Receptor-like properties of the 26 kDa transmembrane form of TNF

Most members of the TNF family of proteins exist as transmembrane proteins with relatively long intracellular domains, and a number of them are involved in the ill-defined phenomenon of "reverse signaling". We have identified a putative nuclear localization signal in the cytoplasmic domain of TNF which proved to be functional in two assays. Western analysis identified an approximately 10 kDa peptide corresponding to the transmembrane and cytoplasmic domains of TNF after the proteolytic liberation of the 17 kDa, soluble form of TNF. This 10 kDa peptide was enriched in internal membranes and nuclear fractions of disrupted cells. Immune electron-microscopic studies proved its localization in transport vesicles and the nucleus. The nuclear transport of the intracellular segment of TNF resembles the signaling process through the Notch-type of receptors. Indeed, the presence of the 10 kDa peptide seems to influence the expression of another inflammatory cytokine, interleukin-1 beta. These findings suggest that the transmembrane form of TNF has receptor-like properties and its interaction with the receptors initiates a bidirectional signaling.     

Medicago truncatula IPD3 is a member of the common symbiotic signaling pathway required for rhizobial and mycorrhizal symbioses

Legumes form endosymbiotic associations with nitrogen-fixing bacteria and arbuscular mycorrhizal (AM) fungi which facilitate nutrient uptake. Both symbiotic interactions require a molecular signal exchange between the plant and the symbiont, and this involves a conserved symbiosis (Sym) signaling pathway. In order to identify plant genes required for intracellular accommodation of nitrogen-fixing bacteria and AM fungi, we characterized Medicago truncatula symbiotic mutants defective for rhizobial infection of nodule cells and colonization of root cells by AM hyphae. Here, we describe mutants impaired in the interacting protein of DMI3 (IPD3) gene, which has been identified earlier as an interacting partner of the calcium/calmodulin-dependent protein, a member of the Sym pathway. The ipd3 mutants are impaired in both rhizobial and mycorrhizal colonization and we show that IPD3 is necessary for appropriate Nod-factor-induced gene expression. This indicates that IPD3 is a member of the common Sym pathway. We observed differences in the severity of ipd3 mutants that appear to be the result of the genetic background. This supports the hypothesis that IPD3 function is partially redundant and, thus, additional genetic components must exist that have analogous functions to IPD3. This explains why mutations in an essential component of the Sym pathway have defects at late stages of the symbiotic interactions.     

Treatment with insulin inhibits poly(ADP-ribose)polymerase activation in a rat model of endotoxemia

In critically ill patients various conditions may lead to the activation of poly(ADP-ribose) polymerase (PARP). By promoting cellular energetic dysfunction, and by enhancing pro-inflammatory gene expression, PARP activation significantly contributes to the pathogenesis of shock. PARP activation is usually triggered by DNA strand breakage, which is typically the result of the overproduction of various reactive oxidant species. One of the pathophysiological conditions associated with PARP activation is hyperglycemia, where the reactive species are produced from the mitochondria and other cellular sources. In the present study we tested whether endotoxin-induced PARP activation and pro-inflammatory mediator production can be modified by insulin therapy. Rats subjected to bacterial lipopolysaccharide (LPS) with or without insulin co-treatment were studied. LPS-induced PARP activation in circulating lymphocytes was measured by flow cytometry, tumor necrosis factor alpha (TNF-alpha) production was measured by ELISA. The direct effect of insulin on the PARP activity of mononuclear leukocytes and human umbilical vein endothelial cells (HUVEC) in elevated glucose conditions was tested in vitro. LPS-induced significant hyperglycemic response activated PARP in circulating lymphocytes and induced TNF-alpha production. Insulin treatment prevented LPS-induced hyperglycemic response, blocked PARP activation and blunted LPS-induced TNF-alpha response. Insulin treatment caused a slight reduction in the PARP activity of mononuclear cells and HUVECs in vitro. We demonstrate that insulin treatment blocks LPS-induced PARP activation in vivo. We propose that this effect is mainly indirect, and occurs due to the prevention of stress induced hyperglycemia, with a direct cellular effect of insulin playing a potential minor supplemental role. The current findings may have significant implications in the context of the emerging concept of tight glycemic control and insulin treatment for critically ill patients.     

ON THE POSSIBILITY OF LYSOPHOSPHATIDE FORMATION DURING WALLERIAN DEGENERATION

—During an extensive decomposition of phospholipids, at the end of the second week of Wallerian degeneration, the decomposition of glycerophosphatides were studied in detail. In a degenerative process lasting for 2 weeks about one-third of the choline phosphatides, two-thirds of the ethanolamine phosphatides, one-third of the serine phosphatides and one-quarter of the inositol phosphatides, were destroyed. The amount of lysophosphatidylcholine decreased proportionally to the destruction of choline phosphatide. On the other hand, the amount of lysophosphatides formed from‘kephalin'-containing fatty aldehyde, during the marked destruction of these phospholipids, remained constant or increased to a small extent and its percentage distribution increased 2 or 3 times compared with other phospholipids. Ethanolamine phosphatides having a high fatty aldehyde content can be regarded as mainly responsible for the relative accumulation of lysophosphatides.     

Remodeling of phosphatidylglycerol in Synechocystis PCC6803

The phosphatidylglycerol deficient ΔpgsA mutant of Synechocystis PCC6803 provided a unique experimental system for investigating in vivo retailoring of exogenously added dioleoylphosphatidylglycerol in phosphatidylglycerol-depleted cells. Gas chromatographic analysis of fatty acid composition suggested that diacyl-phosphatidylglycerols were synthesized from the artificial synthetic precursor. The formation of new, retailored lipid species was confirmed by negative-ion electrospray ionization–Fourier-transform ion cyclotron resonance and ion trap tandem mass spectrometry. Various isomeric diacyl-phosphatidylglycerols were identified indicating transesterification of the exogenously added dioleoylphosphatidyl-glycerol at the sn-1 or sn-2 positions. Polyunsaturated fatty acids were incorporated selectively into the sn-1 position. Our experiments with Synechocystis PCC6803/ΔpgsA mutant cells demonstrated lipid remodeling in a prokaryotic photosynthetic bacterium. Our data suggest that the remodeling of diacylphosphatidylglycerol likely involves reactions catalyzed by phospholipase A₁ and A₂ or acyl-hydrolase, lysophosphatidylglycerol acyltransferase and acyl-lipid desaturases.     

Identification of thylakoid membrane thermal transitions in Synechocystis sp. PCC6803 photosynthetic mutants

We used differential scanning calorimetry (DSC) as a technique capable of identifying photosynthetic complexes on the basis of their calorimetric transitions. Annotation of thermal transitions was carried out with thylakoid membranes isolated from various photosynthetic mutants of Synechocystis sp. PCC6803. The thylakoid membranes exhibited seven major DSC bands between 40 and 85°C. The heat sorption curves were analyzed both by mathematical deconvolution of the overall endotherms and by a subsequent annealing procedure. The successive annealing procedure proved to be more reliable technique than mathematical deconvolution in assigning thermal transitions. The main DSC band, around 47°C, resulting from the high enthalpy change that corresponds to non-interacting complex of PSII, was assigned using the PSI-less/apcE(-) mutant cells. Another band around 68-70°C relates to the denaturation of PSII surrounded by other proteins of the photosynthetic complexes in wild type and PSI-less/apcE(-) cells. A further major transition found at 82-84°C corresponds to the PSI core complex of wild type and PSII-deficient BE cells. Other transition bands between 50-67 and 65-75°C are believed to relate to ATP synthase and cytochrome b(6)f, respectively. These thermal transitions were obtained with thylakoids isolated from PSI(-)/PSII(-) mutant cells. Some minor bands determined at 59 and 83-84°C correspond to an unknown complex and NADH dehydrogenase, respectively. These annotations were done by PSI-less/apcE(-) and PSI(-)/PSII(-) mutants.     

Phosphatidylglycerol Depletion Induces an Increase in Myxoxanthophyll Biosynthetic Activity in Synechocystis PCC6803 Cells

Phosphatidylglycerol (PG) depletion suppressed the oxygen-evolvingactivity of Synechocystis PCC6803 pgsA mutant cells. Shortageof PG led to decreased photosynthetic activity, which, similarto the effect of high light exposure, is likely to generatethe production of reactive oxygen species (ROS) or free radicals.Protection of the PG-depleted cells against light-induced damageincreased the echinenone and myxoxanthophyll content of thecells. The increased carotenoid content was localized in a solublefraction of the cells as well as in isolated thylakoid and cytoplasmicmembranes. The soluble carotenoid fraction contained carotenederivatives, which may bind to proteins. These carotene–proteincomplexes are similar to orange carotenoid protein that is involvedin yielding protection against free radicals and ROS. An increasein the content of myxoxanthophyll and echinenone upon PG depletionsuggests that PG depletion regulates the biosynthetic pathwayof specific carotenoids.