Ca2+-induced polyphosphoinositide breakdown due to phosphomonoesterase activity in chicken erythrocytes.
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ABSTRACT:
SUBMITTER: Raval PJ
PROVIDER: S-EPMC1152719 | biostudies-other | 1985 Oct
REPOSITORIES: biostudies-other
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Project description:An investigation was made of the modifications in phospholipids that occur during the exocytotic event known as the 'sperm acrosome reaction'. Phospholipids were prelabelled with 32P, and exocytosis was induced with Ca2+ and the ionophore A23187. When incubated with [32P]Pi in various media suitable for supporting sperm survival or fertilization in vitro, spermatozoa from all five species examined (ram, boar, guinea pig, mouse and human) incorporated 32P rapidly into the components of the phosphoinositide cycle. There were differences both between species and between media with respect to the actual rate of incorporation of label, and also between species with respect to other phospholipids labelled. Treatment of spermatozoa with Ca2+ and A23187 to induce the acrosome reaction resulted in a rapid breakdown of phosphatidylinositol 4, 5-bisphosphate and phosphatidylinositol 4-phosphate, which was complete within 3 min; there was also a great increase in labelling of phosphatidate. Occurrence of acrosome reactions in the sperm population was only observed after 5-10 min and reached a maximum response of greater than 90% after more than 30 min. The phosphoinositide breakdown was related to subsequent exocytosis: after EGTA/ionophore treatment, neither inositide breakdown nor exocytosis took place; however, later addition of Ca2+ resulted in immediate inositide breakdown, and exocytosis followed, with a delay relative to Ca2+ addition exactly similar to that following standard Ca2+/ionophore treatment. Neomycin inhibited both inositide breakdown and subsequent exocytosis provided it was added together with Ca2+ and ionophore; however, if the drug was added 3 min after Ca2+ and ionophore (by which time inositide breakdown was already complete), exocytosis was not inhibited. Ca2+ seemed to have several consecutive roles in the acrosome reaction. Low (micromolar) levels of free Ca2+ were needed both for phosphoinositide breakdown and for an event downstream of this breakdown; no other bivalent cation could substitute for Ca2+ in either event, and inositide breakdown was actually inhibited by Mg2+. In addition, millimolar levels of Ca2+ were needed for later stages of exocytosis, although this requirement could be satisfied by Sr2+. We conclude that breakdown of polyphosphoinositides is an essential early process after Ca2+ entry in the chain of events that lead to exocytosis in the mammalian sperm acrosome reaction.
| S-EPMC1138523 | biostudies-other
Project description:1. Both the Ca(2+)-pump ATPase and the polyphosphoinositide phosphodiesterase of the erythrocyte membrane can, when assayed under appropriate conditions, be activated by Ca(2+) in the micromolar range. We have therefore compared the mechanisms and affinities for Ca(2+) activation of the two enzymes in human erythrocyte membranes, to see whether the polyphosphoinositide phosphodiesterase would be active in normal healthy erythrocytes. 2. At physiological ionic strength and in the presence of calmodulin, the Ca(2+)-pump ATPase was activated by Ca(2+) in a highly co-operative manner, with half-maximal activation occurring at about 0.3mum-Ca(2+). At an optimal Ca(2+) concentration, calmodulin stimulated the Ca(2+)-sensitive ATPase activity about 10-fold. 3. Ca(2+) activated the polyphosphoinositide phosphodiesterase in a non-co-operative manner. The Ca(2+) requirements for breakdown of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were identical, which supports our previous conclusion that Ca(2+) activates a single polyphosphoinositide phosphodiesterase that degrades both lipids with equal facility. Added calmodulin did not affect the activity of the polyphosphoinositide phosphodiesterase. 4. At low ionic strength in the absence of Mg(2+), half-maximal activation of the phosphodiesterase was at about 3mum-Ca(2+). The presence of 1mm-Mg(2+) shifted the Ca(2+) activation curve to the right, as did elevation of the ionic strength. When the Ca(2+)-pump ATPase and the polyphosphoinositide phosphodiesterase were assayed in the same incubations and under conditions of intracellular ionic strength and Mg(2+) concentration, the ATPase was fully activated at 3mum-Ca(2+), whereas no polyphosphoinositide phosphodiesterase activity was detected below 100mum-Ca(2+). 5. The Ca(2+)-pump ATPase of the erythrocyte membrane normally maintains the Ca(2+) concentration of healthy erythrocytes below approx. 0.1mum. It therefore seems unlikely that the polyphosphoinositide phosphodiesterase of the erythrocyte membrane ever expresses its activity in a healthy erythrocyte.
| S-EPMC1158073 | biostudies-other
Project description:Because Plasmodium falciparum replicates inside of a parasitophorous vacuole (PV) within a human erythrocyte, parasite egress requires the rupture of two limiting membranes. Parasite Ca2+ , kinases, and proteases contribute to efficient egress; their coordination in space and time is not known. Here, the kinetics of parasite egress were linked to specific steps with specific compartment markers, using live-cell microscopy of parasites expressing PV-targeted fluorescent proteins, and specific egress inhibitors. Several minutes before egress, under control of parasite [Ca2+ ]i , the PV began rounding. Then after ~1.5 min, under control of PfPKG and SUB1, there was abrupt rupture of the PV membrane and release of vacuolar contents. Over the next ~6 min, there was progressive vacuolar membrane deterioration simultaneous with erythrocyte membrane distortion, lasting until the final minute of the egress programme when newly formed parasites mobilised and erythrocyte membranes permeabilised and then ruptured-a dramatic finale to the parasite cycle of replication.
| S-EPMC6368063 | biostudies-literature
Project description:BACKGROUND:Chicken erythrocytes are involved in immunity through binding of toll-like receptors (TLRs) with their ligands to activate downstream signaling and lead to cytokine production in erythrocytes. Some avian ?-defensins (AvBDs) are constitutively expressed in tissues and some others can be induced by various bacteria and viruses. However, the expression of AvBDs in erythrocytes has not yet been studied extensively. RESULTS:The transcripts of eight AvBDs (AvBD1 to AvBD7, and AvBD9) and liver-expressed antimicrobial peptide-2 (LEAP-2) were found in normal chicken erythrocytes. The expression levels of AvBD2, 4 and 7 were significantly increased (P?<?0.01), whereas the levels of AvBD1, 6 and 9 were significantly decreased (P?<?0.01) after Marek's disease virus (MDV) infection. The mRNA expression level of LEAP-2 was not significantly changed after MDV infection. Highest viral nucleic acid (VNA) of MDV in the feather tips among the tested time points was found at 14 days post-infection (d.p.i.). In addition, 35 MD5-related gene segments were detected in the erythrocytes at 14 d.p.i. by transcriptome sequencing. CONCLUSIONS:These results suggest that the AvBDs in chicken erythrocytes may participate in MDV-induced host immune responses.
| S-EPMC6249751 | biostudies-literature
Project description:Muscarinic-receptor stimulation by 0.1 mM-carbachol in longitudinal muscle of the guinea-pig ileum increases the incorporation of [3H]inositol into inositol-containing phospholipid. This effect was blocked by 16 microM-atropine. After 60 min incubation, carbachol increased the accumulation of total inositol phosphates 20-fold in the presence of 10 mM-Li+. Less than 20% of the total inositol phosphate corresponded to inositol 1-phosphate by ion-exchange chromatography, whereas of the remainder about two-thirds corresponded to inositol bisphosphate and one third to inositol trisphosphate. It is concluded that stimulation of muscarinic receptors in guinea-pig ileum enhances breakdown of polyphosphoinositides, suggesting that this may be a primary event associated with Ca2+ mobilization in the guinea-pig ileum.
| S-EPMC1144327 | biostudies-other
Project description:Erythrocytes represent the most abundant cell type of the bloodstream in all vertebrates. The principal feature that differenciates erythrocytes of non-mammals is that unlike mammals they mantain the nucleus in their terminal differentiation. While the main function associated to erythrocytes is the oxygen and carbon dioxide transport, some other functions, no less important, have been attributed to these cells. The focus of this study was to investigate the response of cultured chicken erythrocytes to bacterial lipopolysaccharide (LPS), bacterial peptidoglycan (PGN) and to the analog viral dsRNA Poly(I:C) using a comercial microarray platform from Agilent.
2011-11-22 | GSE33813 | GEO
Project description:H4R3me2a and H3R2me2s often track together, locate to transcriptionally active DNA and are at regulatory regions of expressed genes.
2020-03-09 | GSE145398 | GEO
Project description:1. Human erythrocytes were treated with Ca2+ and ionophore A23187 and measurements were made of K+ efflux, polyphosphoinositide breakdown, 1,2-diacylglycerol accumulation, phosphatidate synthesis, changes in membrane polypeptide pattern and release of microvesicles. 2. It was shown that neither transamidase-mediated protein cross-linking, proteolysis of polypeptides 2.1 (ankyrin) or 4.1, nor accumulation of diacylglycerol or phosphatidate appeared to be necessary for microvesiculation to occur. 3. Microvesicles were released only under conditions where KCl efflux leading to cell shrinkage occurred and where polyphosphoinositides were broken down. These circumstances were sufficient to cause microvesiculation only in the presence of increased intracellular concentrations of Ca2+.
| S-EPMC1163286 | biostudies-other
Project description:The enzyme Tpt1 removes the 2'-PO4 at the splice junction generated by fungal tRNA ligase; it does so via a two-step reaction in which (i) the internal RNA 2'-PO4 attacks NAD+ to form an RNA-2'-phospho-ADP-ribosyl intermediate; and (ii) transesterification of the ribose O2″ to the 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1″,2″-cyclic phosphate products. The role that Tpt1 enzymes play in taxa that have no fungal-type RNA ligase remains obscure. An attractive prospect is that Tpt1 enzymes might catalyze reactions other than internal RNA 2'-PO4 removal, via their unique NAD+-dependent transferase mechanism. This study extends the repertoire of the Tpt1 enzyme family to include the NAD+-dependent conversion of RNA terminal 2' and 3' monophosphate ends to 2'-OH and 3'-OH ends, respectively. The salient finding is that different Tpt1 enzymes vary in their capacity and positional specificity for terminal phosphate removal. Clostridium thermocellum and Aeropyrum pernix Tpt1 proteins are active on 2'-PO4 and 3'-PO4 ends, with a 2.4- to 2.6-fold kinetic preference for the 2'-PO4 The accumulation of a terminal 3'-phospho-ADP-ribosylated RNA intermediate during the 3'-phosphotransferase reaction suggests that the geometry of the 3'-p-ADPR adduct is not optimal for the ensuing transesterification step. Chaetomium thermophilum Tpt1 acts specifically on a terminal 2'-PO4 end and not with a 3'-PO4 In contrast, Runella slithyformis Tpt1 and human Tpt1 are ineffective in removing either a 2'-PO4 or 3'-PO4 end.
| S-EPMC6573784 | biostudies-literature
Project description:Erythrocytes represent the most abundant cell type of the bloodstream in all vertebrates. The principal feature that differenciates erythrocytes of non-mammals is that unlike mammals they mantain the nucleus in their terminal differentiation. While the main function associated to erythrocytes is the oxygen and carbon dioxide transport, some other functions, no less important, have been attributed to these cells. The focus of this study was to investigate the response of cultured chicken erythrocytes to bacterial lipopolysaccharide (LPS), bacterial peptidoglycan (PGN) and to the analog viral dsRNA Poly(I:C) using a comercial microarray platform from Agilent. Chicken erythrocytes were isolated from blood by twice Ficoll 1.007 density gradient centrifugations. For primary cell culture, erythrocytes were resuspended in DMEM (PAA Laboratories) supplemented with 10% heat-inactivated fetal bovine serum (FBS, PAA Laboratories) and the antibiotic Primocin (100 μg/ml, Invivogen) at a density of 7.5x106 cells/ml in six well cell culture plates (NUNC) and cultured at 37ºC and 5% CO2. Erythrocytes were stimulated with LPS from E. coli (serotype 0111:B4, Sigma) and Poly(I:C) (Invivogen) at 50 μg/ml, and with peptidoglycan from E.coli (0111:B4, Invivogen) at 5 μg/ml. All culture plates were incubated for 12h. Total RNA was extracted from the cultures using 1 ml of Tri Reagent (Sigma) following the manufacturer’s instructions with minor modifications. Quantity and integrity was analyzed by Nanodrop1000 (ThermoScientific) and Bioanalyzer 2100 (Agilent Technologies) respectively. Three biological replicates were used.
2011-11-22 | E-GEOD-33813 | biostudies-arrayexpress