase (MMP) (MMP25) is a glycosylphosphatidylinositol-anchored matrix metalloproteinase (MMP) that is highly expressed in leukocytes and in some cancer tissues. We previously showed that natural MT6-MMP is expressed on the cell surface as a major reduction-sensitive form of M(r) 120, likely representing enzyme homodimers held by disulfide bridges. Among the membrane type-MMPs, the stem region of MT6-MMP contains three cysteine residues at positions 530, 532, and 534 which may contribute to dimerization. A systematic site-directed mutagenesis study of the Cys residues in the stem region shows that Cys(532) is involved in MT6-MMP dimerization by forming an intermolecular disulfide bond. The mutagenesis data also suggest that Cys(530) and Cys(534) form an intramolecular disulfide bond. The experimental observations on cysteines were also investigated by computational studies of the stem peptide, which validate these proposals. Dimerization is not essential for transport of MT6-MMP to the cell surface, partitioning into lipid rafts or cleavage of alpha-1-proteinase inhibitor. However, monomeric forms of MT6-MMP exhibited enhanced autolysis and metalloprotease-dependent degradation. Collectively, these studies establish the stem region of MT6-MMP as the dimerization interface, an event whose outcome imparts protease stability to the protein.

Platelet-Derived Factor Va Expressed on the Surface of the Activated Platelet Is GPI-Anchored

Coagulation factor V exists in two pools in human blood. One is plasma-derived, originating from synthesis in the liver. The other is platelet-derived, originating from endocytosis of the plasma-derived cofactor by megakaryocytes and consisting of both unactivated and activated cofactor. Studies have demonstrated the presence of a non-dissociable, membrane-bound form of platelet-derived factor Va. When washed platelets were activated with thrombin and subjected to additional washing in the presence of EDTA to disrupt the calcium-dependent interaction between the factor Va heavy and light chains, western blotting and flow cytometric analyses revealed that ~35% of the heavy chain could not be removed from the platelet surface. Similarly, in a prothrombin time-based clotting assay, ~25% of the factor Va cofactor activity remained on the activated platelet surface after extensive washing, demonstrating that this platelet-bound cofactor pool functions in Prothrombinase. The mechanism by which this factor Va pool is bound to the membrane was investigated. Sequence analysis of factor Va has revealed a consensus sequence for glycosylphosphatidylinositol (GPI) anchor addition at Ser692 in the C-terminus of the heavy chain. Lipid raft domains in cell membranes are enriched in GPI-anchored proteins and are resistant to solubilization in Triton X-100 at 4°C but are soluble at 37°C. Compared to lysis at 4°C, ~50% more factor Va heavy chain was solubilized when platelets were lysed at 37°C. When cells are solubilized in Triton X-114, membrane-anchored and trans-membrane proteins segregate to the detergent phase, and when activated platelets were subjected to this procedure, a portion of the factor Va heavy chain segregated to the detergent phase. Flow cytometric analyses of activated platelets have demonstrated that phosphatidylinositol-specific phospholipase C (PI-PLC), which can cleave GPI-anchored proteins from cell surfaces, is able to remove ~45% of the non-dissociably bound factor Va heavy chain from the platelet surface in a dose-dependent manner. Subsequent analysis of these samples by Triton X-114 phase separation corroborated these results, indicating that ~50% of the heavy chain was removed from the detergent phase upon treatment with PI-PLC. After cleavage by PI-PLC, GPI-anchored proteins express an epitope known as the cross-reacting determinant (CRD), which encompasses the remainder of the GPI anchor on the modified protein. Western blotting analyses of platelet supernatants after PI-PLC treatment have demonstrated that the platelet-derived factor Va heavy chain contains the CRD epitope. To investigate the origin of the non-dissociably bound pool of factor Va on the platelet surface, platelets were isolated from a factor V-deficient individual with undetectable levels of the cofactor, who receives therapeutic transfusions of fresh frozen plasma. When the individual's platelets were subjected to Triton X-114 phase separation, a population of factor Va heavy chain was detected in the detergent phase. These results are consistent with the non-dissociable portion of the platelet-derived factor Va pool being formed by post-translational modification of factor V from plasma subsequent to its endocytosis by megakaryocytes. The presence of a GPI anchor consensus sequence in the factor Va heavy chain, combined with the washing, solubilization, phase separation, PI-PLC treatment, and anti-CRD immunoblotting data strongly suggest that non-dissociably bound, platelet-derived factor Va is linked to the activated platelet membrane via a GPI anchor on its heavy chain. GPI-anchored, platelet-derived factor Va is functional in Prothrombinase, and, as it is retained on the platelet surface at the site of vascular injury, it is likely a physiologically significant source of cofactor activity.

Detection, Purification and Identification of An Endogenous Inhibitor of l-Dopa Decarboxylase Activity from Human Placenta

An endogenous inhibitor of L-Dopa decarboxylase activity was identified and purified from human placenta. The endogenous inhibitor of L-Dopa decarboxylase (Ddc) was localized in the membrane fraction of placental tissue. Treatment of membranes with phosphatidylinositol-specific phospholipase C or proteinase K did not affect membrane-associated Ddc inhibitory activity, suggesting that a population of the inhibitor is embedded within membranes. Purification was achieved by extraction from a nondenaturing polyacrylamide gel. The purification scheme resulted in the isolation of a single 35 kDa band, bearing L-Dopa decarboxylase inhibitory activity. The purified inhibitor was identified as Annexin V. The elucidation of the biological importance of the presence of an L-Dopa decarboxylase activity inhibitor in normal human tissues could provide us with new information leading to the better understanding of the biological pathways that Ddc is involved in.

The ability of Listeria monocytogenes PI-PLC to facilitate escape from the macrophage phagosome is dependent on host PKCβ

Listeria monocytogenes are facultative intracellular pathogenic bacteria that can infect macrophages as well as non-professional phagocytes. After entry in the host cell, the bacteria escape from the phagosome into the cytoplasm. In murine macrophages and in cell lines derived from these cells, escape of L. monocytogenes from the phagosome is absolutely dependent on listeriolysin O (LLO) and facilitated by a secreted phosphatidylinositol-specific phospholipase C (PI-PLC). Work in this laboratory has previously demonstrated a LLO and PI-PLC-dependent translocation of host PKCβ isoforms. Pharmacological inhibition of PKCβ resulted in a significant reduction in permeabilization of the phagosome, and in the number of bacteria reaching the cytosol. These findings led to the prediction that the bacterial PI-PLC promotes escape through the production of diacylglycerol leading to the activation of host PKCβ. To test this hypothesis, bone marrow-derived macrophages (BMMf) obtained from PKCβ knockout (PKCβKO) or C57Bl/6 mice were infected with L. monocytogenes. We observed that wild-type L. monocytogenes escapes from the phagosome of PKCβKO BMMf as well as from C57Bl/6 BMMf. However, in PKCβKO BMMf, L. monocytogenes uses a PI-PLC-independent, but phosphatidylcholine-preferring PLC (PC-PLC)-dependent pathway to facilitate escape. These findings strongly support the hypothesis that PI-PLC promotes escape through mobilization of host PKCβ.

A 106-kDa Aminopeptidase Is a Putative Receptor for Bacillus thuringiensis Cry11Ba Toxin in the Mosquito Anopheles gambiae

Bacillus thuringiensis (Bt) insecticidal toxins bind to receptors on midgut epithelial cells of susceptible insects, and binding triggers biochemical events that lead to insect mortality. Recently, a 100-kDa aminopeptidase N (APN) was isolated from brush border membrane vesicles (BBMV) of Anopheles quadrimaculatus and shown to bind Cry11Ba toxin with surface plasmon resonance (SPR) detection [Abdullah et al. (2006) BMC Biochem. 7, 16]. In our study, a 106-kDa APN, called AgAPN2, released by phosphatidylinositol-specific phospholipase C (PI-PLC) from Anopheles gambiae BBMV was extracted by Cry11Ba bound to beads. The AgAPN2 cDNA was cloned, and analysis of the predicted AgAPN2 protein revealed a zinc-binding motif (HEIAH), three potential N-glycosylation sites, and a predicted glycosylphosphatidylinositol (GPI) anchor site. Immunohistochemistry localized AgAPN2 to the microvilli of the posterior midgut. A 70-kDa fragment of the 106-kDa APN was expressed in Escherichia coli. When purified, it competitively displaced 125I-Cry11Ba binding to An. gambiae BBMV and bound Cry11Ba on dot blot and microtiter plate binding assays with a calculated K d of 6.4 nM. Notably, this truncated peptide inhibited Cry11Ba toxicity to An. gambiae larvae. These results are evidence that the 106-kDa GPI-anchored APN is a specific binding protein, and a putative midgut receptor, for Bt Cry11Ba toxin.

Placental Folate Transport during Pregnancy

The aim of this study was to elucidate the mechanism of folate transport in the placenta over the course of pregnancy. We found that folate receptor alpha (FRalpha) and reduced folate carrier (RFC) localized on the apical side of human placental villi. Since folate binding to placental brush-border membrane vesicles (BBMVs) was strongly inhibited by phosphatidylinositol-specific phospholipase C (PI-PLC) treatment, it is possible that FRalpha, a glycosyl phosphatidylinositol linked glycoprotein, is a candidate for folate uptake from maternal blood to the placenta. Moreover, additional inhibitory effects of thiamine pyrophosphate (TPP) and hemin on folate uptake after PI-PLC treatment suggested that not only FRalpha but also RFC and heme carrier protein 1 (HCP1) are involved in the folate transport mechanism in the human placenta. It was also found that accumulation of folate after intravenous injection increased with the progress of gestation in the rat placenta and the fetus. Furthermore, increases in the expression levels of mRNA of rFRalpha, rRFC, and rHCP1 in the rat placenta during pregnancy were observed. These findings suggest that FRalpha, RFC, and HCP1 are important carriers of folate in the placenta during pregnancy. The results of this study suggest that increases in the expression levels of FRalpha, RFC, and HCP1 in the placenta play an important role in the response to increased need for folate for the placenta and fetus during development with the progress of gestation.

Involvement of phosphatidylcholine-specific phospholipase C in thromboxane A2-induced activation of mitogen-activated protein kinase in astrocytoma cells.

Thromboxane A2 (TXA2) receptor-mediated signal transduction was investigated in 1321N1 human astrocytoma cells. 9,11-Epithio-11,12-methano-TXA2 (STA2), a TXA2 receptor agonist, induced Ca2+ mobilization and phosphoinositide hydrolysis in a concentration-dependent manner. These responses were inhibited by treatment with U73122, an inhibitor of phosphatidylinositol-specific phospholipase C, or by culturing in 0.5% fetal calf serum containing 0.5 mM dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP) for 2 days. However, the dbcAMP treatment augmented the TXA2 receptor-mediated phosphorylation of mitogen-activated protein kinase (MAPK). These results were confirmed by a functional MAPK assay measuring the incorporation of 32P into the MAPK substrate peptide. The TXA2 receptor-mediated MAPK activation was inhibited by SQ29548, a TXA2 receptor antagonist, and GF109203X, an inhibitor of protein kinase C. Although U73122 did not inhibit or only slightly inhibited the activation of MAPK, D-609, an inhibitor of phosphatidylcholine-specific phospholipase C, potently attenuated the activation in a concentration-dependent manner. Furthermore, STA2 accelerated the release of [3H]choline metabolites from the cells prelabeled with [3H]choline chloride. This release was inhibited by treatment with D-609. These results suggest that phosphatidylcholine-specific phospholipase C and protein kinase C, but not phosphatidylinositol-specific phospholipase C, are involved in TXA2 receptor-mediated MAPK activation in 1321N1 human astrocytoma cells.

The nuclear ceramide/diacylglycerol balance depends on the physiological state of thyroid cells and changes during UV-C radiation-induced apoptosis

Intranuclear lipid metabolism modifications in relation to cell proliferation and/or apoptosis were demonstrated in hepatocytes. The aim of this study was to establish whether nuclear lipid metabolites influence cell function in different experimental models using a rat thyroid cell line (FRTL-5) treated with UV-C radiation. After UV-C irradiation cells proliferate and undergo apoptosis in the presence of thyrotropin, are quiescent and resistant to radiation-induced apoptosis in its absence and finally are proapoptotic for nutrition withdrawal. In nuclei purified from proliferating cells, irradiation stimulates neutral-sphingomyelinase activity and inhibits sphingomyelin-synthase, phosphatidylcholine-specific phospholipase C and phosphatidylinositol-specific phospholipase C activity with a consequent increase in the ceramide/diacylglycerol ratio. This effect is marked in proapoptotic cell nuclei and low in quiescent cell nuclei. In conclusion, UV-C radiation induces apoptosis, modifying nuclear lipid metabolism in relation to the physiological state of cells.

Role and mechanism of phosphatidylinositol‐specific phospholipase C in survival and virulence of Cryptococcus neoformans

Phospholipase B1 (Plb1) is secreted after release from its glycosylphosphatidylinositol anchor and is implicated in initiation and dissemination of infection of the pathogenic fungus, Cryptococcus neoformans. To investigate the role of phosphatidylinositol-specific phospholipase C (PI-PLC) in Plb1 secretion, we identified two putative PI-PLC-encoding genes in C. neoformans var. grubii (PLC1 and PLC2), and created Deltaplc1 and Deltaplc2 deletion mutants. In Deltaplc1, which expressed less PI-PLC activity than wild type (WT), three major cryptococcal virulence traits, Plb1 secretion, melanin production and growth at host temperature (37 degrees C) were abolished and absence of Plb1 secretion coincided with Plb1 accumulation in plasma membranes. In addition, Deltaplc1 cell walls were defective, as indicated by cell clumping and irregular morphology, slower growth and an inability to activate mitogen-activated protein kinase (MAPK) in the presence of cell wall-perturbing agents. In contrast to Deltaplc2, which was as virulent as WT, Deltaplc1 was avirulent in mice and exhibited attenuated killing of Caenorhabditis elegans at 25 degrees C, demonstrating that mechanism(s) independent of the 37 degrees C growth defect contribute to the virulence composite. We conclude that Plc1 is a central regulator of cryptococcal virulence, acting through the protein kinase C/MAPK pathway, that it regulates release of Plb1 from the plasma membrane and is a candidate antifungal drug target.

UVA-induced calcium oscillations in rat mast cells

UVA is a major bio-active component in solar irradiation, and is shown to have immunomodulatory and anti-inflammatory effects. The detailed molecular mechanism of UVA action in regard to calcium signaling in mast cells, however, is not fully understood. In this study, it was found that UVA induced ROS formation and cytosolic calcium oscillations in individual rat mast cells. Exogenously added H2O2 and hypoxanthine/xanthine oxidase (HX/XOD) mimicked UVA effects on cytosolic calcium increases. Regular calcium oscillation induced by UVA irradiation was inhibited completely by the phosphatidylinositol-specific phospholipase C inhibitor U73122, but U73343 was without effect. Tetrandrine, a calcium entry blocker, or calcium-free buffer abolished UVA-induced calcium oscillations. L-type calcium channel blocker nifedipine and stores-operated calcium channel blocker SK&F96365 had no such inhibitory effect. ROS induction by UVA was abolished after pre-incubation with anti-oxidant NAC or with NAD(P)H oxidase inhibitor DPI; such treatment also made UVA-induced calcium oscillation to disappear. UVA irradiation did not increase mast cell diameter, but it made mast cell structure more granular. Spectral confocal imaging revealed that the emission spectrum of the endogenous fluorophore in single mast cell contained a sizable peak which corresponded to that of NAD(P)H. Taken together, these data suggest that UVA in rat mast cells could activate NAD(P)H oxidase, to produce ROS, which in turn activates phospholipase C signaling, to trigger regular cytosolic calcium oscillation.

Distinct Domains of Complexins Bind SNARE Complexes and Clamp Fusion in Vitro

In regulated exocytosis, the core membrane fusion machinery proteins, the SNARE proteins, are assisted by a group of regulatory factors in order to couple membrane fusion to an increase of intracellular calcium ion (Ca(2+)) concentration. Complexin-I and synaptotagmin-I have been shown to be key elements for this tightly regulated process. Many studies suggest that complexin-I can arrest the fusion reaction and that synaptotagmin-I can release the complexin-I blockage in a calcium-dependent manner. Although the actual molecular mechanism by which they exert their function is still unknown, recent in vivo experiments postulate that domains of complexin-I produce different effects on neurotransmitter release. Herein, by using an in vitro flipped SNARE cell fusion assay, we have identified and characterized the minimal functional domains of complexin-I necessary to couple calcium and synaptotagmin-I to membrane fusion. Moreover, we provide evidence that other isoforms of complexin, complexin-II, -III, and -IV, can also be functionally coupled to synaptotagmin-I and calcium. These correspond closely to results from in vivo experiments, providing further validation of the physiological relevance of the flipped SNARE system.

Functional activity of the membrane-associated complement inhibitor CD59 in a pig-to-human in vitro model for hyperacute xenograft rejection.

Hyperacute rejection triggered by activation of the recipient's complement system represents the major barrier to successful xenotransplantation. Transfer of human membrane-associated complement regulators to donor organs has been suggested as one strategy to interfere with complement-mediated hyperacute xenograft rejection. Pigs are discussed as potential organ donors. We therefore investigated a putative protective function of the membrane-bound complement inhibitor CD59 in a pig-to-human in vitro model of hyperacute xenograft rejection. Aortic porcine endothelial cells were transfected with human CD59 cDNA. Expression of human CD59 was demonstrated by cytofluorimetric and RNA analysis. Removal of CD59 from the cell surface by phosphatidylinositol-specific phospholipase C (PI-PLC) demonstrated its production as a glycosyl phosphatidylinositol (GPI)-anchored protein. Functional activity of the transfected CD59 was tested by a lactate dehydrogenase (LDH) release assay for complement-mediated lysis. Porcine endothelial cells expressing human CD59 were significantly protected from lysis by human serum complement compared with CD59- cells. The protective effect was abolished by preincubating the cells with anti-CD59 antibodies or PI-PLC. We calculated by Scatchard analysis that the established CD59+ cell line expressed a CD59 level comparable to that of human endothelial cells. Our results recommend the production of pigs transgenic for CD59.

Characterization of homologous restriction factor (HRF20) in human skin and leucocytes

Homologous restriction factor with a molecular weight of 20 kD (HRF20) is a membrane protein that inhibits assembly of the membrane attack complex of homologous complement. Distribution of HRF20 in normal human skin was studied. The plasma membrane of keratinocytes was stained, and the intensity of the staining pattern was higher in the basal cell layer than in the granular layer. Endothelial cells of blood vessels in the dermis were also stained. The molecular weight of HRF20 on erythrocytes and epidermis is 16 kD, determined by Western blot analysis. Those of polymorphonuclear cells and lymphocytes appeared as two bands, a major band of 20 kD and a minor band of 16 kD. Susceptibility of HRF20 to phosphatidylinositol-specific phospholipase C (PIPLC) was examined. After PIPLC treatment of the sections, HRF20 was not detected on the epidermis and was very slightly expressed on the blood vessels. These results indicate that HRF20 attaches to keratinocytes and blood vessels via phosphatidylinositol, regulating the formation of membrane attack complexes of homologous complement on the cell membrane.