Effect of various solubilizers on angiotensinII receptors in bovine adrenocortical plasma membranes

Summary 3 monionic detergents, Triton X-100, Lubrol WX and NP-40, inhibited binding of [³H]-AT_II to bovine adrenocortical plasma membranes. This effect appeared to be direct and not due to solubilization of the AT_II receptor by these agents. Sodium deoxycholate and the chaotropic ions, ClO ₄ ^– and Br^–, produced effects similar to the nonionic detergents.Supported by grants from the Quebec Heart Foundation and the Medical Research Council of Canada. The authors acknowledge the technical assistance of Mrs D. Michaud and the secretarial aide of Mrs D. Huot-Blais, and Miss L. Leblanc.     

Metabolism and signaling activities of nuclear lipids

Apart from the lipids present in the nuclear envelope, the nucleus also contains lipids which are located further inside and are resistant to treatment with nonionic detergents. Evidence is being accumulated on the importance of internal nuclear lipid metabolism. Nuclear lipid metabolism gives rise to several lipid second messengers that function within the nucleus. Moreover, it is beginning to emerge that nuclear lipids not only act as precursors of bioactive second messengers but may be directly involved in regulation of nuclear structure and gene expression. Over the last 10years, especially the role of the inositol lipid cycle in nuclear signal transduction has been extensively studied. This cycle is activated following a variety of stimuli and is regulated independently from the inositide cycle located at the plasma membrane. However, the nucleus contain other lipids, such as phosphatidylcholine, sphingomyelin, fatty acids and eicosanoids. There are numerous reports which suggest that these classes of nuclear lipids may play roles in the nucleus as important as those of phosphoinositides. This review aims at highlighting the most important aspects regarding the metabolism and signaling activities of nuclear phosphatidylcholine, sphingomyelin, fatty acids and eicosanoids.Received 7 November 2003; received after revision 18 December 2003; accepted 29 December 2003     

The effects of 4 detergents on the respiration of mitochondria isolated from rat liver]

The effects of these four detergents (two non-ionic: Titron X100 and Tween 80, two ionic: sodium cholate and sodium dedeoxycholate) upon the respiratory intensities of mitochondria and upon the ADP/0 and respiratory control ratios were observed. At low concentrations and in the absence of exogenous ADP, non ionic as well as ionic detergents provoked a threefold (or fourfold) increase of the respiratory intensities of mitochondria. At higher concentrations, the four detergents were inhibitory for mitochondrial oxidations in the order: Triton X 100 greater than DOC greater than cholate greater than Tween 80. At increasing doses, the four detergents progressively decreased the phosphorylating capacities of mitochondria.     

Membrane lipids and electron transfer. Effects of four detergents on NADH-ferricyanide reductase and NADH-cytochrome c reductase activities of potato tuber microsomes]

The NADH-ferricyanure reductase activity of Potato microsomes is stimulated by non ionic detergents (Triton X100 and Tween80) and is partially inhibited by ionic detergents (sodium-cholate and deoxycholate). All these four detergents progressively decreased the NADH-cytochrome c reductase in the following order: sodium deoxycholate greater than Triton X100 greater than sodium cholate greater than Tween80.     

The activity of Newcastle disease virus-envelope proteins after treatment with detergents.

Treatment of NDV with anionic detergents or lipid solvents destroys the activities of hemagglutinin and neuraminidase. After disruption of the virus with non-ionic detergents, the activities of envelope proteins remain unchanged. It is suggested that the phosholipids are very important for the biological activity of NDV-envelope proteins.     

The quantitative nature of the reaction between aminoglycoside and polymyxin class antibiotics with polyanionic detergents

Summary Using classical tube and gel precipitation techniques aminoglycoside and polymyxin class antibiotics were found to quantitatively react with polyanionic detergents.     

Purification of a protein binding quinacrine and histrionicotoxin from membrane fragments rich in cholinergic receptors in Torpedo marmorata]

A protein is purified by differential centrifugation from membrane fragments rich in acetylcholine receptor prepared from Torpedo marmorata electric organ after dissolution by a mixture of non denaturing detergents. After polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and Coomassie blue staining the purified protein yields a single band of apparent molecular weight 43,000. Spectroscopic experiments carried out in the absence of Ca++ and detergents reveal that the 43 K protein interacts with the fluorescent local anesthetic quinacrine and with the frog toxin histrionicotoxin (apparent KD : 7 X 10(-7) M) but not with carbamylcholine and the alpha toxin from N. nigricollis.     

Amphipols: polymeric surfactants for membrane biology research

Membrane proteins classically are handled in aqueous solutions as complexes with detergents. The dissociating character of detergents, combined with the need to maintain an excess of them, frequently results in more or less rapid inactivation of the protein under study. Over the past few years, we have endeavored to develop a novel family of surfactants, dubbed amphipols (APs). APs are amphiphilic polymers that bind to the transmembrane surface of the protein in a noncovalent but, in the absence of a competing surfactant, quasi-irreversible manner. Membrane proteins complexed by APs are in their native state, stable, and they remain water-soluble in the absence of detergent or free APs. An update is presented of the current knowledge about these compounds and their demonstrated or putative uses in membrane biology.     

Über den Einfluss von Alkylbenzolsulfonaten auf Froschlarven

Summary The toxicity of two important detergents was studied in tadpoles. Dodecyl benzenesulphonate was found to be much more toxic than tetrapropylene benzenesulphonate; the median lethal concentrations are 7.6 and 21.6 mg/l respectively.     

Purification of a membrane protein modifying the sensitivity of Na+/K+ ATPase to ouabain from a supernatant of plasma membrane treated with EDTA]

A membrane protein of M. W. 32,000 D and pI 5 has been purified from EDTA-treated plasma membrane supernatants by Page without detergents. These membranes are purified from the MF2S cell line issued from the murine plasmacytoma MOPC 173. The purified protein was able to induce a 300 fold increase in the Na+/K+ ATPase resistance to ouaba?n thus mimicking the original resistance of the enzyme to the drug.     

Evidence of protein kinase activity in 2 murine oncornaviruses]

A protein kinase activity has been detected in two strains of murine Oncornaviruses, MSV/MLV and EFV. This activity phosphorylates not only endogenous viral proteins but also exogenous substrates (histones and phosvitin). The stimulation of enzyme activity by detergents along with the increase of specific activity in viruses treated with trypsin during purification suggest that the enzyme is located in the viral particle.     

Induction of proteolytic activity in serum by treatment with anionic detergents and organic solvents

Using casein plates as a sensitive assay for proteolytic activity, it was observed that sodium-dodecyl sulfate (SDS) and other anionic detergents induce caseinolysis when mixed with sera and plasma. Caseinolysis was dependent on the presence of plasminogen in the fluids and could be blocked by inhibitors of serine proteases and antibody to plasminogen. Similarly, organic solvents such as isopropanol induced caseinolysis after mixing with plasma, but not normal serum. Isopropanol dissociated complexes of alpha 1-antitrypsin or alpha 2-macroglobulin with trypsin preformed in vitro. As both SDS and organic solvents are widely used in biochemical investigations of biological fluids, attention should be paid to the possible induction of proteolysis.     

Long-term changes in tyrosine phosphorylation of the abundant nuclear proteins during granulocytic differentiation of HL-60 cells

The two-dimensional electrophoretic patterns of nuclear proteins and their tyrosine phosphorylation were compared for HL-60 cells before and after differentiation induction to granulocytes by dimethyl sulfoxide, all-trans retinoic acid and N ⁶,O ²-dibutyryl adenosine 3′5′-cyclic monophosphate. Regardless of the inducer used, some nuclear proteins, which are tyrosine-phosphorylated in proliferating HL-60 cells, undergo gradual dephosphorylation 12–72 h after induction of differentiation, followed by drastic dephosphorylation during maturation to granulocytes. At least 13 nuclear proteins with a molecular mass of 35–110 kDa are dephosphorylated, and 6 nuclear proteins undergo tyrosine phosphorylation. Analysis of the nuclear proteins differentially extracted by salt and detergents indicates that changes in their tyrosine phosphorylation during the maturation stage of differentiating granulocytes occur mainly in proteins which are abundant in nucleoplasm, chromatin and residual nuclear structures. The abundance of these proteins, residing in the nuclear structures, and their long-term modification in phosphorylation during the maturation stages of differentiation strongly suggest that tyrosine phosphorylation of these proteins is involved in reorganization of the differentiating cell nucleus. Received 21 September 1998; received after revision 24 November 1998; accepted 3 December 1998     

The regulation of trehalose metabolism in insects

Trehalose is a non-reducing disaccharide comprising two glucose molecules. It is present in high concentration as the main haemolymph (blood) sugar in insects. The synthesis of trehalose in the fat body (an organ analogous in function to a combination of liver and adipose tissue in vertebrates) is stimulated by neuropeptides (hypertrehalosaemic hormones), released from the corpora cardiaca, a neurohaemal organ associated with the brain. The peptides cause a decrease in the content of fructose 2,6-bisphosphate in fat body cells. Fructose 2,6-bisphosphate, acting synergistically with AMP, is a potent activator of the glycolytic enzyme 6-phosphofructokinase-1 and a strong inhibitor of the gluconeogenic enzyme fructose 1,6-bisphosphatase. This indicates that fructose 2,6-bisphosphate is a key metabolic signal in the regulation of trehalose synthesis in insects. Trehalose is hydrolysed by trehalase (E.C. 3.2.1.28). The activity of this enzyme is regulated in flight muscle, but the mechanism by which this is achieved is unknown. Trehalase from locust, flight muscle is a glycoprotein bound to membranes of the microsomal fraction. The enzyme can be activated by detergents in vitro and by short flight intervals in vivo, which indicates that changes in the membrane environment modulate trehalase activity under physiological conditions.     

A sensitive protein assay method using micro-titer plates

Summary A commercially available protein-assay, based on the reaction between bicinchoninic acid (4,4-dicarboxy-2,2-biquinoline) and copper was adapted for use in microtiter plates. The assay is sensitive between 10 m/ml and several mg/ml, and it uses only 10 l of sample material. The results obtained with the method were in accordance with other protein determination methods. The assay was shown to be reproducible, reliable and insensitive to non-ionic detergents.Acknowledgments. The authors are grateful to Miss Barbara Moser who skillfully performed a number of these experiments. This research was supported by the Swiss National Science Foundation, Grant No 3.300–0.82, to U.B.     

ABCB4 missense mutations D243A,K435T,G535D,I490T,R545C,and S978P significantly impair the lipid floppase and likely predispose to secondary pathologies in the human population

Bile salts are natural detergents required to solubilise dietary fat and lipid soluble vitamins. They are synthesised in hepatocytes and secreted into the luminal space of the biliary tree by the bile salt export pump (BSEP), an ATP-binding cassette (ABC) transporter in the canalicular membrane. BSEP deficiency causes cytotoxic accumulation of bile salts in the hepatocyte that results in mild-to-severe forms of cholestasis. The resulting inflammation can also progress to hepatocellular cancer via a novel mechanism involving upregulation of proliferative signalling pathways. A second ABC transporter of the canalicular membrane is also critical for bile formation. ABCB4 flops phosphatidylcholine into the outer leaflet of the membrane to be extracted by bile salts in the canalicular space. These mixed micelles reduce the detergent action of the bile salts and protect the biliary tree from their cytotoxic activity. ABCB4 deficiency also causes cholestasis, and might be expected to cause cholangitis and predispose to liver cancer. Non-synonymous SNPs in ABCB4 have now been described in patients with liver cancer or with inflammatory liver diseases that are known to predispose to cancer, but data showing that the SNPs are sufficiently deleterious to be an etiological factor are lacking. Here, we report the first characterisation at the protein level of six ABCB4 variants (D243A, K435T, G535D, I490T, R545C, and S978P) previously found in patients with inflammatory liver diseases or liver cancer. All significantly impair the transporter with a range of phenotypes exhibited, including low abundance, intracellular retention, and reduced floppase activity, suggesting that ABCB4 deficiency is the root cause of the pathology in these cases.     

Requirements for leukocyte transmigration via the transmembrane chemokine CX3CL1

The surface-expressed transmembrane CX3C chemokine ligand 1 (CX3CL1/fractalkine) induces firm adhesion of leukocytes expressing its receptor CX3CR1. After shedding by the disintegrins and metalloproteinases (ADAM) 10 and 17, CX3CL1 also acts as soluble leukocyte chemoattractant. Here, we demonstrate that transmembrane CX3CL1 expressed on both endothelial and epithelial cells induces leukocyte transmigration. To investigate the underlying mechanism, we generated CX3CR1 variants lacking the intracellular aspartate-arginine-tyrosine (DRY) motif or the intracellular C-terminus which led to a defect in intracellular calcium response and impaired ligand uptake, respectively. While both variants effectively mediated firm cell adhesion, they failed to induce transmigration and rather mediated retention of leukocytes on the CX3CL1-expressing cell layer. Targeting of ADAM10 led to increased adhesion but reduced transmigration in response to transmembrane CX3CL1, while transmigration towards soluble CX3CL1 was not affected. Thus, transmembrane CX3CL1 mediates leukocyte transmigration via the DRY motif and C-terminus of CX3CR1 and the activity of ADAM10.     

E-cadherin and plakoglobin recruit plakophilin3 to the cell border to initiate desmosome assembly

A decrease in the levels of the desmosomal plaque protein, plakophilin3 (PKP3), leads to a decrease in desmosome size and cell-cell adhesion. To test the hypothesis that PKP3 is required for desmosome formation, the recruitment of desmosomal components to the cell surface was studied in the PKP3 knockdown clones. The PKP3 knockdown clones showed decreased cell border staining for multiple desmosomal proteins, when compared to vector controls, and did not form desmosomes in a calcium switch assay. Further analysis demonstrated that PKP3, plakoglobin (PG) and E-cadherin are present at the cell border at low concentrations of calcium. Loss of either PG or E-cadherin led to a decrease in the levels of PKP3 and other desmosomal proteins at the cell border. The results reported here are consistent with the model that PG and E-cadherin recruit PKP3 to the cell border to initiate desmosome formation.     

Death receptor 3 mediates necroptotic cell death

Death receptor 3 (DR3) was initially identified as a T cell co-stimulatory and pro-inflammatory molecule, but further studies revealed a more complex role of DR3 and its ligand TL1A. Although being a death receptor, DR3 gained to date predominantly attention as a contributor to inflammation-driven diseases. In our study, we investigated the cell death pathways associated with DR3. We show that in addition to apoptosis, DR3 can robustly trigger necroptotic cell death and provide evidence for TL1A-induced, DR3-mediated necrosome assembly. DR3-mediated necroptosis critically depends on receptor-interacting protein 1 (RIP1) and RIP3, the core components of the necroptotic machinery, which activate the pseudo-kinase mixed lineage kinase domain-like, the prototypic downstream effector molecule of necroptosis. Moreover, we demonstrate that DR3-mediated necroptotic cell death is accompanied by, but does not depend on generation of reactive oxygen species. In sum, we identify DR3 as a novel necroptosis-inducing death receptor and thereby lay ground for elucidating the (patho-) physiological relevance of DR3-mediated necroptotic cell death in vitro and in vivo.