Lipid alterations in transient forebrain ischemia: possible new mechanisms of CDP-choline neuroprotection

We have previously demonstrated that cytidine 5'-diphosphocholine (CDP-choline or citicoline) attenuated arachidonic acid (ArAc) release and provided significant protection for the vulnerable hippocampal CA(1) neurons of the cornu ammonis after transient forebrain ischemia of gerbil. ArAc is released by the activation of phospholipases and the alteration of phosphatidylcholine (PtdCho) synthesis. Released ArAc is metabolized by cyclooxygenases/lipoxygenases to form eicosanoids and reactive oxygen species (ROS). ROS contribute to neurotoxicity through generation of lipid peroxides and the cytotoxic byproducts 4-hydroxynonenal and acrolein. ArAc can also stimulate sphingomyelinase to produce ceramide, a potent pro-apoptotic agent. In the present study, we examined the changes and effect of CDP-choline on ceramide and phospholipids including PtdCho, phosphatidylethanolamine (PtdEtn), phosphatidylinositol (PtdIns), phosphatidylserine (PtdSer), sphingomyelin, and cardiolipin (an exclusive inner mitochondrial membrane lipid essential for electron transport) following ischemia/1-day reperfusion. Our studies indicated significant decreases in total PtdCho, PtdIns, PtdSer, sphingomyelin, and cardiolipin and loss of ArAc from PtdEtn in gerbil hippocampus after 10-min forebrain ischemia/1-day reperfusion. CDP-choline (500 mg/kg i.p. immediately after ischemia and at 3-h reperfusion) significantly restored the PtdCho, sphingomyelin, and cardiolipin levels as well as the ArAc content of PtdCho and PtdEtn but did not affect PtdIns and PtdSer. These data suggest multiple beneficial effects of CDP-choline: (1) stabilizing the cell membrane by restoring PtdCho and sphingomyelin (prominent components of outer cell membrane), (2) attenuating the release of ArAc and limiting its oxidative metabolism, and (3) restoring cardiolipin levels.     

Effect of calmodulin antagonists on lysosomal enzyme secretion and phospholipid metabolism in guinea-pig macrophages

The effects of calmodulin antagonists on the secretion of lysosomal enzyme and lipid metabolism in guinea-pig peritoneal macrophages were studied. Calmodulin antagonists, such as trifluoperazine, dibucaine and quinacrine, inhibited the secretion of N-acetyl-β-d-glucosaminidase from cytochalasin B-treated macrophages when the macrophages were stimulated by the chemotactic peptide, formylmethionyl-leucyl-phenylalanine (f Met-Leu-Phe) or the Ca²⁺ ionophore A23187. The effect of calmodulin antagonists on the incorporation of [³²P]P_i or [³H]glycerol into glycerolipids as well as on the redistribution of [¹⁴C]glycerol or [³H]arachidonic acid in [¹⁴C]glycerol- or [³H]arachidonic acid-prelabelled lipids were examined. Trifluoperazine, dibucaine or quinacrine stimulated [³²P]P_i incorporation into phosphatidic acid (PtdA) and phosphatidylinositol (PtdIns) without significant effect on the labelling of phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), lysophosphatidylcholine (lyso-PtdCho) and lysophosphatidylethanolamine (lyso-PtdEtn). The incorporation of [³²P]P_i into phosphatidylcholine (PtdCho) was, on the contrary, inhibited. When calmodulin antagonists were added to macrophages stimulated by fMet-Leu-Phe, [³²P]P_i incorporation into PtdIns and PtdA was synergistically increased compared with that induced only by calmodulin antagonists. Trifluoperazine inhibited the incorporation of [³H]glycerol into PtdCho, triacylglycerol and PtdEtn. Also in this case, the incorporation of [³H]glycerol into PtdA and PtdIns was greatly enhanced. But [³H]glycerol incorporation into PtdSer, lyso-PtdEtn and lyso-PtdCho was not affected by the drug. On the other hand, diacylglycerol labelling with [³H]glycerol was maximally activated by 10μm-trifluoperazine and levelled off with the increasing concentration. When the effect of calmodulin antagonists on the redistribution of [¹⁴C]glycerol among lipids was examined in pulse-chase experiments, no significant effect on [¹⁴C]glycerol redistribution in PtdEtn, PtdCho, PtdIns, PtdSer, PtdA and tri- and di-acylglycerol could be detected. When macrophages prelabelled with [³H]arachidonic acid were treated with trifluoperazine, dibucaine or quinacrine, the [³H]arachidonic acid moiety in PtdEtn and PtdCho was decreased and that in PtdA was increased. The formation of [arachidonate-³H]diacylglycerol and non-esterified [³H]-arachidonic acid was also enhanced, but the increase in [³H]arachidonic acid was only observed at concentrations between 1 and 50μm. [Arachidonate-³H]PtdIns was not significantly affected. The activated formation of [arachidonate-³H]PtdA, diacylglycerol and non-esterified arachidonic acid by these drugs was synergistically enhanced in the presence of fMet-Leu-Phe.     

Phosphatidylthreonine and Lipid-Mediated Control of Parasite Virulence

The major membrane phospholipid classes, described thus far, include phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), and phosphatidylinositol (PtdIns). Here, we demonstrate the natural occurrence and genetic origin of an exclusive and rather abundant lipid, phosphatidylthreonine (PtdThr), in a common eukaryotic model parasite, Toxoplasma gondii. The parasite expresses a novel enzyme PtdThr synthase (TgPTS) to produce this lipid in its endoplasmic reticulum. Genetic disruption of TgPTS abrogates de novo synthesis of PtdThr and impairs the lytic cycle and virulence of T. gondii. The observed phenotype is caused by a reduced gliding motility, which blights the parasite egress and ensuing host cell invasion. Notably, the PTS mutant can prevent acute as well as yet-incurable chronic toxoplasmosis in a mouse model, which endorses its potential clinical utility as a metabolically attenuated vaccine. Together, the work also illustrates the functional speciation of two evolutionarily related membrane phospholipids, i.e., PtdThr and PtdSer.     

Phospholipid Composition and Electric Charge in Healthy and Cancerous Parts of Human Kidneys

Phospholipids are ubiquitous in nature and are essential for the lipid bilayer of cell membranes. Their structural and functional properties are pivotal for the survival of the cell. In this study the phospholipids of healthy and cancerous human renal tissues from the same patients are compared with special reference to the electric charge of the membrane. A simple and highly effective normal-phase method is described for analyzing phospholipids content. This work is focused on changes of phospholipids content (PtdIns, phosphatidylinositol; PtdSer, phosphatidylserine; PtdEtn, phosphatidylethanoloamine; PtdCho, phosphatidylcholine) in cell membranes of renal cancer of pT1 stage, G2 grade, without metastasis. Surface charge density of healthy and cancerous human renal tissues was measured by electrophoresis. The measurements were carried out at various pH of solution. Depending on the surface charge density as a function of pH, acidic (C _TA) and basic (C _TB) functional group concentrations and their average association constants with hydrogen (K _AH) or hydroxyl (K _BOH) ions were evaluated. The process of cancer transformation was accompanied by an increase in total amount of phospholipids as well as an increase in C _TA and K _BOH, whereas K _AH and C _TB were decreased compared with unchanged tumor cells.     

Myelination process in the rat sciatic nerve during regeneration and development: Molecular species composition and acyl group biosynthesis of choline-, ethanolamine-, and serine-glycerophospholipids of myelin fractions

The content of alkenyl-acyl, alkyl-acyl and diacyl types of the three major myelin glycerophospholipids such as PtdCho, PtdEtn and PtdSer was determined in myelin fractions prepared from sciatic nerve segments of rats at 12, 25 and 45 days after birth, and of adult rats (6-month-old) 90 days after crush injury. The biosynthesis and metabolic heterogeneity of lipid classes and types were also studied by incubation with [1-¹⁴C] acetate of nerve segments of young rats at different ages as well as crushed and sham-operated control nerve segments of adult rats. The analysis of composition and positional distribution in major individual molecular species extracted from light myelin and myelin-related fraction suggest that the metabolism of alkenyl-acyl-glycerophosphorylethanolamines and unsaturated species of PtdCho and PtdSer may not be regulated in the same manner during peripheral nerve myelination of developing rat and remyelination of regenerating nerve in the adult animal. The¹⁴C-radioactivity incorporation into lipid classes and alkyl and acyl moieties of the three major phospholipids of sciatic nerve segments during the developmental period investigated revealed that Schwann cells were capable of synthesizing acyl-linked fatty acids in both myelin fractions at a decreasing rate and with different patterns during development. In regenerating sciatic nerve of adult animals the labeling of myelin lipid classes and types of remyelinating nerve segment distal to the crush site was markedly higher than that of sham-operated normal one; however, the magnitude and the pattern of the specific radioactivity never approached those observed during active myelination of the nerve in young animals. These observations show that the remyelinating process of injured nerve during regeneration seems not to recapitulate nerve myelin ensheathment occurring during development.Abbreviations used PtdEtn Phosphatidylethanolamine - PtdCho Phosphatidylcholine - PtdSer Phosphatidylserine - GPE Glycero(3)phosphoethanolamine - GPC Glycero(3)phosphocholine - GPS Glycero(3)phosphoserine - DG-acetates 1,2-diradyl-3-acetyl-sn-glycerols - HPLC High performance liquid chromatography - TLC Thin-layer chromatography - BHT 2,6-di-tert-butyl-4-methylphenol     

Some characteristics of cytochromec-555 isolated from sulfide oxidizingXanthomonas sp. DY44

From a heterotrophic bacterium,Xanthomonas sp. DY44 which was previously reported to oxidize hydrogen sulfide (H₂S) to polysulfide, cytochromec-555 (cyt.c-555) responsible for oxidation of sulfide was purified by DEAE-Toyopearl and Sepadex G-75 column chromatography. Cyt.c-555 with a molecular weight of 12,500 showed maximum absorption at 555 nm (α-peak), 522 nm (β-peak) and 417 nm (γ-peak) for the reduced form which was prepared by addition of Na₂S₂O₄. Cyt.c-555 was also reduced by addition of sulfide (Na₂S and H₂S), and the oxidized products of sulfide by cyt.c-555 was identified as polysulfide. The reduced form of cyt.c-555 was suggested to be oxidized coupled with cyt.c oxidase which is tolerant to sulfide.     

Glucose metabolism determines resistance of cancer cells to bioenergetic crisis after cytochrome‐c release

Many anticancer drugs activate caspases via the mitochondrial apoptosis pathway. Activation of this pathway triggers a concomitant bioenergetic crisis caused by the release of cytochrome‐c (cyt‐c). Cancer cells are able to evade these processes by altering metabolic and caspase activation pathways. In this study, we provide the first integrated system study of mitochondrial bioenergetics and apoptosis signalling and examine the role of mitochondrial cyt‐c release in these events. In accordance with single‐cell experiments, our model showed that loss of cyt‐c decreased mitochondrial respiration by 95% and depolarised mitochondrial membrane potential ΔΨ_m from ?142 to ?88 mV, with active caspase‐3 potentiating this decrease. ATP synthase was reversed under such conditions, consuming ATP and stabilising ΔΨ_m. However, the direction and level of ATP synthase activity showed significant heterogeneity in individual cancer cells, which the model explained by variations in (i) accessible cyt‐c after release and (ii) the cell's glycolytic capacity. Our results provide a quantitative and mechanistic explanation for the protective role of enhanced glucose utilisation for cancer cells to avert the otherwise lethal bioenergetic crisis associated with apoptosis initiation.     

Cytochrome <Emphasis Type="Italic">c</Emphasis> is rapidly reduced in the cytosol after mitochondrial outer membrane permeabilization

Visible spectroscopy was used to measure real-time changes in the oxidation state of cytochrome c (cyt c) and the a-cytochromes (cyt aa₃) of cytochrome oxidase during mitochondrial outer membrane permeabilization (MOMP) initiated by anisomycin in HL-60 cells. The oxidation state of mitochondrial cyt c was found to be ≈62% oxidized before MOMP and became ≈70% oxidized after MOMP. In contrast, the cytosolic pool of cyt c was found to be almost fully reduced. This oxidation change allows cyt c release to be continuously and quantitatively monitored in real time. Anoxia and antimycin were used to fully reduce and fully oxidize, respectively, the mitochondrial pool of cyt c and it was found that the release of cyt c was independent of it oxidation state consistent with a simple model of cyt c passively diffusing down a concentration gradient through a pore or tear in the outer membrane. After MOMP was complete, the flux of cyt c diffusing back into the mitochondria was measured from the residual mitochondrial oxygen consumption after complete inhibition of the bc₁ with antimycin and myxothiazol. The outer membrane was found to be highly permeable after MOMP implying that the reduction of cyt c in the cytosol must be very rapid. The permeability of the outer membrane measured in this study would result in the release of cyt c with a time constant of less than 1 s.     

Phorbol ester stimulates the synthesis of sphingomyelin in NIH 3T3 cells A diminished response in cells transformed with human A-raf carrying retrovirus

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated the synthesis of sphingomyelin (CerPCho) from a [¹⁴C]choline-labelled phosphatidylcholine (PtdCho) pool in NIH 3T3 cells. Maximal stimulation (68%) of CerP-Cho synthesis, accompanied by an increase (38%) in its cellular content, required only 2 nM TPA. Higher concentrations of TPA (2–100 nM) had progressively less effect on CerPCho synthesis which correlated with increased hydrolysis of precursor PtdCho. In cells transformed with human or mouse A-raf carrying retroviruses TPA-stimulated PtdCho hydrolysis, but not CerPCho synthesis, suggesting independent regulation of these processes by the TPA-stimulated signal transduction system.     

Flavin oxidation in flavin‐dependent N‐monooxygenases

Siderophore A (SidA) from Aspergillus fumigatus is a flavin‐containing monooxygenase that hydroxylates ornithine (Orn) at the amino group of the side chain. Lysine (Lys) also binds to the active site of SidA; however, hydroxylation is not efficient and H₂O₂ is the main product. The effect of pH on steady‐state kinetic parameters was measured and the results were consistent with Orn binding with the side chain amino group in the neutral form. From the pH dependence on flavin oxidation in the absence of Orn, a pK_a value >9 was determined and assigned to the FAD‐N5 atom. In the presence of Orn, the pH dependence displayed a pK_a value of 6.7 ±0.1 and of 7.70 ±0.10 in the presence of Lys. Q102 interacts with NADPH and, upon mutation to alanine, leads to destabilization of the C4a‐hydroperoxyflavin (FAD_OOH). Flavin oxidation with Q102A showed a pK_a value of ~8.0. The data are consistent with the pK_a of the FAD N5‐atom being modulated to a value >9 in the absence of Orn, which aids in the stabilization of FAD_OOH. Changes in the FAD‐N5 environment lead to a decrease in the pK_a value, which facilitates elimination of H₂O₂ or H₂O. These findings are supported by solvent kinetic isotope effect experiments, which show that proton transfer from the FAD N5‐atom is rate limiting in the absence of a substrate, however, is significantly less rate limiting in the presence of Orn and or Lys.     

Evolutionary alkaline transition in human cytochrome <Emphasis Type="Italic">c</Emphasis>

Conformational transitions in cytochrome c (cyt c) are being realized to be responsible for its multi-functions. Among a number of conformational transitions in cyt c, the alkaline transition has attracted much attention. The cDNA of human cyt c is cloned by RT-PCR and a high-effective expression system for human cyt c has been developed in this study. The equilibrium and kinetics of the alkaline transition of human cyt c have been systematically investigated for the first time, and compared with those of yeast and horse cyt c from an evolutionary perspective. The pK_a value for the alkaline transition of human cyt c is apparently higher than that of yeast and horse. Kinetic studies suggest that it is increasingly difficult for the alkaline transition of cyt c from yeast, horse and human. Molecular modeling of human cyt c shows that the omega loop where the lysine residue is located apparently further away from heme in human cyt c than in yeast iso-1 and horse heart cyt c. These results regarding alkaline conformational transition provide valuable information for understanding the molecular basis for the biological multi-functions of cyt c.     

Arranged marriage in lipid signalling? The limited choices of PtdIns(4,5)P2 in finding the right partner

Inositol‐containing phospholipids (phosphoinositides, PIs) control numerous cellular processes in eukaryotic cells. For plants, a key involvement of PIs has been demonstrated in the regulation of membrane trafficking, cytoskeletal dynamics and in processes mediating the adaptation to changing environmental conditions. Phosphatidylinositol‐4,5‐bisphosphate (PtdIns(4,5)P₂) mediates its cellular functions via binding to various alternative target proteins. Such downstream targets of PtdIns(4,5)P₂ are characterised by the possession of specific lipid‐binding domains, and binding of the PtdIns(4,5)P₂ ligand exerts effects on their activity or localisation. The large number of potential alternative binding partners – and associated cellular processes – raises the question how alternative or even contrapuntal effects of PtdIns(4,5)P₂ are orchestrated to enable cellular function. This article aims to provide an overview of recent insights and new views on how distinct functional pools of PtdIns(4,5)P₂ are generated and maintained. The emerging picture suggests that PtdIns(4,5)P₂ species containing different fatty acids influence the lateral mobility of the lipids in the membrane, possibly enabling specific interactions of PtdIns(4,5)P₂ pools with certain downstream targets. PtdIns(4,5)P₂ pools with certain functions might also be defined by protein–protein interactions of PI4P 5‐kinases, which pass PtdIns(4,5)P₂ only to certain downstream partners. Individually or in combination, PtdIns(4,5)P₂ species and specific protein–protein interactions of PI4P 5‐kinases might contribute to the channelling of PtdIns(4,5)P₂ signals towards specific functional effects. The dynamic nature of PI‐dependent signalling complexes with specific functions is an added challenge for future studies of plant PI signalling.     

The recognition of membrane‐bound PtdIns3P by PX domains

Phox‐homology (PX) domains target proteins to the organelles of the secretary and endocytic systems by binding to phosphatidylinositol phospholipids (PIPs). Among all the structures of PX domains that have been solved, only three have been solved in a complex with the main physiological ligand: PtdIns3P. In this work, molecular dynamic simulations have been used to explore the structure and dynamics of the p40^phox–PX domain and the SNX17–PX domain and their interaction with membrane‐bound PtdIns3P. In the simulations, both PX domains associated spontaneously with the membrane‐bound PtdIns3P and formed stable complexes. The interaction between the p40^phox–PX domain and PtdIns3P in the membrane was found to be similar to the crystal structure of the p40^phox–PX–PtdIns3P complex that is available. The interaction between the SNX17–PX domain and PtdIns3P was similar to that observed in the p40^phox–PX–PtdIns3P complex; however, some residues adopted different orientations. The simulations also showed that nonspecific interactions between the β1–β2 loop and the membrane play an important role in the interaction of membrane bound PtdIns3P and different PX domains. The behaviour of unbound PtdIns3P within a 2‐oleoyl‐1‐palmitoyl‐sn‐glycero‐3‐phosphocholine (POPC) membrane environment was also examined and compared to the available experimental data and simulation studies of related molecules. Proteins 2014; 82:2332–2342. © 2014 Wiley Periodicals, Inc.     

Synthesis,Characterization, and Anti‐Amoebic Activity of N‐(Pyrimidin‐2‐yl)benzenesulfonamide Derivatives

A new series of N‐(pyrimidin‐2‐yl)benzenesulfonamide derivatives, 3a – 3i and 4a – 4i , was synthesized from pyrimidin‐2‐amines, 2a – 2i , with the aim to explore their effects on in vitro growth of Entamoeba histolytica. The chemical structures of the compounds were elucidated by elemental analysis, FT‐IR, ¹H‐ and ¹³C‐NMR, and ESI mass‐spectral data. In vitro anti‐amoebic activity was evaluated against HM1 : IMSS strain of Entamoeba histolytica. The IC₅₀ values were calculated by using the double dilution method. The results were compared with the IC₅₀ value of the standard drug ‘metronidazole’. The selected compounds were tested for their cytotoxic activities by cell‐viability assay using H9C2 cardiac myoblasts cell line, and the results indicated that all the compounds displayed remarkable >80% viabilities to a concentration of 100 μg/ml.     

Identification of Two Molecular Species of Rat Brain Phosphatidylcholine that Rapidly Incorporate and Turn Over Arachidonic Acid In Vivo

Abstract: In vivo rates of arachidonic acid incorporation and turnover were determined for molecular species of rat brain phosphatidylcholine (PtdCho) and phosphatidylinositol (PtdIns). [³H]Arachidonic acid was infused intravenously in pentobarbital-anesthetized rats at a programmed rate to maintain constant plasma specific activity for 2–10 min. At the end of infusion, animals were killed by microwave irradiation, and brain phospholipids were isolated, converted to diacylglycerobenzoates, and resolved as molecular species by reversed-phase HPLC. Most [³H]arachidonate (>87%) was incorporated into PtdCho and PtdIns, with arachidonic acid at the sn -2 position and with oleic acid (18:1), palmitic acid (16:0), or stearic acid (18:0) at the sn -1 position. However, 10–15% of labeled brain PtdCho eluted in a small peak containing two molecular species with arachidonic acid at the sn -2 position and palmitoleic acid (16:1) or linoleic acid (18:2) at the sn -1 position. Analysis demonstrated that tracer was present in both the 16:1–20:4 and 18:2–20:4 PtdCho species at specific activities 10–40 times that of the other phospholipids. Based on the measured mass of arachidonate in each phospholipid molecular species, half-lives were calculated for arachidonate of <10 min in 16:1–20:4 and 18:2–20:4 PtdCho and 1–3 h in 16:0–20:4, 18:0–20:4, and 18:1–20:4 PtdCho and PtdIns. The very short half-lives for arachidonate in the 16:1–20:4 and 18:2–20:4 PtdCho molecular species suggest important roles for these molecules in brain phospholipid metabolism and signal transduction.     

Dual 4‐ and 5‐phosphatase activities regulate SopB‐dependent phosphoinositide dynamics to promote bacterial entry

Salmonella are able to invade non‐phagocytic cells such as intestinal epithelial cells by modulating the host actin cytoskeleton to produce membrane ruffles. Two type III effector proteins SopB and SopE play key roles to this modulation. SopE is a known guanine nucleotide exchange factor (GEF) capable of activating Rac1 and CDC42. SopB is a phosphatidylinositol 4‐phosphatase and 5‐phosphatase promoting membrane ruffles and invasion of Salmonella through undefined mechanisms. Previous studies have demonstrated that the 4‐phosphatase activity of SopB is required for PtdIns‐3‐phosphate (PtdIns(3)P) accumulation and SopB‐mediated invasion. We show here that both the 4‐phosphatase as well as the 5‐phosphatase activities of SopB are essential in ruffle formation and subsequent invasion. We found that the 5‐phosphatase activity of SopB is likely responsible for generating PtdIns‐3,4‐bisphosphate (PtdIns(3,4)P₂) and subsequent recruitment of sorting nexin 9 (SNX9), an actin modulating protein. Intriguingly, the 4‐phosphatase activity is responsible for the dephosphorylation of PtdIns(3,4)P₂ into PtdIns(3)P. Alone, neither activity is sufficient for ruffling but when acting in conjunction with one another, the 4‐phosphatase and 5‐phosphatase activities led to SNX9‐mediated ruffling and Salmonella invasion. This work reveals the unique ability of bacterial effector protein SopB to utilize both its 4‐ and 5‐phosphatase activities to regulate phosphoinositide dynamics to promote bacterial entry.     

Serine and ethanolamine incorporation into different plasmalogen pools: Subcellular analyses of phosphoglyceride synthesis in cultured glioma cells

In cultured glioma cells, plasma membrane (PM) is enriched in phosphatidylserine (PtdSer) and plasmalogens (1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine). Serine can be a precursor of headgroups of both ptdSer and ethanolamine phosphoglycerides (PE) including plasmalogens and non-plasmalogen PE (NP-PE). Synthesis of phospholipids was investigated at the subcellular level using established fractionation procedures and incorporation of [³H(G)]L-serine and [1,2-¹⁴C]ethanolamine. Specific radioactivity of PtdSer from [³H]serine was 2-fold greater in PM than in microsomes, reaching maximum by 2–4 h. Labeled plasmalogen from [³H]serine appeared in PM by 4 h and increased to 48 h, whereas almost no plasmalogen accumulated in microsomes within 12 h. In contrast, labeled plasmalogen from [1,2-¹⁴C]ethanolamine appeared in both PM and microsomes at early incubation times and became enriched in PM beyond 12 h. Thus, in glioma cells: (1) greater and faster accumulation of labeled PtdSer in PM may reflect direct synthesis from serine within PM; (2) PM is a major source of PtdSer for decarboxylation and PE synthesis; (3) NP-PE in both PM and microsome provides headgroup for synthesis of plasmalogen; and, (4) plasmalogen synthesis may involve different intracellular pools depending on headgroup origin.Abbreviations NP-PE nonplasmenylethanolamine phosphoglycerides including both diacyl and alkylacyl species - PE total ethanolamine phosphoglycerides: plasmalogen-plasmenylethanolamine or alkenylacyl ethanolamine phosphoglyceride (1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) - PL phospholipid - PM plasma membrane - PtdCho phosphatidylcholine - PtdSer phosphatidylserine     

Production of phosphatidylinositol 5‐phosphate via PIKfyve and MTMR3 regulates cell migration

Although phosphatidylinositol 5‐phosphate (PtdIns5P) is present in many cell types and its biogenesis is increased by diverse stimuli, its precise cellular function remains elusive. Here we show that PtdIns5P levels increase when cells are stimulated to move and we find PtdIns5P to promote cell migration in tissue culture and in a Drosophila in vivo model. First, class III phosphatidylinositol 3‐kinase, which produces PtdIns3P, was shown to be involved in migration of fibroblasts. In a cell migration screen for proteins containing PtdIns3P‐binding motifs, we identified the phosphoinositide 5‐kinase PIKfyve and the phosphoinositide 3‐phosphatase MTMR3, which together constitute a phosphoinositide loop that produces PtdIns5P via PtdIns(3,5)P₂. The ability of PtdIns5P to stimulate cell migration was demonstrated directly with exogenous PtdIns5P and a PtdIns5P‐producing bacterial enzyme. Thus, the identified phosphoinositide loop defines a new role for PtdIns5P in cell migration.     

Selective Cytochrome c Displacement by Phosphate and Ca2+ in Brain Mitochondria

In brain mitochondria, phosphate- and Ca²⁺-dependent cytocrome c (cyt c) release reveals pools that interact differently with the inner membrane. Detachment of the phosphate-dependent pool did not influence the pool released by Ca²⁺. Cyt c pools were also detected in a system of cyt c reconstituted in cardiolipin (CL) liposomes. Gradual binding of cyt c (1 nmol) to CL/2–[12-(7-nitrobenz- 2-oxa-1,3-diazol-4-yl)amino]dodecanoyl-1-hexadecan oyl-sn-glycero-3-phosphocholine (NBDC₁₂-HPC) liposomes (10 nmol) produced NBD fluorescence quenching up to 0.4 nmol of added protein. Additional bound cyt c did not produce quenching, suggesting that cyt c-CL interactions originate distinct cyt c pools. Cyt c was removed from CL/NBDC₁₂-HPC liposomes by either phosphate or Ca²⁺, but only Ca²⁺ produced fluorescence dequenching and leakage of encapsulated 8-aminonaphthalene-1,3,6-trisulfonic acid/p-xylene-bis-pyridinium bromide. In mitochondria, complex IV activity and mitochondrial membrane potential (Δψ_m) were not affected by the release of the phosphate-dependent cyt c pool. Conversely, removal of cyt c by Ca²⁺ caused inhibition of complex IV activity and impairment of Δψ_m. In a reconstituted system of mitochondria, nuclei and supernatant, cyt c detached from the inner membrane was released outside mitochondria and triggered events leading to DNA fragmentation. These events were prevented by enriching mitochondria with exogenous CL or by sequestering released cyt c with anti-cyt c antibody.