Phosphatidylinositol Phospholipase C Research Articles

Phospholipase cocktail: A new degumming technique for crude soybean oil

Enzymatic degumming (EDG) is an emerging alternative process for decreasing the phosphorus content, increasing the oil yield, and preserving the oil quality. Purifine® 3G is a cocktail of phospholipases composed of phospholipase A2 (PLA2), phospholipase C (PLC), and phosphatidylinositol phospholipase C (PI-PLC). In this study, Purifine® 3G was applied to crude soybean oil, and the optimum degumming conditions (enzyme concentration, temperature, and water dosage) were determined using a central composite rotatable design (CCRD). The contents of diacylglycerols (DAGs) and free fatty acids (FFAs) in the studied system considerably increased at temperatures below 64 °C and enzyme concentrations above 100 mg/kg, while the phosphorus content decreased with increasing water amount and enzyme concentration. In particular, EDG with 200 mg/kg of Purifine® 3G conducted for 120 min at a temperature of 60 °C and water concentration of 3% (w/w) lowered the residual phosphorus content to 8.9 mg/kg and increased the FFA and DAG concentrations by 0.17% and 0.72%, respectively. Meanwhile, EDG retained the tocopherol content in crude soybean oil, maintaining its quality. Hence, Purifine® 3G increases the neutral oil yield (FFA and DAG), decreases the phosphorus content, and preserves the oil quality, which make it a commercially viable degumming agent.

Sphingomyelin synthase‐related protein generates diacylglycerol via hydrolysis of phosphatidic acid, phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine without ceramide

Introduction Sphingomyelin synthase-related protein (SMSr) produces diacylglycerol (DG) and ceramide phosphoethanolamine (CPE) by the transfer of phosphoethanolamine from phosphatidylethanolamine (PE) to ceramide. We previously reported that SMSr functionally interacts with DG kinase (DGK) δ via steraile α motif domains (SAMD), protein-protein interaction module [Murakami et al. J. Biol. Chem. 295, 2932–47, 2020]. Moreover, SMSr provided DG to DGKδ. Puzzlingly, mammalian SMSr produces only trace amounts of CPE and DG. However, we previously reported that DG levels, particularly saturated fatty acid (SFA)- and/or monounsaturated fatty acid (MUFA)-containing DG molecular species, were significantly increased in COS-7 cells overexpressing human SMSr. To clarify this discrepancy, we purified human SMSr and determined whether purified SMSr showed other DG-generating activity via hydrolyis of phospholipids without ceramide in the present study. Results and Conclusion Human SMSr was expressed using the baculovirus-insect cell expression system and highly purified. Interestingly, purified SMSr showed DG-generating activity by hydrolysis of PE, phosphatidylcholine (PC), phosphatidylinositol (PI) and phosphatidic acid (PA) in the absence of ceramide. PA phosphatase (PAP) activity of SMSr was about 300-fold stronger than CPE synthase activity. Moreover, SMSr showed substrate selectivity for SFA- and/or MUFA-containing PA molecular species, but not arachidonic acid-containing PA, which is exclusively generated in the PI(4,5)P2 cycle. We confirmed that SMSr expressed in COS-7 cells showed PAP and PI-phospholipase C (PLC) activities. Collectively, our study indicated that SMSr possesses previously unrecognized enzyme activities (multi-glycerophospholipid PLC hydrolase) and constitutes a novel DG signaling pathway together with DGKd independent of the PI(4,5)P2 cycle.

Role of glypican-1 in endothelial NOS activation under various steady shear stress magnitudes

Blood flow patterns in proatherogenic and antiatherogenic regions are rather different. We hypothesize that the laminar flow with steady shear stress increased nitric oxide (NO) bioavailability while disturbed flow with low shear stress reduced it, which is mediating by glypican-1. Thus, we detected the expression of glypican-1 under different shear stress magnitudes, and tested whether the magnitude of shear stress determines the level of endothelial NO synthase (eNOS) via glypican-1 by using phosphatidylinositol phospholipase C (PI-PLC). Results revealed that the expression of glypican-1 depends on the magnitude and duration of shear stress loading. Activation of eNOS in HUVECs is downregulated by 4dyn/cm2 of shear stress, but is upregulated by 15dyn/cm2. Removal of glypican-1 significantly suppressed the 15dyn/cm2 shear stress-induced eNOS activity, and further reduced the 4dyn/cm2-inhibited eNOS activity. Therefore, eNOS activation depends on shear stress magnitudes and is mediated by glypican-1. The role of glypican-1 in mediating the eNOS activation under shear stress might involve in protecting the endothelial function against disturbed flow and enhancing the sensitive of the endothelial cell to laminar flow, supporting a potential role of glypican-1 against atherosclerosis.

Tissue Factor Pathway Inhibitor Isoforms of Macro- and Microvascular Endothelial Cells

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that inhibits both FXa and TF-FVIIa and is an important physiological inhibitor of the extrinsic coagulation pathway. The main portion (~80%) of TFPI in humans is reportedly associated with endothelial cells (ECs). At least 2 different isoforms of TFPI exist in humans, namely, TFPI alpha (α) and TFPI beta (β). In contrast to TFPIα, which consists of 3 Kunitz domains (KD) and a basic C-terminal part, TFPIβ lacks the third KD (KD3) and the basic C–terminal region. In TFPIβ, these 2 domains are replaced, due to alternative splicing, by a sequence that adds a glycosylphosphatidylinositol (GPI) anchor to the protein, linking it to the cell membrane. The present study aimed to identify possible differences in the TFPI level of macro- and microvascular ECs of various tissues to obtain further insight into the relevance of TFPI isoforms α and β at the cellular level.Primary macro- and microvascular ECs of various tissues were treated with phosphatidylinositol phospholipase C (PI-PLC) to remove the GPI-anchored TFPI isoform β from the cell surface. After detachment with an enzyme-free cell dissociation solution, living cells were washed and stained with a polyclonal anti human TFPI antibody and subsequently used for fluorescence activated cell sorting (FACS). TFPIα, the non-GPI anchored TFPI isoform, is not affected by PI-PLC treatment and therefore remains on the cell surface. Measuring the fluorescence intensities of these cells by FACS allowed us to determine the relative amount of cell surface TFPIβ. The supernatants and similarly treated cells were used in two different ELISA assays to quantify TFPI antigen. The first ELISA quantifies total TFPI, consisting of both TFPIα and β, whereas the second ELISA specifically determines the TFPIα content using another capture antibody. Subtraction of TFPIα from the total TFPI amount enabled us to determine the concentration of TFPIβ.Three macrovascular (HUVEC, HAoEC and HPAEC) and four microvascular (HDMEC, HDBEC, HCMEC and HPMEC) cell types were used for TFPI analysis. Based on availability, ECs from more than one donor were analyzed to explore individual differences in TFPI expression. FACS results of living cells revealed that ~85% of the cell surface TFPI of all analyzed ECs represents TFPIβ. ELISA measurements of supernatants and cell lysates showed that TFPIα was not affected by PI-PLC treatment, whereas TFPIβ increased in the supernatant and decreased in the total cell lysate after PI-PLC treatment. These results were obtained with all tested cell types. Differences in the absolute TFPI level of individual donors were also detected and microvascular cells were shown to exhibit more total TFPI than macrovascular cells.In conclusion, both TFPIα and β appear to be present on micro- and macrovascular ECs and ~85% of cell surface TFPI to represent TFPIβ. Furthermore, TFPI levels in the various vasculatures were shown to be dependent on the size of the blood vessel and on the individual donor. DisclosuresDockal:Baxter Innovations GmbH, Vienna, Austria: Employment. Pachlinger:Baxter Innovations GmbH, Vienna, Austria: Employment. Baldin-Stoyanova:Baxter Innovations GmbH, Vienna, Austria: Employment. Knofl:Baxter Innovations GmbH, Vienna, Austria: Employment. Ullrich:Baxter Innovations GmbH, Vienna, Austria: Employment. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment.

Detection of Plasmid in Listeria monocytogenes Isolated from Fresh Raw Milk in Nigeria

Aims: To determine the presence of plasmids in Listeria monocytogenes strains isolated from fresh raw milk from healthy cattle in Nigeria and also determine its role in antibiotics resistance since this organism is a food borne pathogen of public health importance. Methods and results: Isolation of Listeria monocytogenes was done using selective enrichment and isolation protocol, as recommended by United States Department of Agriculture (USDA) and Association of Analytical Chemists/ International Dairy Federation (AOAC/ IDF). Identification was by phenotypic characterization and confirmed using β-haemolytic activity, phosphatidyl inositol phospholipase-C (PIPLC) assay and polymerase chain reaction (PCR) . Strains identified as Listeria monocytogenes from the six zones were determined and results expressed as rate of prevalence in graph. Twenty-three strains of Listeria monocytogenes isolated from 305 samples of fresh raw milk were screened for the presence of plasmid DNA using the method of Ehrenfeld and Clewel and agarose gel electrophoresis. One isolate was found to harbour one plasmid with molecular weight of 14.62kb. The plasmid was cured using different concentrations of acridine orange. Antibiotic sensitivity test was carried out on both the wild strain (with plasmid) and cured strain using disc diffusion method of Bauer-Kirby and it was found that the wild strain was resistant to seven of the twelve antibiotics used while the cured strain was resistant to only one. Conclusions: There is a possibility of the presence of strains of Listeria monocytogenes that can harbour plasmids. This could confer antibiotic resistance to the strains and thus make antibiotic therapy unsuccessful. The increase in the number of antibiotics which the cured strain of Listeria monocytogenes was susceptible to in this study showed that plasmid may play a significant role in antibiotics resistance. The Fulani herdsmen who take fresh raw milk without boiling or pasteurization may serve as carriers of resistant strains of Listeria monocytogenes and help in its spread in the environment through faecal pollution.

A Computational Module Assembled from Different Protease Family Motifs Identifies PI PLC from Bacillus cereus as a Putative Prolyl Peptidase with a Serine Protease Scaffold

Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a β-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

Glycosylphosphatidylinositol Anchored TFPI As Main Regulator of Factor X Activation On the Surface of Endothelial Cells.

Abstract 2210Tissue factor pathway inhibitor (TFPI) is a key regulator of factor X (FX) activation in the extrinsic pathway of blood coagulation. TFPI inhibits FXa generation by formation of a quaternary complex consisting of factor VIIa (FVIIa), tissue factor (TF), FXa and TFPI. The main portion (∼80%) of TFPI in humans is reportedly associated with endothelial cells. We used human umbilical vein endothelial cells (HUVECs) as a model to obtain further insight into the function of TFPIα and the glycosylphosphatidylinositol (GPI) anchored TFPI form, which represents TFPIα bound to GPI-anchored surface proteins and/or TFPIβ. In contrast to TFPIα, which consists of 3 Kunitz domains (KD) and a basic C-terminal part, GPI-anchored TFPIβ lacks the third Kunitz domain (KD3) and the basic C–terminal region due to alternative splicing. In TFPIβ these two domains are replaced by a sequence that adds a GPI anchor to the protein linking it to the cell membrane.Treatment of HUVECs with phosphatidylinositol phospholipase C (PI-PLC) that cleaves GPI-anchors and subsequent fluorescence activated cell sorting (FACS) on living cells showed that GPI-anchored TFPI represents about 70–80% of cell surface TFPI. Staining of TFPI on and in fixed and permeabilized cells (total TFPI) demonstrated that GPI-anchored cell surface TFPI contributes to ∼20% of total cellular TFPI. Enzyme-linked immunosorbent assay (ELISA) showed that PI-PLC treatment released a TFPI protein lacking the KD3 and basic C-terminus. These findings strongly suggest that TFPIβ is the predominant GPI-anchored form of TFPI on HUVECs. FX activation assays performed on the cell surface of PI-PLC treated living HUVECs showed the importance of GPI-anchored TFPI on extrinsic Xase complex activity. PI-PLC treatment resulted in increased FX activation. Although GPI-anchored TFPI displays ∼70–80% of cell surface TFPI, overall FXa generation was increased only by ∼50%. In conclusion, HUVEC surface TFPI is predominantly TFPIβ, and GPI-anchored TFPI is the main but not sole regulator of FX activation on the surface of HUVECs. Disclosures:No relevant conflicts of interest to declare.

Failure of Prion Protein Oxidative Folding Guides the Formation of Toxic Transmembrane Forms

The mechanism by which pathogenic mutations in the globular domain of the cellular prion protein (PrP(C)) increase the likelihood of misfolding and predispose to diseases is not yet known. Differences in the evidences provided by structural and metabolic studies of these mutants suggest that in vivo folding could be playing an essential role in their pathogenesis. To address this role, here we use the single or combined M206S and M213S artificial mutants causing labile folds and express them in cells. We find that these mutants are highly toxic, fold as transmembrane PrP, and lack the intramolecular disulfide bond. When the mutations are placed in a chain with impeded transmembrane PrP formation, toxicity is rescued. These results suggest that oxidative folding impairment, as on aging, can be fundamental for the genesis of intracellular neurotoxic intermediates key in prion neurodegenerations.

Phospholipase C and Protein Kinase C-β 2 Mediate Insulin-Like Growth Factor II-Dependent Sphingosine Kinase 1 Activation

We recently reported that IGF-II binding to the IGF-II/mannose-6-phosphate (M6P) receptor activates the ERK1/2 cascade by triggering sphingosine kinase 1 (SK1)-dependent transactivation of G protein-coupled sphingosine 1-phosphate (S1P) receptors. Here, we investigated the mechanism of IGF-II/M6P receptor-dependent sphingosine kinase 1 (SK1) activation in human embryonic kidney 293 cells. Pretreating cells with protein kinase C (PKC) inhibitor, bisindolylmaleimide-I, abolished IGF-II-stimulated translocation of green fluorescent protein (GFP)-tagged SK1 to the plasma membrane and activation of endogenous SK1, implicating PKC as an upstream regulator of SK1. Using confocal microscopy to examine membrane translocation of GFP-tagged PKCα, β1, β2, δ, and ζ, we found that IGF-II induced rapid, transient, and isoform-specific translocation of GFP-PKCβ2 to the plasma membrane. Immunoblotting of endogenous PKC phosphorylation confirmed PKCβ2 activation in response to IGF-II. Similarly, IGF-II stimulation caused persistent membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, which does not dissociate from the membrane after translocation. IGF-II stimulation increased diacylglycerol (DAG) levels, the established activator of classical PKC. Interestingly, the polyunsaturated fraction of DAG was increased, indicating involvement of phosphatidyl inositol/phospholipase C (PLC). Pretreating cells with the PLC inhibitor, U73122, attenuated IGF-II-dependent DAG production and PKCβ2 phosphorylation, blocked membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, and reduced sphingosine 1-phosphate production, suggesting that PLC/PKCβ2 are upstream regulators of SK1 in the pathway. Taken together, these data provide evidence that activation of PLC and PKCβ2 by the IGF-II/M6P receptor are required for the activation of SK1.

Pro-prion Binds Filamin A, Facilitating Its Interaction with Integrin β1, and Contributes to Melanomagenesis

Filamin A (FLNA) is an integrator of cell mechanics and signaling. The spreading and migration observed in FLNA sufficient A7 melanoma cells but not in the parental FLNA deficient M2 cells have been attributed to FLNA. In A7 and M2 cells, the normal prion (PrP) exists as pro-PrP, retaining its glycosylphosphatidyl-inositol (GPI) anchor peptide signal sequence (GPI-PSS). The GPI-PSS of PrP has a FLNA binding motif and binds FLNA. Reducing PrP expression in A7 cells alters the spatial distribution of FLNA and organization of actin and diminishes cell spreading and migration. Integrin β1 also binds FLNA. In A7 cells, FLNA, PrP, and integrin β1 exist as two independent, yet functionally linked, complexes; they are FLNA with PrP or FLNA with integrin β1. Reducing PrP expression in A7 cells decreases the amount of integrin β1 bound to FLNA. A PrP GPI-PSS synthetic peptide that crosses the cell membrane inhibits A7 cell spreading and migration. Thus, in A7 cells FLNA does not act alone; the binding of pro-PrP enhances association between FLNA and integrin β1, which then promotes cell spreading and migration. Pro-PrP is detected in melanoma in situ but not in melanocyte. Invasive melanoma has more pro-PrP. The binding of pro-PrP to FLNA, therefore, contributes to melanomagenesis.

Low density lipoprotein metabolism, hepatic lipase and coronary disease: Intriguing findings from genome wide association studies

Blood flow patterns in proatherogenic and antiatherogenic regions are rather different. We hypothesize that the laminar flow with steady shear stress increased nitric oxide (NO) bioavailability while disturbed flow with low shear stress reduced it, which is mediating by glypican-1. Thus, we detected the expression of glypican-1 under different shear stress magnitudes, and tested whether the magnitude of shear stress determines the level of endothelial NO synthase (eNOS) via glypican-1 by using phosphatidylinositol phospholipase C (PI-PLC). Results revealed that the expression of glypican-1 depends on the magnitude and duration of shear stress loading. Activation of eNOS in HUVECs is downregulated by 4 dyn/cm2 of shear stress, but is upregulated by 15 dyn/cm2. Removal of glypican-1 significantly suppressed the 15 dyn/cm2 shear stress-induced eNOS activity, and further reduced the 4 dyn/cm2-inhibited eNOS activity. Therefore, eNOS activation depends on shear stress magnitudes and is mediated by glypican-1. The role of glypican-1 in mediating the eNOS activation under shear stress might involve in protecting the endothelial function against disturbed flow and enhancing the sensitive of the endothelial cell to laminar flow, supporting a potential role of glypican-1 against atherosclerosis.

Hypoxia-induced mitogenic factor/FIZZ1 induces intracellular calcium release through the PLC-IP3 pathway

Hypoxia-induced mitogenic factor (HIMF), also known as "found in inflammatory zone 1" (FIZZ1) or resistin-like molecule-alpha (RELMalpha), is a profound vasoconstrictor of the pulmonary circulation and a strong mitogenic factor in pulmonary vascular smooth muscle. To further understand the mechanism of these contractile and mitogenic responses, we examined the effect of HIMF on intracellular Ca(2+) in human pulmonary artery smooth muscle cells (SMC). Ca(2+) imaging in fluo 4-loaded human pulmonary artery SMC revealed that recombinant murine HIMF increased intracellular Ca(2+) concentration ([Ca(2+)](i)) in a sustained and oscillatory manner. This increase occurred independent of extracellular Ca(2+) influx. Pretreatment of human pulmonary artery SMC with U-73122, a specific inhibitor of phosphatidylinositol-phospholipase C (PLC) completely prevented the HIMF-induced Ca(2+) signal. The [Ca(2+)](i) increase was also abolished by pretreatment with 2-aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-trisphosphate (IP(3)) receptor antagonist. Ryanodine pretreatment did not affect initiation of [Ca(2+)](i) activation or internal release but reduced [Ca(2+)](i) at the plateau phase. Pretreatment with the Galpha(i)-specific inhibitor pertussis toxin and the Galpha(s)-specific inhibitor NF-449 did not block the Ca(2+) signal. Knockdown of Galpha(q/11) expression did not prevent Ca(2+) release, but the pattern of Ca(2+) release changed from the sustained oscillatory transients with prolonged plateau to a series of short [Ca(2+)](i) transients that return to baseline. However, pretreatment with the tyrosine kinase inhibitor genistein completely inhibited the internal Ca(2+) release. These results demonstrate that HIMF can stimulate intracellular Ca(2+) release in human pulmonary artery SMC through the PLC signaling pathway in an IP(3)- and tyrosine phosphorylation-dependent manner and that Galpha(q/11) protein-coupled receptor and ryanodine receptor contribute to the increase of [Ca(2+)](i).

Use of a phospholipase-C assay, in vivo pathogenicity assays and PCR in assessing the virulence of Listeria spp.

Listeria spp. was isolated from 19.8% of animals with a history of reproductive disorders. A total of 333 faecal, genital swab and blood samples from 111 animals (cattle, buffaloes, sheep and goats) were subjected to PCR to detect virulence-associated genes (prfA, plcA, hlyA, actA and iap) and pathogenicity testing by the phosphatidylinositol phospholipase-C (PI-PLC) assay, and by mouse and chick embryo inoculation. One isolate of Listeria ivanovii recovered from a genital swab from a sheep was found to be pathogenic. Virulence assessment was then carried out on two L. ivanovii and 29 Listeria monocytogenes isolates from various sources using these assays. Haemolytic L. monocytogenes isolates lacking the plcA gene and PI-PLC activity were deemed non-pathogenic when assessed by mouse and chick embryo inoculation tests, in spite of having the hlyA gene. The results suggested that the PI-PLC and PCR assays are reliable in vitro alternatives to in vivo pathogenicity tests for L. monocytogenes.

High Expression of Decoy Receptor TRAIL-R3 on AML Blasts Is Associated to Shortened Overall Survival. TRAIL-R3 Induced Apoptosis Resistance Can Be Overcome by Specifically Targeting TRAIL-R2 in Vitro

In vivo, effector T cells and NK cells do express membrane-bound TRAIL (TNF-related apoptosis-inducing ligand). T cells also secrete a soluble form of TRAIL (sTRAIL). Binding of TRAIL to one of the receptors on target cells can induce apoptosis. In human, 4 membranebound receptors for TRAIL have been identified: two of them (TRAIL-R1 and TRAIL-R2) contain a functional death domain, while the two other (TRAIL-R3 and TRAIL-R4) lack a functional death domain and function as decoy receptors. Most normal cells express TRAIL-R3 and TRAIL-R4, where many tumor cells express TRAIL-R1 and TRAIL-R2, making activation of the TRAIL mediated apoptotic pathway a possible new model for targeted therapy. We investigated bone-marrow samples from 119 patients with de novo AML and from 11 healthy donors for TRAIL receptor expression on CD45dim/SSClow blasts by flow-cytometry. Expression profiles of patients were correlated to survival data. Functional data were obtained by incubation of 6 myeloid leukemic cell lines and fresh AML samples with different concentrations of sTRAIL/Apo2L (n=10) and specific antibodies directly targeting R1 and R2 (HGS-ETR1 and HGS-ETR2) (n=18). Apoptosis was assessed by flow–cytometry after staining with 7AAD and annexinV. R3 expression on the cell surface of one cell line (MM6) was down-modulated with PI-PLC (Phosphatidyl-inositol phospholipase C ).When comparing AML and normal blasts, significantly more pro-apoptotic TRAIL-R1 and TRAIL-R2 receptor expression on myeloid leukemic blasts (p<0.0005) was measured however without association with outcome. About 30% of AML patients had a high anti-apoptotic TRAIL-R3 expression, which was strongly correlated to a shortened overall survival (log rank, p=0.0051). In multivariate analysis, R3 expression remained a significant prognostic factor next to cytogenetics (p=0.03 and p= 0.015 respectively). In vitro, studies on 6 myeloid leukemic cell lines confirmed sTRAIL sensitivity in cell lines that expressed presumably TRAIL-R1 and TRAIL-R2. Moreover, down-modulation of TRAIL-R3 by treatment with PI-PLC (TRAIL-R3 is the only GPI-linked TRAIL receptor) on cell lines improved sTRAIL sensitivity. Fresh myeloid leukemic samples were treated with sTRAIL. Mean apoptosis induction was 14% (0–54%). sTRAIL can bind to both the pro-apoptotic as the decoy receptors. Bypassing of the decoy R3 expression by treatment with the antibody directly targeting TRAIL-R2 (anti-TRAIL-R2) resulted in much higher rates of apoptosis (mean 30%, 0–80%). Apoptosis induction via direct targeting of TRAIL-R2 was, as expected, independent of TRAIL-R3 expression. All leukemic blasts that showed apoptosis with anti-TRAIL-R2 expressed the R2 receptor, but not all blasts that expressed TRAIL-R2 were sensitive for anti-TRAIL-R2, which could be due to inhibitory factors downstream the apoptotic cascade. In conclusion, AML blasts do express pro-apoptotic TRAIL receptors. However, co-expression with the decoy receptor TRAIL-R3 results in significant shortened overall survival. AML blasts are sensitive to targeting the pro-apoptotic TRAIL-R2 receptor, yielding a new therapeutic option for AML patients.

Glial Cell Line-derived Neurotrophic Factor Reverses Ethanol-mediated Increases in Tyrosine Hydroxylase Immunoreactivity via Altering the Activity of Heat Shock Protein 90

We previously found that glial cell line-derived neurotrophic factor (GDNF) in the midbrain ventral tegmental area (VTA) negatively regulates alcohol drinking (He, D. Y., McGough, N. N., Ravindranathan, A., Jeanblanc, J., Logrip, M. L., Phamluong, K., Janak, P. H., and Ron, D. (2005) J. Neurosci. 25, 619-628). Several studies suggest a role for GDNF in the regulation of tyrosine hydroxylase (TH) levels in the midbrain (Georgievska, B., Kirik, D., and Bjorklund, A. (2004) J. Neurosci. 24, 6437-6445). Up-regulation of TH levels has been reported as a hallmark of biochemical adaptations to in vivo chronic exposure to drugs of abuse, including ethanol (Ortiz, J., Fitzgerald, L. W., Charlton, M., Lane, S., Trevisan, L., Guitart, X., Shoemaker, W., Duman, R. S., and Nestler, E. J. (1995) Synapse 21, 289-298). We hypothesized that GDNF plays an important role in regulating prolonged ethanol-mediated increases in TH protein levels. Using the SH-SY5Y dopaminergic-like cell line, we found that the increase in TH levels in the presence of ethanol required the activation of the cAMP/PKA pathway and was reversed by GDNF. Ethanol treatment did not alter the mRNA level or protein translation of TH, but enhanced the stability of the protein that was decreased by GDNF. Interestingly, we observed that ethanol treatment resulted in an increase in TH association with the chaperone heat shock protein (HSP90) that was mediated by the cAMP/PKA pathway and inhibited by GDNF. Taken together, these data suggest that prolonged ethanol exposure leads to increased association of TH and HSP90 via the cAMP/PKA pathway, resulting in the stabilization and subsequent accumulation of TH. GDNF reverses this ethanol-mediated adaptation by inhibiting the interaction of TH with HSP90.

Multiple point mutations in virulence genes explain the low virulence of Listeria monocytogenes field strains

In order to understand the causes of the low virulence of Listeria monocytogenes field strains, five low-virulence strains were analysed. These five strains showed changes in relation to invasion, phosphatidyl-inositol phospholipase C (PI-PLC) activity, plaque formation and in vivo virulence. Molecular analyses revealed the same mutations in the plcA, inlA and inlB genes in all five strains. The Thr262Ala substitution in the PI-PLC protein was responsible for the absence of PI-PLC activity. This residue, conserved in certain L. monocytogenes species, is located at the outer rim of the active site pocket and could impair the cleavage activity of the enzyme. The low invasion rate of these strains was due to a nonsense codon leading to a lack of InlA protein synthesis, and to an Ala117Thr substitution in the leucine-rich repeat of InlB, which altered the interaction with the Met receptor. Single trans complementation with the inlA(EGDe), inlB(EGDe) or plcA(EGDe) genes restored the capacity of low-virulence strains either to enter epithelial and fibroblastic cells or to express PI-PLC activity. Complementation by allelic exchange of the plcA(EGDe) gene on the chromosome and trans complementation with either the inlA(EGDe) or the inlB(EGDe) gene restored the ability to form plaques, but only partly restored the in vivo virulence, suggesting that there were other gene mutation(s) with consequences that could mainly be observed in vivo. These results indicate that the low virulence of L. monocytogenes strains can be explained by point mutations in a number of virulence genes; these could therefore be important for detecting low-virulence strains. Moreover, the fact that all the strains had the same substitutions suggests that they have a common evolutionary pathway.

Tumor Necrosis Factor-α Enhances Neutrophil Adhesiveness: Induction of Vascular Cell Adhesion Molecule-1 via Activation of Akt and CaM Kinase II and Modifications of Histone Acetyltransferase and Histone Deacetylase 4 in Human Tracheal Smooth Muscle Cells

Up-regulation of vascular cell adhesion molecule-1 (VCAM-1) involves adhesions between both circulating and resident leukocytes and the human tracheal smooth muscle cells (HTSMCs) during airway inflammatory reaction. We have demonstrated previously that tumor necrosis factor (TNF)-alpha-induced VCAM-1 expression is regulated by mitogen-activated protein kinases, nuclear factor-kappaB, and p300 activation in HTSMCs. In addition to this pathway, phosphorylation of Akt and CaM kinase II has been implicated in histone acetyltransferase and histone deacetylase 4 (HDAC4) activation. Here, we investigated whether these different mechanisms participated in TNF-alpha-induced VCAM-1 expression and enhanced neutrophil adhesion. TNF-alpha significantly increased HTSMC-neutrophil adhesions, and this effect was associated with increased expression of VCAM-1 on the HTSMCs and was blocked by the selective inhibitors of Src [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1)], epidermal growth factor receptor [EGFR; 4-(3'-chloroanilino)-6,7-dimethoxy-quinazoline, (AG1478)], phosphatidylinositol 3-kinase (PI3K) [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride(LY294002) and wortmannin],calcium[1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester; BAPTA-AM], phosphatidylinositol-phospholipase C (PLC) [1-[6-[[17beta-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122)], protein kinase C (PKC) [12-(2-cyanoethyl)-6,7,12, 13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Gö6976), rottlerin, and 3-1-[3-(amidinothio)propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl) maleimide (bisindolylmaleimide IX) (Ro 31-8220)], CaM (calmidazolium chloride), CaM kinase II [(8R(*),9S(*),11S(*))-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-14-n-propoxy-2,3,9, 10-tetrahydro-8,11-epoxy, 1H,8H, 11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde]trinden-1-one (KT5926) and 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN62)], p300 (curcumin), and HDAC (trichostatin A) or transfection with short interfering RNAs for Src, Akt, PKCalpha, PKCmu, and HDAC4. At gene regulation level, reverse-transcriptase polymerase chain reaction and promoter assays revealed that expression of VCAM-1 was also attenuated by these signaling molecule inhibitors. Moreover, TNF-alpha induced Akt and CaM kinase II phosphorylation via cascades through Src/EGFR/PI3K and PLC/calcium/CaM, respectively. Finally, activation of Akt and CaM kinase II may eventually lead to the acetylation of histone residues and phosphorylation of histone deacetylase. These findings revealed that TNF-alpha induced VCAM-1 expression via multiple signaling pathways. Blockade of these pathways may be selectively targeted to reduce neutrophil adhesion via VCAM-1 suppression and attenuation of the inflammatory responses in airway diseases.

Phospholipase C, phosphatidylinositol 3-kinase, and intracellular [Ca 2+] mediate the activation of chicken HD11 macrophage cells by CpG oligodeoxynucleotide

The activation of phospholipases is one of the earliest key events in receptor-mediated cellular responses to a number of extracellular signaling molecules. Oligodeoxynucleotides containing CpG motifs (CpG ODN) mimic microbial DNA and are immunostimulatory to most vertebrate species. In the present study, we used the production of nitric oxide (NO) as an indicator to evaluate the involvement of the signaling cascades of phospholipases and phosphatidylinositol 3-kinase (PI3K) in the activation of chicken HD11 macrophage cells by CpG ODN. Using selective inhibitors, we have identified the involvement of phosphatidylinositol (PI)-phospholipase C (PI-PLC), but not phosphatidylcholine (PC)-phospholipase C (PC-PLC) and PC-phospholipase D (PC-PLD), in CpG ODN-induced NO production in HD11 cells. Preincubation with PI-PLC selective inhibitors (U-73122) completely abrogated CpG ODN-induced NO production in HD11 cells, whereas PC-PLC inhibitor (D609) and PC-PLD inhibitor (n-butanol) had no inhibitory effects. Additionally, inhibition of PI3K and protein kinase C (PKC) with selective inhibitors and chelation of intracellular [Ca(2+)] also significantly attenuated NO production in CpG ODN-activated HD11 cells. Our results demonstrate that PI-PLC, PI3 K, PKC, and intracellular [Ca(2+)] are important components of the CpG ODN-induced signaling pathway that leads to the production of NO in avian macrophage cells.

Protein and lipid signaling in membrane fusion: nuclear envelope assembly

AbstractMembrane fusion is important in many cell processes including membrane trafficking, mitotic reconstitution of organelles, viral infection and fertilization. Several fusion events occur just prior and subsequent to fertilization in the sea urchin, including the sperm acrosomal reaction, fusion of sperm and egg plasma membranes, exocytosis of cortical granules, reassembly of the sperm nuclear envelope and fusion of the male and female pronuclear envelopes leading to the zygote nucleus of the one‐cell embryo. The study of male pronuclear membrane dynamics with cell‐free extracts of fertilized sea urchin eggs has revealed several novel features, in particular a structural role arising from altering phospholipids prior to nuclear membrane formation. Fusion of chromatin‐bound membrane vesicles in vitro can be triggered by either GTP hydrolysis or exogenous phosphatidyl inositol phospholipase C (PI‐PLC). Recent data strongly implicate a role for diacylglycerol in nuclear envelope formation as a structural destabilizing lipid in membrane fusion. Moreover, the endogenous enzyme, PI‐PLCγ, is &gt;100‐fold enriched in a nuclear envelope precursor vesicle population (MV1) that is required for nuclear envelope assembly. NMR and mass spectrometry analyses show that MV1 contains high levels of phosphoinositides, including the substrate of PLCγ, compared to the other nuclear envelope precursor membranes. MV1 exists in eggs as vesicles in the cortex distinct from the endoplasmic reticulum which contributes most of the nuclear envelope membrane. PLCγ is activated by a tyrosine kinase in response to GTP hydrolysis at an early stage of nuclear envelope formation suggesting a role in initiation of fusion and revealing aspects of a signaling mechanism leading to fusion. The binding of MV1 to two poles of the sperm nucleus offers spatial as well as temporal control of the initiation phase.

A Novel Follicle-Stimulating Hormone-Induced Gαh/Phospholipase C-δ1 Signaling Pathway Mediating Rat Sertoli Cell Ca2+-Influx

FSH is known to activate Gs/cAMP signaling pathway in Sertoli cells (SCs) to support spermatogenesis. However, the molecular mechanism of FSH-induced Gs/cAMP-independent Ca2+-influx in SCs is not clear. In this study, FSH indeed induced an immediate and dose-dependent intracellular Ca2+-elevation in rat SCs. In the presence of EDTA (2.5 mm) or in the absence of extracellular Ca2+, the FSH-induced intracellular Ca2+-elevation was abolished. The confocal microscopic observation of Ca2+ image revealed that the SC cellular Ca2+ level was gradually increased after 50 sec of FSH treatment. Dantrolene, a blocker of intracellular Ca2+ release, did not affect this FSH-induced intracellular Ca2+ elevation. The pretreatment of rat SCs with phosphatidylinositol-phospholipase C (PLC)-specific inhibitor, U73122 (3 and 10 microm), inhibited the FSH-induced Ca2+-influx in a dose-dependent manner, but treatment with Gs-specific inhibitor, NF449 (0.1 and 0.3 microm), did not. On the other hand, the activation of G alpha h was immediately induced by FSH in the rat SCs within 5 sec of treatment. The translocation of PLC-delta1 from cytosol to cell membrane and the formation of G alpha h /PLC-delta1 complexes occurred within 5 and 10 sec, respectively, of FSH exposure. The intracellular inositol 1,4,5-triphosphate (IP3) production was also detected after 30 sec of FSH treatment. The synthetic peptide of PLC-delta1 (TIPWNSLKQGYRHVHLL), not Gs inhibitor, predominantly inhibited the FSH-induced PLC-delta1 translocation, formation of G alpha h /PLC-delta1 complex, intracellular IP3 production, and Ca2+ influx. In contrast, the peptide did not interfere with FSH-induced intracellular cAMP accumulation. In conclusion, the FSH-induced immediate Ca2+ influx is unambiguously mediated by an alternative G alpha h /PLC-delta1/IP3 pathway that is distinct from the Gs/cAMP pathway in rat SCs.