Proposed three-phenylalanine motif involved in magnetoreception signalling of an Actinopterygii protein expressed in mammalian cells.

Studies at the cellular and molecular level of magnetoreception-sensing and responding to magnetic fields-are a relatively new research area. It appears that different mechanisms of magnetoreception in animals evolved from different origins, and, therefore, many questions about its mechanisms remain left open. Here we present new information regarding the Electromagnetic Perceptive Gene (EPG) from Kryptopterus vitreolus that may serve as part of the foundation to understanding and applying magnetoreception. Using HaloTag coupled with fluorescent ligands and phosphatidylinositol specific phospholipase C we show that EPG is associated with the membrane via glycosylphosphatidylinositol anchor. EPG's function of increasing intracellular calcium was also used to generate an assay using GCaMP6m to observe the function of EPG and to compare its function with that of homologous proteins. It was also revealed that EPG relies on a motif of three phenylalanine residues to function-stably swapping these residues using site directed mutagenesis resulted in a loss of function in EPG. This information not only expands upon our current understanding of magnetoreception but may provide a foundation and template to continue characterizing and discovering more within the emerging field.

Phosphatidylinositol-specific phospholipase C can decrease Müller cell viability and suppress its phagocytic activity by modulating PI3K/AKT signaling pathway.

Bacillus cereus endophthalmitis is a devastating eye infection that causes rapid blindness through the release of extracellular tissue-destructive exotoxins. The phagocytic and antibacterial functions of ocular cells are the keys to limiting ocular bacterial infections. In a previous study, we identified a new virulence gene, plcA-2 (different from the original plcA-1 gene), that was strongly associated with the plcA gene of Listeria monocytogenes. This plcA gene had been confirmed to play an important role in phagocytosis. However, how the Bc-phosphatidylinositol-specific phospholipase C (PI-PLC) proteins encoded by the plcA-1/2 genes affect phagocytes remains unclear in B. cereus endophthalmitis. Here, we found that the enzymatic activity of Bc-PI-PLC-A2 was approximately twofold higher than that of Bc-PI-PLC-A1, and both proteins inhibited the viability of Müller cells. In addition, PI-PLC proteins reduced phagocytosis of Müller cells by decreasing the phosphorylation levels of key proteins in the PI3K/AKT signaling pathway. In conclusion, we showed that PI-PLC proteins contribute to inhibit the viability of and suppress the phagocytosis of Müller cells, providing new insights into the pathogenic mechanism of B. cereus endophthalmitis.

Design of Staphylococcus aureus New Vaccine Candidates with B and T Cell Epitope Mapping, Reverse Vaccinology, and Immunoinformatics.

An effective vaccine against Staphylococcus aureus infection is a major planetary heath priority, particularly with increasing antibiotic resistance worldwide. Previous efforts for a highly effective S. aureus vaccine were largely unsuccessful, in part, because the vaccine designs have tended to target mainly the B cell immunity and development of opsonic antibodies. In contrast, recent observations suggest that cell mediated immunity may be critical for protection against S. aureus. In addition, the S. aureus surface proteins are among the key immunodominant antigens because they are the first molecules to interact with the host organism cells and tissues. We report here an original vaccinomics study in which we used a reverse vaccinology and immunoinformatics in silico strategy integrated with genomics. After analyzing 2767 proteins, we defined 16 proteins of S. aureus as promising subunit vaccine candidates. Phosphatidylinositol phosphodiesterase (Plc) is secreted by extracellular pathogens such as S. aureus. We mapped the B and T cell epitopes for the Plc protein, tested the reactivity of the synthesized epitopes by Western blotting, and verified our findings in a pilot study of 10 patients with S. aureus infection. The peptides were then tested for their protective effect in groups of mice challenged with pathogenic S. aureus strain, which showed high protection level. These findings warrant further translational research for development of novel vaccines against S. aureus infection. Reverse vaccinology is an advanced approach that can be applied to identify new vaccine candidates against a host of microorganisms, including S. aureus.

Search and Subvert: Minimalist Bacterial Phosphatidylinositol-Specific Phospholipase C Enzymes.

Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes from Gram-positive bacteria are secreted virulence factors that aid in downregulating host immunity. These PI-PLCs are minimalist peripheral membrane enzymes with a distorted (ßα)8 TIM barrel fold offering a conserved and stable scaffold for the conserved catalytic amino acids while membrane recognition is achieved mostly through variable loops. Decades of experimental and computational research on these enzymes have revealed the subtle interplay between molecular mechanisms of catalysis and membrane binding, leading to a semiquantitative model for how they find, bind, and cleave their respective substrates on host cell membranes. Variations in sequence and structure of their membrane binding sites may correlate with how enzymes from different Gram-positive bacteria search for their particular targets on the membrane. Detailed molecular characterization of protein-lipid interactions have been aided by cutting-edge methods ranging from 31P field-cycling NMR relaxometry to monitor protein-induced changes in phospholipid dynamics to molecular dynamics simulations to elucidate the roles of electrostatic and cation-π interactions in lipid binding to single molecule fluorescence measurements of dynamic interactions between PI-PLCs and vesicles. This toolkit is readily applicable to other peripheral membrane proteins including orthologues in Gram-negative bacteria and more recently discovered eukaryotic minimalist PI-PLCs.

A function for a specific zinc metalloprotease of African trypanosomes.

The Trypanosoma brucei genome encodes three groups of zinc metalloproteases, each of which contains approximately 30% amino acid identity with the major surface protease (MSP, also called GP63) of Leishmania. One of these proteases, TbMSP-B, is encoded by four nearly identical, tandem genes transcribed in both bloodstream and procyclic trypanosomes. Earlier work showed that RNA interference against TbMSP-B prevents release of a recombinant variant surface glycoprotein (VSG) from procyclic trypanosomes. Here, we used gene deletions to show that TbMSP-B and a phospholipase C (GPI-PLC) act in concert to remove native VSG during differentiation of bloodstream trypanosomes to procyclic form. When the four tandem TbMSP-B genes were deleted from both chromosomal alleles, bloodstream B (-/-) trypanosomes could still differentiate to procyclic form, but VSG was removed more slowly and in a non-truncated form compared to differentiation of wild-type organisms. Similarly, when both alleles of the single-copy GPI-PLC gene were deleted, bloodstream PLC (-/-) cells could still differentiate. However, when all the genes for both TbMSP-B and GPI-PLC were deleted from the diploid genome, the bloodstream B (-/-) PLC (-/-) trypanosomes did not proliferate in the differentiation medium, and 60% of the VSG remained on the cell surface. Inhibitors of cysteine proteases did not affect this result. These findings demonstrate that removal of 60% of the VSG during differentiation from bloodstream to procyclic form is due to the synergistic activities of GPI-PLC and TbMSP-B.

Membrane-associated phosphoinositides-specific phospholipase C forms from Catharanthus roseus transformed roots.

We have previously reported that Catharanthus roseus transformed roots contain at least two phosphatidylinositol 4,5-bisphosphate-phospholipase C (PLC) activities, one soluble and the other membrane associated. Detergent, divalent cations, and neomycin differentially regulate these activities and pure protein is required for a greater understanding of the function and regulation of this enzyme. In this article we report a partia purification of membrane-associated PLC. We found that there are at least two forms of membraneassociated PLC in transformed roots of C. roseus. These forms were separated on the basis of their affinity for heparin. One form shows an affinity for heparin and elutes at approx 600 mM KCl. This form has a molecular mass of 67 kDa by size exclusion chromatography and Western blot analysis, whereas the other form does not bind to heparin and has a molecular mass of 57 kDa. Possible differential regulation of these forms during transformed root growth is discussed.

Roles of brain phosphatidylinositol-specific phospholipase C and diacylglycerol lipase in centrally administered histamine-induced adrenomedullary outflow in rats.

Recently, we reported that intracerebroventricularly (i.c.v.) administered histamine evokes the secretion of noradrenaline and adrenaline from adrenal medulla by brain cyclooxygenase-1- and thromboxane A2-mediated mechanisms in rats. These results suggest the involvement of brain arachidonic acid cascade in the histamine-induced activation of the central adrenomedullary outflow. Arachidonic acid is released mainly by phospholipase A2 (PLA2)-dependent pathway or phospholipase C (PLC)/diacylglycerol lipase-dependent pathway. In the present study, histamine (27 nmol/animal, i.c.v.) -induced elevation of plasma noradrenaline and adrenaline was dose-dependently reduced by U-73122 (PLC inhibitor) (10 and 100 nmol/animal, i.c.v.), ET-18-OCH3 (phosphatidylinositol-specific PLC inhibitor) (10 and 30 nmol/animal, i.c.v.) and RHC-80267 (diacylglycerol lipase inhibitor) (1.3 and 2.6 micromol/animal, i.c.v.). However, mepacrine (PLA2 inhibitor) (1.1 and 2.2 micromol/animal, i.c.v.) and D609 (phosphatidylcholine-specific PLC inhibitor) (30, 100 and 300 nmol/animal, i.c.v.) had no effect. These results suggest the involvement of brain phosphatidylinositol-specific PLC and diacylglycerol lipase in the centrally administered histamine-induced activation of the adrenomedullary outflow in rats.

Switch activation of PI-PLC downstream signals in activated macrophages with wortmannin.

Phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P(2)) has been known to serve as a substrate for phosphatidylinositol 3-kinase (PI(3)K) and phosphoinositide-specific phospholipase C (PI-PLC), which can produce PtdIns(3,4,5)P(3) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) and diacylglycerol (DAG), respectively. In this study, we elucidated the role of PI-PLC during the LPS-activated mouse macrophages RAW264.7 treated with PI(3)K inhibitor wortmannin. First, wortmannin treatment enhanced Ins(1,4,5)P(3) production and iNOS expression in LPS-activated macrophages. Inhibition of PI(3)K by p85 siRNA also showed an enhancement of iNOS expression. On the other hand, overexpression of PI(3)K by ras-p110 expression plasmid significantly decreased iNOS expression in LPS-activated macrophages. In addition, overexpression of wild-type or dominant-negative Akt expression plasmid did not affect the iNOS expression in LPS-activated macrophages. Second, treatment of PI-PLC inhibitor U73122 reversed the enhancement of iNOS expression, the increase of phosphorylation level of ERK, JNK and p38, and the increase of AP-1-dependent gene expression in wortmannin-treated and LPS-activated macrophages. However, NF-kappaB activity determined by EMSA assay and reporter plasmid assay did not change during LPS-activated macrophages with or without wortmannin. We propose that the inhibition of PI(3)K by wortmannin in mouse macrophages enhances the PI-PLC downstream signals, and subsequently increases the LPS induction of iNOS expression independently of Akt pathway.

Two waves of the nuclear phospholipase C activity in serum-stimulated HL-60 cells during G(1) phase of the cell cycle.

Phosphatidylinositol-specific phospholipase C (PI-PLC) is activated in cell nuclei during the cell cycle progression. We have previously demonstrated two peaks of an increase in the nuclear PI-PLC activities in nocodazole-synchronized HL-60 cells. In this study, the activity of nuclear PI-PLC was investigated in serum-stimulated HL-60 cells. In serum-starved HL-60 cells, two peaks of the activity of nuclear PI-PLC were detected at 30 min and 11 h after the re-addition of serum with no parallel increase in PLC activity in cytosol, postnuclear membranes or total cell lysates. An increase in the serine phosphorylation of b splicing variant of PI-PLCbeta(1) was detected with no change in the amount of PI-PLCbeta(1b) in nuclei isolated at 30 min and 11 h after the addition of serum. PI-PLC inhibitor ET-18-OCH(3) and MEK inhibitor PD 98059 completely abolished serum-mediated increase at both time-points. The addition of inhibitors either immediately or 6 h after the addition of serum had inhibitory effects on the number of cells entering S phase. These results demonstrate that two waves of nuclear PI-PLCbeta(1b) activity occur in serum-stimulated cells during G(1) phase of the cell cycle and that the later increase in the PLC activity is equally important for the progression into the S phase.

Dimer structure of an interfacially impaired phosphatidylinositol-specific phospholipase C.

The crystal structure of the W47A/W242A mutant of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis has been solved to 1.8A resolution. The W47A/W242A mutant is an interfacially challenged enzyme, and it has been proposed that one or both tryptophan side chains serve as membrane interfacial anchors (Feng, J., Wehbi, H., and Roberts, M. F. (2002) J. Biol. Chem. 277, 19867-19875). The crystal structure supports this hypothesis. Relative to the crystal structure of the closely related (97% identity) wild-type PI-PLC from Bacillus cereus, significant conformational differences occur at the membrane-binding interfacial region rather than the active site. The Trp --> Ala mutations not only remove the membrane-partitioning aromatic side chains but also perturb the conformations of the so-called helix B and rim loop regions, both of which are implicated in interfacial binding. The crystal structure also reveals a homodimer, the first such observation for a bacterial PI-PLC, with pseudo-2-fold symmetry. The symmetric dimer interface is stabilized by hydrophobic and hydrogen-bonding interactions, contributed primarily by a central swath of aromatic residues arranged in a quasiherringbone pattern. Evidence that interfacially active wild-type PI-PLC enzymes may dimerize in the presence of phosphatidylcholine vesicles is provided by fluorescence quenching of PI-PLC mutants with pyrene-labeled cysteine residues. The combined data suggest that wild-type PI-PLC can form similar homodimers, anchored to the interface by the tryptophan and neighboring membrane-partitioning residues.

Streptococcus agalactiae CAMP factor binds to GPI-anchored proteins.

CAMP factor (protein B) is a pore-forming protein secreted by Streptococcus agalactiae. It causes lysis of sheep red blood cells when these have been sensitized with staphylococcal sphingomyelinase. We here show that CAMP factor binds to GPI-anchored proteins, and that this interaction involves the carbohydrate core of the GPI-anchor. Enzymatic cleavage of GPI-anchors with phosphatidylinositol-specific phospholipase C strongly reduces the sensitivity of erythrocytes to CAMP factor. Incorporation of alkaline phosphatase, a model GPI-anchored protein, into liposome membranes renders the latter susceptible to permeabilization by CAMP factor. GPI-anchored proteins therefore function as cellular receptors for CAMP factor.

Calcium signaling in human airway goblet cells following purinergic activation.

Despite the general importance of Ca(2+) signaling in signal transduction, and of goblet cell mucin hypersecretion in inflammatory pulmonary diseases, measurement of airway goblet cell intracellular Ca(2+) (Ca(i)(2+)) has not been reported. In this article, we describe the results of experiments measuring Ca(i)(2+) in primary cultures of human bronchial goblet cells after stimulation with the purinergic agonist adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) and phorbol 12-myristate 13-acetate (PMA). Ca(2+) signaling in human goblet cells after purinergic stimulation follows the classic paradigm of a Ca(i)(2+) transient from a basal activity of 110 nM to a peak response of 260.1 +/- 41.2 nM within 2 min, followed by a long superbasal plateau (155.3 +/- 0.2 nM) between 10 and 15 min. The rise in Ca(i)(2+) appears to result from a mobilization of intracellular stores, because the transient was nearly abolished by inhibition of PLC with the phosphatidylinositol-specific PLC inhibitor U-73122, and it was not affected significantly by removal of extracellular Ca(2+). Loading goblet cells with BAPTA inhibited the ATPgammaS-induced Ca(2+) transient by 86.0 +/- 13.1%, relative to control. Finally, in contrast to the massive effects of high doses of PMA (300 nM) on mucin secretion from goblet cells, phorbol ester stimulated a small (27.1 +/- 7% of the ATPgammaS control peak), brief rise in Ca(i)(2+). This diminutive signal likely denotes a local Ca(2+) gradient, which may be associated with the mucin granule exocytotic process.

Detection of multiple virulence-associated genes in Listeria monocytogenes isolated from bovine mastitis cases.

Clinical samples (n=725) were collected from bovines (n=243) which were positive for mastitis using the California mastitis test (CMT) and somatic cell count (SCC). The clinical samples comprising blood (n=239), milk (n=243), and faecal swabs (n=243) were examined for the presence of pathogenic Listeria spp. Isolation of the pathogen was done using selective enrichment in University of Vermont Medium and plating onto Dominguez-Rodriguez isolation agar. Confirmation of the isolates was based on biochemical tests and Christie, Atkins, Munch-Petersen (CAMP) test followed by pathogenicity testing. Pathogenicity of the isolates was tested by phosphatidylinositol-specific phospholipase C (PI-PLC) assay as well as in vivo tests namely, chick embryo and mice inoculation tests. The isolates were subjected to PCR assay for five virulence-associated genes, plcA, prfA, hlyA, actA and iap. Listeria spp. were isolated from 12 (1.66%) samples. Of these 4 (0.55%) and 1 (0.14%) were confirmed as Listeria monocytogenes and Listeria ivanovii, respectively. L. monocytogenes and L. ivanovii were recovered from milk samples (2) and faecal (3) of mastitic cattle (3) and buffaloes (2). L. monocytogenes recovered from the milk of mastitic cattle and L. ivanovii from the faecal swab of buffalo turned out to be pathogenic. However, the remaining three hemolytic isolates exhibiting positive CAMP test turned out to be negative in PI-PLC assay, chick embryo and mice inoculation. L. monocytogenes and L. ivanovii isolates characterized as pathogenic by PI-PLC assay and in vivo pathogenicity tests were found to possess all the five virulence-associated genes and three genes, plcA, prfA and actA respectively. The remaining three hemolytic but non-pathogenic L. monocytogenes isolates were negative for plcA by PCR. It seems that the plcA gene and its expression (in the PI-PLC assay) have an important role as virulence determinants in pathogenic Listeria spp. In conclusion, the PI-PLC assay and virulence genes targeted PCR (plcA, prfA and hlyA genes for L. monocytogenes and plcA, prfA and actA genes for L. ivanovii) hold a good promise as rapid and reliable in vitro alternatives to in vivo pathogenicity tests.

Expression of phosphoinositide-specific phospholipase C isoenzymes in cultured astrocytes.

Signal transduction from plasma membrane to cell nucleus is a complex process depending on various components including lipid signaling molecules, in particular phosphoinositides and their related enzymes, which act at cell periphery and/or plasma membrane as well as at nuclear level. As far as the nervous system may concern the inositol lipid cycle has been hypothesized to be involved in numerous neural as well as glial functions. In this context, however, a precise panel of glial PLC isoforms has not been determined yet. In the present experiments we investigated astrocytic PLC isoforms in astrocytes obtained from foetal primary cultures of rat brain and from an established cultured (C6) rat astrocytoma cell line, two well known cell models for experimental studies on glia. Identification of PLC isoforms was achieved by using a combination of RT-PCR and immunocytochemistry experiments. While in both cell models the most represented PI-PLC isoforms were beta4, gamma1, delta4, and epsilon, isoforms PI-PLC beta2 and delta3 were not detected. Moreover, in primary astrocyte cultures PI-PLC delta3 resulted well expressed in C6 cells but was absent in astrocytes. Immunocytochemistry performed with antibodies against specific PLC isoforms substantially confirmed this pattern of expression both in astrocytes and C6 glioma cells. In particular while some isoenzymes (namely isoforms beta3 and beta4) resulted mainly nuclear, others (isoforms delta4 and epsilon) were preferentially localized at cytoplasmic and plasma membrane level.

Apolipoprotein CIII-induced THP-1 cell adhesion to endothelial cells involves pertussis toxin-sensitive G protein- and protein kinase C alpha-mediated nuclear factor-kappaB activation.

OBJECTIVE: Plasma apolipoprotein CIII (apoCIII) independently predicts risk for coronary heart disease (CHD). We recently reported that apoCIII directly enhances adhesion of human monocytes to endothelial cells (ECs), and identified the activation of PKC alpha as a necessary upstream event of enhanced monocyte adhesion. This study tested the hypothesis that apoCIII activates PKC alpha in human monocytic THP-1 cells, leading to NF-kappaB activation. METHODS AND RESULTS: Among inhibitors specific to PKC activators, phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor D609 limited apoCIII-induced PKC alpha activation and THP-1 cell adhesion. ApoCIII increased PC-PLC activity in THP-1 cells, resulting in PKC alpha activation. Pertussis toxin (PTX) inhibited apoCIII-induced PC-PLC activation and subsequent PKC alpha activation, implicating PTX-sensitive G protein pathway. ApoCIII further activated nuclear factor-kappaB (NF-kappaB) through PKC alpha in THP-1 cells and augmented beta1-integrin expression. The NF-kappaB inhibitor peptide SN50 partially inhibited apoCIII-induced beta1-integrin expression and THP-1 cell adhesion. ApoCIII-rich VLDL had similar effects to apoCIII alone. CONCLUSIONS: PTX-sensitive G protein pathway participates critically in PKC alpha stimulation in THP-1 cells exposed to apoCIII, activating NF-kappaB, and increasing beta1-integrin. This action causes monocytic cells to adhere to endothelial cells. Furthermore, because leukocyte NF-kappaB activation contributes to inflammatory aspects of atherogenesis, apoCIII may stimulate diverse inflammatory responses through monocyte activation.

Preparation of polyclonal antibody specific for AtPLC4, an Arabidopsis phosphatidylinositol-specific phospholipase C in rabbits.

Phosphoinositide-specific phospholipase Cs (PI-PLCs) are important enzymes in eukaryotes, which catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate into the two second messengers inositol 1,4,5-trisphosphate and diacylglycerol. The Arabidopsis genome contains nine putative PI-PLC genes. AtPLC4, an abiotic stress induced gene, has been reported to encode an active PI-PLC isoform. However, the exact roles of putative AtPLC4 in plant remain to be elicited. The first 108 amino acid residues of the N-terminal of AtPLC4, referred to as AtPLC4 N, was expressed as a recombinant protein in Escherichia coli and used as antigen in generating antibody. Purified recombinant proteins including AtPLC1 to AtPLC5, AtPLC8, AtPLC9 and AtPLC4 N were transferred onto the same blot to test specificity of the prepared antibody. Western blot result shows that only AtPLC4 and AtPLC4 N can be recognized by the antibody. The antibody recognized a protein of approximately 68kDa in the plasma membrane fraction and cytosolic fractions prepared from Arabidopsis thaliana plants. This corresponds very well with the calculated molecular weight of AtPLC4. The results suggest that AtPLC4 may encode a plasma membrane-associated protein.

Over-expression of the truncated ghrelin receptor polypeptide attenuates the constitutive activation of phosphatidylinositol-specific phospholipase C by ghrelin receptors but has no effect on ghrelin-stimulated extracellular signal-regulated kinase 1/2 activity.

In addition to regulating growth hormone release from the pituitary, ghrelin receptors also influence cell proliferation and apoptosis. By studying mitogen-activated protein kinase activity in human embryonic kidney 293 cells over-expressing ghrelin receptors, we aimed to identify the specific cell signalling pathways used by ghrelin receptors, and to determine if the truncated ghrelin receptor polypeptide had any influence on the functional activity of ghrelin receptors. We found that ghrelin activated extracellular signal-regulated kinases 1/2 with an EC50 value of 10 nM, and that this response was inhibited by the ghrelin receptor antagonists D-Lys3-GHRP-6 and [D-Arg1,D-Phe5,D-Trp(7,9),Leu11]-substance P. Ghrelin had little or no effect on the activity of c-Jun N-terminal kinase, p38 kinase or Akt. Ghrelin appeared to activate extracellular signal-regulated kinases 1/2 through a calcium-independent novel protein kinase C isoform which may utilize diacylglycerol derived from hydrolysis of phosphatidylcholine rather than from phosphatidylinositol. Ghrelin-stimulated extracellular signal-regulated kinases 1/2 activity was independent of transactivation of epidermal growth factor receptors, and even when ghrelin receptor internalization was blocked by concanavalin A or a beta-arrestin mutant, there was no decrease in phosphorylated extracellular signal-regulated kinases 1/2, suggesting this is a G protein-dependent process. The truncated ghrelin receptor polypeptide had no effect on ghrelin receptor signalling to extracellular signal-regulated kinases 1/2, but decreased the constitutive activation of phosphatidylinositol-specific phospholipase C by ghrelin receptors. In conclusion, our results suggest that any up-regulation of the truncated ghrelin receptor polypeptide might preferentially attenuate functional activity dependent on the constitutive activation of ghrelin receptors, while leaving ghrelin-dependent signalling unaffected.

Co-expression of alpha(1,3)galactosyltransferase and Bacillus thuringiensis PIPLC enhances hyperacute rejection of tumor cells.

The use of alpha(1,3)galactosyltransferase (alphaGT) as a method of inducing hyperacute rejection of tumors has been gaining interest recently. However, the approach is based in part on the sensitivity of each tumor line to the effects of complement lysis. Tumors expressing complement resistance factors such as membrane cofactor (CD46), decay accelerating factor (CD55) and protectin (CD59) have been shown to be more resistant to complement mediated lysis. Anchored to the membrane by a glycosylphosphoinositol moiety (GPI-anchored), CD55 and CD59 can be cleaved by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PIPLC). Complement resistant A549 human lung carcinoma cells were engineered to express both the murine alphaGT gene and the B. thuringiensis PIPLC gene to alleviate complement resistance and enhance alphagal-mediated cancer killing. The PIPLC native signal sequence was replaced with the human epidermal growth factor signal sequence, EGFssPIPLC, to induce secretion from A549. Expression of EGFssPIPLC resulted in complete removal of CD55 and CD59 while sparing the non-GPI-anchored CD46. Results demonstrated that A549 cells transduced with two recombinant retroviral vectors carrying the alphaGT and EGFssPIPLC genes expressed high levels of alphagal epitope and exhibited a 5-fold increase in sensitivity to anti-alphagal mediated complement lysis.

Sphingosine-1-phosphate and calcium signaling in cerebellar astrocytes and differentiated granule cells.

S1P is involved in the regulation of multiple biological processes (cell survival, growth, migration and differentiation) both in neurons and glial cells. The study was aimed at investigating the possible effects of S1P on calcium signaling in cerebellar astrocytes and differentiated granule cells. In cerebellar astrocytes S1P is able to mediate calcium signaling mainly through Gi protein coupled receptors, whereas in differentiated neurons it failed to evoke any calcium signaling, despite acting both extracellularly and intracellularly. The data indicate strict cell specificity in S1P-evoked calcium response, which could be relevant to communication between neurons and glial cells in the cerebellum.