Arachidonate incorporation into phospholipids in rat brain: A comparison between slice and membrane preparation

The incorporation of [³H]arachidonic acid ([³H]AA) into cerebral phospholipids was studied in slices and membranes of rat brain cortex. Effects of preincubation, different washing procedures with or without bovine serum albumin, or incubation in the presence of neurotransmitters were investigated. Over 60% of the phospholipid-bound [³H]arachidonic acid was recovered in phosphatidylinositol in both slice and membrane preparations. Preincubation or addition of bovine serum albumin resulted in slower incorporation of the isotope to membrane as well as to slice phospholipids, but removal of the added serum albumin enhanced the incorporation in the membrane preparation. Preincubation of slices in the presence of norepinephrine (1 mM) or serotonin (1 or 2.5 mM) resulted in a marked increase of [³H]arachidonate incorporation into phosphatidylinositol concomitant with decreased incorporation into other phosopholipids. Analysis of [³H]arachidonate incorporation as a function of time, in accordance with a compartmental model, indicated that observed differences in the accumulation of [³H]arachidonate in various phospholipids were probably due to different rates of acylation by arachidonyl-transferases with no appreciable changes in the rates of deacylation by enzymes of the phospholipase A type. Further, the pool of phospholipid-bound arachidonate which is readily available for deacylation-reacylation processes appears to be distributed differentially among various phospholipids with more than 50% of this pool located in phosphatidylinositol and only 0.75% of the phosphatidylinositol molecules appear to be active in deacylation-reacylation processes involving arachidonyl groups.     

Effects of chronic ethanol administration and withdrawal on incorporation of arachidonate into membrane phospholipids

A plasma membrane fraction isolated from cerebral cortex of control and ethanol-treated rats was used to study the effects of chronic ethanol administration on uptake of arachidonate by membrane phospholipids. Upon incubation of the membranes with [¹⁴C] arachidonic acid in the presence of ATP, Mg²⁺, and CoA, radioactivity was incorporated into all of the phospholipids, although a large proportion of the label was found in phosphatidylinositols (PI, 60%) and phosphatidylcholines (PC, 20%). Rats given ethanol (8–10 g/kg body wt) via intubation in the form of a liquid diet for 4 weeks showed an increase (17–20%) in arachidonate incorporation into PI and PC as compared to phosphatidylethanolamines (PE) and phosphatidylserines (PS). A similar increase in uptake activity was observed at 2 or 24 h upon withdrawal of ethanol, but uptake activity returned readily to that of control level by 72 h. The method described in this study is a sensitive and reliable procedure for monitoring the arachidonoyl turnover activity in neural membranes with respect to chronic ethanol induction and withdrawal.     

Pro-caspase-8 is predominantly localized in mitochondria and released into cytoplasm upon apoptotic stimulation

The recruitment and cleavage of pro-caspase-8 to produce the active form of caspase-8 is a critical biochemical event in death receptor-mediated apoptosis. However, the source of pro-caspase-8 available for activation by apoptotic triggers is unknown. In human fibroblasts and mouse clonal striatal cells, confocal microscopy revealed that pro-caspase-8 immunofluorescence was colocalized with cytochrome c in mitochondria and was also distributed diffusely in some nuclei. Biochemical analysis of subcellular fractions indicated that pro-caspase-8 was enriched in mitochondria and in nuclei. Pro-caspase-8 was found in the intermembrane space, inner membrane, and matrix of mitochondria after limited digestion of mitochondrial fractions, and this distribution was confirmed by immunogold electron microscopy. Pro-caspase-8 and cytochrome c were released from isolated mitochondria that were treated with an inhibitor of the ADP/ATP carrier atractyloside, which opens the mitochondria permeability transition pore. Release was blocked by the mitochondria permeability transition pore inhibitor cyclosporin A (CsA). After clonal striatal cells were exposed for 6 h to an apoptotic inducer tumor necrosis factor-alpha (TNF-alpha), mitochondria immunoreactive for cytochrome c and pro-caspase-8 became clustered at perinuclear sites. Pro-caspase-8 and cytochrome c levels decreased in mitochondrial fractions and increased, along with pro-caspase-8 cleavage products, in the cytoplasm of the TNF-alpha-treated striatal cells. CsA blocked the TNF-alpha-induced release of pro-caspase 8 but not cytochrome c. Internucleosomal DNA fragmentation started at 6 h and peaked 12 h after TNF-alpha treatment. These results suggest that pro-caspase-8 is predominantly localized in mitochondria and is released upon apoptotic stimulation through a CsA-sensitive mechanism.     

Purification and analysis of phospholipids in the inner mitochondrial membrane fraction of bovine corpus luteum, and properties of cytochrome P-450scc incorporated into vesicles prepared from these phospholipids

Cytochrome P-450scc, which catalyses the conversion of cholesterol to pregnenolone in steroidogenic tissues, can be incorporated into artificial phospholipid vesicles and cholesterol binding to the cytochrome is affected by the composition of the vesicles. We have purified the phospholipids from the inner mitochondrial membrane fraction of the bovine corpus luteum where the cytochrome is located. The composition in mol % was 49% phosphatidylcholine, 34% phosphatidylethanolamine, 8.7% cardiolipin, 6.4% lysophosphatidylethanolamine and 1.5% phosphatidylinositol. The ratio of cholesterol to phospholipid (mol/mol) in the inner membrane fraction was 0.14 to 1. The Km for cholesterol of purified luteal cytochrome P-450scc incorporated into vesicles prepared from the total inner mitochondrial membrane phospholipids was 0.063 mol of cholesterol per mol of phospholipid. Removal of the cardiolipin component of the inner mitochondrial membrane phospholipids prior to preparation of vesicles caused a four fold increase in the Kd of cytochrome P-450 for cholesterol and a two fold increase in Km. The data suggests that in the inner mitochondrial membrane of the bovine corpus luteum the cholesterol concentration is less than saturating for cytochrome P-450scc.     

The meso-angioblast: a multipotent,self-renewing cell that originates from the dorsal aorta and differentiates into most mesodermal tissues

We have previously reported the origin of a class of skeletal myogenic cells from explants of dorsal aorta. This finding disagrees with the known origin of all skeletal muscle from somites and has therefore led us to investigate the in vivo origin of these cells and, moreover, whether their fate is restricted to skeletal muscle, as observed in vitro under the experimental conditions used. To address these issues, we grafted quail or mouse embryonic aorta into host chick embryos. Donor cells, initially incorporated into the host vessels, were later integrated into mesodermal tissues, including blood, cartilage, bone, smooth, skeletal and cardiac muscle. When expanded on a feeder layer of embryonic fibroblasts, the clonal progeny of a single cell from the mouse dorsal aorta acquired unlimited lifespan, expressed hemo-angioblastic markers (CD34, Flk1 and Kit) at both early and late passages, and maintained multipotency in culture or when transplanted into a chick embryo. We conclude that these newly identified vessel-associated stem cells, the meso-angioblasts, participate in postembryonic development of the mesoderm, and we speculate that postnatal mesodermal stem cells may be derived from a vascular developmental origin.     

Glycoprotein M of herpes simplex virus 1 is incorporated into virions during budding at the inner nuclear membrane

It is widely accepted that nucleocapsids of herpesviruses bud through the inner nuclear membrane (INM), but few studies have been undertaken to characterize the composition of these nascent virions. Such knowledge would shed light on the budding reaction at the INM and subsequent steps in the egress pathway. The present study focuses on glycoprotein M (gM), a type III integral membrane protein of herpes simplex virus 1 (HSV-1) that likely contains eight transmembrane domains. The results indicated that gM localized primarily at the perinuclear region, with especially bright staining near the nuclear membrane (NM). Immunogold electron microscopic analysis indicated that, like gB and gD (M. R. Torrisi et al., J. Virol. 66:554-561, 1992), gM localized within both leaflets of the NM, the envelopes of nascent virions that accumulate in the perinuclear space, and the envelopes of cytoplasmic and mature extracellular virus particles. Indirect immunofluorescence studies revealed that gM colocalized almost completely with a marker of the Golgi apparatus and partially with a marker of the trans-Golgi network (TGN), whether or not these markers were displaced to the perinuclear region during infection. gM was also located in punctate extensions and invaginations of the NM induced by the absence of a viral kinase encoded by HSV-1 U(S)3 and within virions located in these extensions. Our findings therefore support the proposition that gM, like gB and gD, becomes incorporated into the virion envelope upon budding through the INM. The localization of viral glycoproteins and Golgi and TGN markers to a perinuclear region may represent a mechanism to facilitate the production of infectious nascent virions, thereby increasing the amount of infectivity released upon cellular lysis.     

Correlation between endogenous noradrenaline and glucose released from the liver upon hepatic sympathetic nerve stimulation in anesthetized dogs

The metabolic role of neurally released noradrenaline (NA) was studied in the liver of anesthetized dogs. Sustained stimulation with various frequencies was directly applied on the anterior plexus of hepatic nerves. Stimulation-induced changes in plasma concentrations of endogenous catecholamines in hepatic venous blood were determined in correlation with concomitant changes in those of glucose (GL). Mean basal values for hepatic venous NA, adrenaline, dopamine, and GL were 0.062, 0.022, 0.032 ng/mL, and 97.9 mg%, respectively. Among these catecholamines, NA was the only one being released significantly during stimulation. While hepatic venous NA increased rapidly during stimulation, being maximum within 3 min, hepatic venous GL increased gradually, reaching a maximum value 5 min after the onset of stimulation. A highly significant correlation (r = 0.90, P less than 0.001) was found between changes in hepatic venous NA and GL concentrations observed during stimulation at various frequencies (2-16 Hz). However, hepatic vasoconstricting responses to stimulation were not correlated with increased hepatic venous GL. An alpha-blockade with phentolamine (2 mg/kg, iv) resulted in diminished release of GL by approximately 50% (P less than 0.05) and reduced hepatic arterial vasoconstriction by approximately 47% (P less than 0.01) upon stimulation (8 Hz, 5 min), even though NA release was markedly enhanced. We conclude that in the dog, NA is the sole catecholamine released within the liver in response to direct hepatic nerve stimulation, and NA thus released mediates the hepatic glycogenolysis via alpha-adrenoceptors.     

The adenosine 2b receptor is recruited to the plasma membrane and associates with E3KARP and Ezrin upon agonist stimulation

We have previously shown that adenosine is formed in the intestinal lumen during active inflammation from neutrophil-derived 5'-AMP. Acting through the adenosine A2b receptor (A2bR), the luminally derived adenosine induces vectorial chloride secretion and a polarized secretion of interleukin-6 to the intestinal lumen. Although some G protein-coupled receptors interact with anchoring or signaling molecules, not much is known in this critical area for the A2bR. We used the model intestinal epithelial cell line, T84, and Caco2-BBE cells stably transfected with GFP-A2b receptor to study the intestinal A2bR. The A2bR is present in both the apical and basolateral membranes of intestinal epithelia. Apical or basolateral stimulation of the A2bR induces recruitment of the receptor to the plasma membrane and caveolar fractions. The A2bR co-immunoprecipitates with E3KARP and ezrin upon agonist stimulation. Ezrin interacts with E3KARP and PKA and the interaction between ezrin and E3KARP is enhanced by agonist stimulation. Our data suggest that the A2bR is recruited to the plasma membrane upon apical or basolateral agonist stimulation and interacts with E3KARP and ezrin. We speculate that such an interaction may not only anchor the A2bR to the plasma membrane but may also function to stabilize the receptor in a signaling complex in the plasma membrane.     

Vinculin is a permanent component of the membrane skeleton and is incorporated into the (re)organising cytoskeleton upon platelet activation

Vinculin, a 130-kDa protein discovered in chicken gizzard smooth-muscle cells and subsequently also described in platelets, is believed to be involved in membrane-cytoskeleton interactions. In this study we investigated vinculin distribution in human blood platelets. Two skeletal fractions and a remaining cytosolic fraction were prepared with a recently described Triton X-100 lysis buffer causing minimal post-lysis breakdown by proteolysis. The presence of vinculin was demonstrated in the membrane skeleton and cytosol of resting and thrombin-activated human platelets. Upon thrombin stimulation vinculin also appeared in the cytoskeleton. this cytoskeletal incorporation was completed during the early stages of platelet aggregation and secretion, when the uptake of myosin, actin-binding protein and talin was still not maximal. We conclude therefore, that vinculin may play an important role in the structural (re)organisation of the human platelet cytoskeleton upon platelet activation.     

A cytochrome c-GFP fusion is not released from mitochondria into the cytoplasm upon expression of Bax in yeast cells

To study Bax-induced release of cytochrome c in vivo, we have expressed a cytochrome c-GFP (green fluorescent protein) fusion in Saccharomyces cerevisiae cells null for the expression of the endogenous cytochrome. We show here that cytochrome c-GFP is efficiently localised to mitochondria and able to function as an electron carrier between complexes III and IV of the respiratory chain. Strikingly, while natural cytochrome c is released into the cytoplasm upon expression of Bax, the cytochrome c-GFP fusion is not. Nevertheless, cells co-expressing Bax and the cytochrome c-GFP fusion die, indicating that mitochondrial release of cytochrome c is not essential for cell death to occur in yeast. The failure to release cytochrome c-GFP is presumed to arise from increased bulk due to the GFP moiety. We propose that in intact yeast cells, Bax-induced release of cytochrome c into the cytoplasm occurs through a selective pore and not as a consequence of the non-specific breakage of the mitochondrial outer membrane.     

Unfolded cholera toxin is transferred to the ER membrane and released from protein disulfide isomerase upon oxidation by Ero1

The toxic effect of cholera toxin (CT) on target cells is caused by its A1 chain. This polypeptide is released from the holotoxin and unfolded in the lumen of the ER by the action of protein disulfide isomerase (PDI), before being retrotranslocated into the cytosol. The polypeptide is initially unfolded by binding to the reduced form of PDI. We show that upon oxidation of the COOH-terminal disulfide bond in PDI by the enzyme Ero1, the A1 chain is released. Both yeast Ero1 and the mammalian Ero1alpha isoform are active in this reaction. Ero1 has a preference for the PDI-toxin complex. We further show that the complex is transferred to a protein at the lumenal side of the ER membrane, where the unfolded toxin is released from PDI by the action of Ero1. Taken together, our results identify Ero1 as the enzyme mediating the release of unfolded CT from PDI and characterize an additional step in retrotranslocation of the toxin.     

Phospholipid metabolism of stimulated lymphocytes: Preferential incorporation of polyunsaturated fatty acids into plasma membrane phospholipid upon stimulation with concanavalin A

Rabbit thymocytes were isolated and incubated for various lengths of time with concanavalin A. The cultures were pulsed for the last 12.5 min of incubation with equimolar mixtures of radioactively labelled fatty acids, either [³H]arachidonate and [¹⁴C]oleate or [³H]arachidonate and [¹⁴C]palmitate, and the uptake of each fatty acid into phospholipid of plasma membrane was determined. Upon binding of the mitogen, the fatty acids were incorporated at an increased rate with a new steady state being reached between 12.5 and 42.5 min after stimulation. Initially after 12.5 min, when the two fatty acids were added together, no preferential incorporation of the polyunsaturated fatty acid arachidonate was seen compared to the saturated or monounsaturated ones, palmitate or oleate. However shortly thereafter arachidonate, when compared to palmitate or oleate, started to be preferentially incorporated into plasma membrane phospholipid so that by 4 h after activation, only arachidonate was incorporated at an increased rate: the uptake of palmitate and oleate had reverted to that of unstimulated cells. In contrast, when palmitate or oleate were added alone, after 4 h of activation incorporation was increased similar to that of arachidonate, suggesting that all long chain fatty acids compete for the same activated enzyme(s). A detailed analysis of incorporation into phospholipid species showed that all fatty acids were taken up with the highest rate into phosphatidylcholine. After activation, fatty acid incorporation was increased by approx. 50% for phosphatidylcholine: the highest stimulation rates were observed with phosphatidylinositol (3–7-fold) and phosphatidylethanolamine (2–3-fold). The data suggest that shortly after stimulation with mitogens, the membrane phospholipids start to change by replacing saturated and monounsaturated fatty acids by polyunsaturated ones, thus creating a new membrane.     

Phospholipid metabolism of stimulated lymphocytes. Preferential incorporation of polyunsaturated fatty acids into plasma membrane phospholipid upon stimulation with concanavalin A

Rabbit thymocytes were isolated and incubated for various lengths of time with concanavalin A. The cultures were pulsed for the last 12.5 min of incubation with equimolar mixtures of radioactively labelled fatty acids, either [3H]arachidonate and [14C]oleate or [3H]arachidonate and [14C]palmitate, and the uptake of each fatty acid into phospholipid of plasma membrane was determined. Upon binding of the mitogen, the fatty acids were incorporated at an increased rate with a new steady state being reached between 12.5 and 42.5 min after stimulation. Initially after 12.5 min, when the two fatty acids were added together, no preferential incorporation of the polyunsaturated fatty acid arachidonate was seen compared to the saturated or monounsaturated ones, palmitate or oleate. However shortly thereafter arachidonate, when compared to palmitate or oleate, started to be preferentially incorporated into plasma membrane phospholipid so that by 4 h after activation, only arachidonate was incorporated at an increased rate: the uptake of palmitate and oleate had reverted to that of unstimulated cells. In contrast, when palmitate or oleate were added alone, after 4 h of activation incorporation was increased similar to that of arachidonate, suggesting that all long chain fatty acids compete for the same activated enzyme(s). A detailed analysis of incorporation into phospholipid species showed that all fatty acids were taken up with the highest rate into phosphatidylcholine. After activation, fatty acid incorporation was increased by approx. 50% for phosphatidylcholine: the highest stimulation rates were observed with phosphatidylinositol (3-7-fold) and phosphatidylethanolamine (2-3-fold). The data suggest that shortly after stimulation with mitogens, the membrane phospholipids start to change by replacing saturated and monounsaturated fatty acids by polyunsaturated ones, thus creating a new membrane.     

Quantitative studies on translocation and metabolic conversion of lysophosphatidylcholine incorporated into the membrane of intact human erythrocytes from the medium

The fate of palmitoyl-lysophosphatidylcholine (lysoPC) incorporated into the membrane of intact human erythrocytes from a medium was investigated under nonhemolytic conditions at 37 degrees C by means of 14C-labeled tracers. The lysoPC was first incorporated into the outer half of the membrane lipid bilayer and then gradually translocated into the inner half during the incubation. At the same time it was metabolically converted into phosphatidylcholine (PC) and free fatty acid (FFA) plus glycerophosphorylcholine by the actions of acyltransferase and lysophospholipase, respectively. The half times of the conversion were about 14 h, while the value of 0.5 h was obtained when the half time was measured with the hemolysate of the lysoPC-loaded erythrocytes. Chymotrypsin treatment of unsealed ghosts caused a definite decrease in lysophospholipase activity, while similar treatment of resealed ghosts did not. This together with other evidence already reported in the literature suggests that both lysophospholipase and acyltransferase may be located in the inner surface of the membrane. The above findings strongly suggest that the most of the lysoPC loaded to the membrane is gradually translocated from the outer to the inner half of the bilayer and soon converted to either PC or FFA.     

Incorporation of the nuclear pore basket protein nup153 into nuclear pore structures is dependent upon lamina assembly: evidence from cell-free extracts of Xenopus eggs

In cell-free extracts of Xenopus eggs that support the assembly of replication-competent nuclei, we found that lamin B(3) specifically associates with four polypeptides (termed SLAPs, soluble lamin associated proteins). Here, one SLAP is identified as the nuclear pore complex protein Nup153, one member of the F/GXFG motif-containing nucleoporins. In vitro translated Nup153 and lamin B(3) co-immunoprecipitate, and lamin B(3) interacts specifically with the C-terminal domain of Nup153. During nuclear envelope assembly, other F/GXFG-containing nucleoporins are incorporated into the nuclear envelope preceding lamina assembly. Incorporation of Nup153 occurs at the same time as lamina assembly. When lamina assembly is prevented using the dominant-negative mutant XlaminB delta 2+, Nup153 does not appear at the nuclear envelope, while other F/GXFG-containing nucleoporins and Nup93 are recruited normally. When the lamina of pre-assembled nuclei is disrupted using the same dominant-negative mutant, the distribution of other nucleoporins is unaffected. However, Nup153 recruitment at the nuclear envelope is lost. Our results indicate that both the recruitment and maintenance of Nup153 at the pore are dependent upon the integrity of the lamina.