The vacuolating cytotoxin (VacA) of the gastric pathogen Helicobacter pylori binds and enters epithelial cells, ultimately resulting in cellular vacuolation. Several host factors have been reported to be important for VacA function, but none of these have been demonstrated to be essential for toxin binding to the plasma membrane. Thus, the identity of cell surface receptors critical for both toxin binding and function has remained elusive. Here, we identify VacA as the first bacterial virulence factor that exploits the important plasma membrane sphingolipid, sphingomyelin (SM), as a cellular receptor. Depletion of plasma membrane SM with sphingomyelinase inhibited VacA-mediated vacuolation and significantly reduced the sensitivity of HeLa cells, as well as several other cell lines, to VacA. Further analysis revealed that SM is critical for VacA interactions with the plasma membrane. Restoring plasma membrane SM in cells previously depleted of SM was sufficient to rescue both toxin vacuolation activity and plasma membrane binding. VacA association with detergent-resistant membranes was inhibited in cells pretreated with SMase C, indicating the importance of SM for VacA association with lipid raft microdomains. Finally, VacA bound to SM in an in vitro ELISA assay in a manner competitively inhibited by lysenin, a known SM-binding protein. Our results suggest a model where VacA may exploit the capacity of SM to preferentially partition into lipid rafts in order to access the raft-associated cellular machinery previously shown to be required for toxin entry into host cells.
1. A method is presented for the determination of the di- and tri-phosphoinositide in animal tissues. 2. The polyphosphoinositides are quantitatively extracted into chloroform-methanol-hydrochloric acid solvent after a preliminary chloroform-methanol (1:1, v/v) extraction to remove the bulk of the other phospholipids. On washing this extract with n-hydrochloric acid the polyphosphoinositides pass completely into the lower chloroform-rich phase. Their concentrations in the lower phase are determined by chromatography on formaldehyde-treated paper or chromatography and ionophoresis of the acid hydrolysis products. 3. When guinea-pig brain is extracted by the method of Folch (1942), considerable hydrolysis of the triphosphoinositide and accumulation of diphosphoinositide occurs during the initial acetone extraction. 4. The tri- and di-phosphoinositide contents of rat and guinea-pig brain decline substantially within a few minutes after death. 5. The concentrations of tri- and di-phosphoinositide in rat brain are not changed by insulin-hypoglycaemia or electrical stimulation. 6. Examination of frozen rat tissues showed that the brain contained the highest concentration of polyphosphoinositides. Much smaller amounts are present in kidney, and only trace quantities in liver and lung. None could be detected in spleen, heart and skeletal muscle.
The effect of chronic streptozotocin-induced diabetes on phospholipid metabolism in rat sciatic nerve in vitro was investigated. In normal nerve incubated for 2 h in Krebs-Ringer-bicarbonate buffer containing [32P]orthophosphate, radioactivity was primarily incorporated into phosphatidylinositol-4,5-bisphosphate and phosphatidylcholine. Smaller amounts were present in phosphatidylinositol-4-phosphate, phosphatidylinositol, and phosphatidic acid. As compared to controls, phosphatidylinositol-4,5-bisphosphate in nerves from animals made diabetic 2, 10, and 20 weeks earlier accounted for 30-46% more of the isotope, expressed as a percentage, incorporated into all phospholipids. In contrast, the proportion of radioactivity in phosphatidylcholine decreased by 10-25%. When the results were expressed as the quantity of phosphorus incorporated into phospholipid, only phosphatidylinositol-4,5-bisphosphate displayed a change. The amount of isotope which entered this lipid increased 60% and 67% for 2- and 10-week diabetic animals, respectively. Increased phosphatidylinositol-4,5-bisphosphate labeling was observed when epineurial-free preparations were used or when the composition of the incubation medium was varied. Sciatic and caudal nerve conduction velocities were decreased after 10 and 20 weeks but were unchanged after 2 weeks. We conclude that an increase in the turnover of phosphatidylinositol-4,5-bisphosphate in sciatic nerve from streptozotocin-diabetic rats appears relatively early and persists throughout the course of the disease. This metabolic alteration may be related to a primary defect responsible for the accompanying deficient peripheral nerve function.
Using a primitive Earth evaporating pond model, the synthesis of phosphatidylcholine was accomplished when a reaction mixture of choline chloride and disodium phosphatidate, in the presence of cyanamide and traces of acid, was evaporated and heated at temperatures ranging from 25 degrees to 100 degrees C for 7 hours. Optimum yields of about 15% were obtained at 80 degrees C. Phosphatidylcholine was identified by chromatographic, chemical and enzymatic degradation methods. On enzymatic hydrolysis with phospholipase A2 and phospholipase C, lysophosphatidylcholine and phosphorylcholine were formed, respectively. Alkaline hydrolysis gave glycerophosphorylcholine. The synthesis of phosphatidylcholine as the major compound was accompanied by the formation of lysophosphatidylcholine in smaller amounts. Cyanamide was found to be essential for the formation of phosphatidylcholine, and only traces of HCl, of the order of that required to convert the disodium phosphatidate to free phosphatidic acid were found necessary for the synthesis. This work suggests that phosphatidylcholine, which is an essential component of most biological membranes, could have been synthesized on the primitive Earth.
Peripheral nerve from experimentally diabetic rats exhibits lowered levels of myo-inositol (MI) and decreased incorporation of [3H]MI into phosphatidylinositol (PI) myo-Inositol (MI) has been suggested to play an important role in the development of experimental diabetic neuropathy (1-3). Mammalian tissues maintain millimolar concentrations of MI, which in peripheral nerve is at least 40-fold higher than in plasma (4, 5). It has long been known that MI content is reduced substantially in peripheral nerve from diabetic animals (1, 4, 6). Evidence from both diabetic nerve preparations and neural cell cultures grown in the presence of elevated medium glucose concentrations indicates that the depletion of cellular MI levels is associated with increased intracellular sorbitol, alterations of inositol phospholipid metabolism, and decreased Na+,K+-ATPase activity (2,7,8), which is critical for the maintenance of resting membrane potential. Impairment of this enzyme has been linked to reduced nerve conduction velocity that is characteristic of both human and experimental diabetes (2). Furthermore, supplementing the diet of diabetic animals with MI tends to restore the cyclitol level, as well as Na',K+-ATPase activity and nerve conduction velocity, to normal (1, 9, 10).There is evidence that both Na+,K+-ATPase activity and a component of phosphatidylinositol (PI) turnover in normal tissues are inhibited by incubation in MI-free medium (11,12). Consequently, reduced MI content in diabetic nerve could reflect depletion of the MI supply that specifically serves as substrate for PI metabolism, which in turn is integral to regulation of Na+,K+-ATPase activity.To investigate this possibility, we have used propranolol, which like several other cationic amphiphilic drugs markedly enhances accumulation of phosphatidyl CMP (CMP-PA) in a number of tissues including iris muscle, pineal gland, and pancreatic islets (13)(14)(15). This occurs because propranolol redirects the metabolism of phospholipids, primarily by inhibiting PA phosphatase (16,17), thereby decreasing the formation of phosphatidylcholine and phosphatidylethanolamine and enhancing the synthesis of PI and phosphatidylglycerol. In the presence of propranolol, the accumulation of CMP-PA, the immediate precursor of the latter two phospholipids, will be determined by its rate of biosynthesis relative to its rate of utilization. The consumption of the liponucleotide is governed by the availability of endogenous MI, since its appearance is prevented by inclusion of sufficient MI in the medium (15,16 9818The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
1. On fractionation of guinea-pig forebrain homogenates by differential and gradient-density centrifugation most of the polyphosphoinositides were recovered in the myelin-rich particles. 2. The phospholipids of pure preparations of myelin contained di- and tri-phosphoinositide in proportions 2-3 times greater than in the whole-brain phospholipids. 3. Di- and tri-phosphoinositide appeared in young rat brain during the period of myelination. 4. After the administration of [(32)P]phosphate to guinea pigs the labelling of the polyphosphoinositides in isolated pure myelin was as great as in the whole brain, whereas little synthesis of the other myelin phospholipids had occurred. 5. When brain subcellular fractions were incubated with [gamma-(32)P]ATP, some triphosphoinositide labelling occurred in the myelin-rich fraction whereas the active labelling of diphosphoinositide was localized mainly in the mitochondrial fraction. 6. The Na(+), K(+) and Mg(2+) plus Ca(2+) concentrations in purified myelin have been determined. The Mg(2+) plus Ca(2+) content present showed close acid-base equivalence to the polyphosphoinositides. 7. It is concluded that di- and tri-phosphoinositide are rapidly-metabolizing components of the myelin sheath or intimately associated structures.
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