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1.
Acta Anaesthesiol Scand ; 59(2): 238-45, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25496028

ABSTRACT

BACKGROUND: The spread of injectate during a saphenous nerve block at the adductor canal has not been clearly described. METHODS: We examined the spread of 15 ml dyed injectate during ultrasound-guided saphenous nerve blocks at the adductor canal in 15 unembalmed cadavers' lower limbs followed by comparative dissections of the same limbs. RESULTS: The spread of the injectates was determined by the fascial limits and the muscles surrounding the adductor canal. The anteromedial limit of the adductor canal (the roof) was found to be a continuous fascia, with a thin proximal part and a thicker distal part (the vastoadductor membrane) covering the canal from the apex of the femoral triangle to the adductor hiatus. The fascial limits of the adductor canal formed a conduit around the femoral neurovascular bundle. The dyed aqueous injectate spread throughout the entire adductor canal to the femoral triangle and reached 1-2 cm into the popliteal fossa. Injections superficial to the adductor canal spread over the femoral artery within the subsartorial fat compartment resembling the injections within the canal but with ultrasonographic distinct features. These injections spread only half the length of the adductor canal. The only nerve observed within the adductor canal was the saphenous nerve. CONCLUSIONS: Injection of 15 ml dye was sufficient to spread throughout the adductor canal and beyond both proximally and distally. Distinct ultrasonographic features could be identified separating a subsartorial injection from an injection within the adductor canal with consequent differences in the spread.


Subject(s)
Nerve Block , Ultrasonography, Interventional , Aged , Aged, 80 and over , Cadaver , Contrast Media , Female , Humans , Leg/diagnostic imaging , Leg/innervation , Male
2.
Biochim Biophys Acta ; 1717(1): 34-40, 2005 Nov 10.
Article in English | MEDLINE | ID: mdl-16236245

ABSTRACT

Cav-p60, a specific and ubiquitous caveolar protein, was immunoprecipitated from solubilized rat adipocyte plasma membranes and identified as similar to a GeneBank entry annotated mouse polymerase transcript release factor (PTRF) by MALDI-TOF and MS-MS of major fragments. Cloning and virtual translation of the corresponding rat adipocyte cDNA sequence revealed 98.7% identity with mouse PTRF. In vitro translation of this sequence produced a protein, which was recognized by antibodies to both cav-p60 and PTRF. EM gold labeling studies showed that a rabbit antiserum against murine PTRF immunolabeled caveolae specifically in adipocytes from both mouse and rat. In view of the reported function of the protein, which is exerted in the cell nucleus, its subcellular localization was investigated. We found that the protein could be purified by differential solubilization of a plasma membrane fraction followed by SDS-PAGE, and that the protein was as abundant as caveolin in this fraction. We were unable to detect the protein in cell nuclei by subcellular fractionation or fluorescence microscopy. The results show that in a large number of cell types, PTRF is essentially located to caveolae, and that each caveola harbors many copies of the protein. Consequently, we suggest the name Cavin for this protein.


Subject(s)
Caveolae/chemistry , Caveolins/chemistry , Cytosol/chemistry , Membrane Proteins/chemistry , Adipocytes/ultrastructure , Amino Acid Sequence , Animals , Cell Membrane/ultrastructure , Humans , Immunoprecipitation , Male , Mass Spectrometry , Membrane Proteins/genetics , Membrane Proteins/isolation & purification , Mice , Microscopy, Fluorescence , Molecular Sequence Data , Protein Biosynthesis , RNA-Binding Proteins/analysis , Rats , Rats, Wistar , Sequence Alignment
3.
in Portuguese | DANTEPAZZANESE, SESSP-IDPCACERVO | ID: dan-1798

Subject(s)
Diagnosis/methods , Anatomy
4.
Acta Physiol Scand ; 179(2): 179-88, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14510782

ABSTRACT

AIMS: In vascular smooth muscle cells caveolae are important for signalling mechanisms regulating vascular contraction. In smooth muscle layer of the renal afferent arteriole juxtaglomerular cells (JG cells) are non-contractile renin producing cells that have the capacity to change their phenotype into smooth muscle cells and back again by metaplastic transformation. Signalling mechanisms in JG cells are not fully understood and we therefore investigated if caveolae were present, and thereby could be involved as integrators of cellular signalling in both of these phenotypes of smooth muscle cells. METHODS: Using electron microscopy we compared the number of caveolae in JG cells and smooth muscle cells in the afferent arteriole of the rat kidney. The expression of caveolin and cav-p60 was examined using a combination of immunogold electron microscopy and immunofluorescence microscopy. RESULTS: We found that JG cells have sixfold less caveolae per cell surface sectional length than smooth muscle cells. The expression of cavolin-1 and cav-p60 correlated with the number of caveolae. An examination of the general distribution of caveolae, cav-p60 and caveolins in the rat kidney showed that cav-p60, like caveolin-1, is a specific maker of caveolae. CONCLUSION: The number of caveolae in JG cells is very low, and this makes it unlikely that caveolae are of major importance for the renin secretion specific for JG cells.


Subject(s)
Caveolae/metabolism , Caveolins/analysis , Kidney/cytology , Muscle, Smooth, Vascular/cytology , Renin/biosynthesis , Animals , Blotting, Western/methods , Immunohistochemistry/methods , Kidney/metabolism , Kidney/ultrastructure , Male , Microscopy, Electron/methods , Microscopy, Fluorescence/methods , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/ultrastructure , Rats , Rats, Wistar
5.
Cell Tissue Res ; 306(2): 265-76, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11702238

ABSTRACT

Caveolae are plasmalemmal invaginations of uncertain function. In view of the large number of hypotheses on caveolar functions, it is important to identify which components of caveolae are tissue specific and which are general. The only well-characterized major protein of caveolae is caveolin, which exists in three tissue-specific isoforms: caveolin-1, -2, and -3. Recently cav-p60 was characterized as a 60-kDa caveola-specific protein in adipocytes. The distributions of cav-p60 and caveolin isoforms in different rat muscle tissues were examined by immunofluorescence and immunoelectron microscopy. Cav-p60 was present in caveolae of skeletal and heart muscle, in vascular and intestinal smooth muscle, and in adipocyte caveolae. Furthermore cav-p60 was present in endothelial cells and cells of perineural sheaths. Caveolin-1 and -2 were present in adipocytes, endothelial cells, and cells of perineural sheaths. In all kinds of vascular and intestinal smooth muscle, caveolin-1 and -2 were present at high levels, whereas caveolin-3 expression was low or undetectable, depending on the specific smooth muscle subtype. High levels of caveolin-3 were found only in caveolae and T tubules of skeletal and heart muscle. We conclude that cav-p60 is a highly specific marker of caveolae in many if not all cell types having caveolae.


Subject(s)
Caveolae/metabolism , Membrane Proteins/metabolism , Muscle, Skeletal/metabolism , Muscle, Smooth/metabolism , Myocardium/metabolism , Adipocytes/chemistry , Adipocytes/metabolism , Adipocytes/ultrastructure , Animals , Caveolae/chemistry , Caveolins/metabolism , Cell Fractionation , Cell Membrane/metabolism , Immunohistochemistry , Male , Muscle, Skeletal/chemistry , Muscle, Skeletal/ultrastructure , Muscle, Smooth/chemistry , Muscle, Smooth/cytology , Myocardium/chemistry , Myocardium/cytology , Protein Isoforms , Rats , Rats, Wistar
6.
Cell Tissue Res ; 305(1): 99-106, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11512676

ABSTRACT

To search for caveolar proteins, mice were immunised with rat adipocyte membranes. Hybridoma supernatants were screened for antibodies to proteins on the cytosolic face of caveolae by indirect immunoelectron microscopy of immunogold-labelled adipocyte plasma membrane sheets adsorbed on electron-microscope (EM) grids. One of the hybridoma supernatants (2F11) produced a specific labelling of caveolae which was much more intense than that obtained with caveolin-1 antibodies. In Western blots of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) separated proteins in crude membrane fractions from different rat tissues, 2F11 labelled a band corresponding to 60 kDa. The intensity of 2F11 labelling was high in adipose tissue and in other tissues varied in parallel to caveolin- labelling. In blots of plasma membrane (PM) and light-microsomal (LM) fractions from a homogenate of adipocytes, prior insulin stimulation of the adipocytes translocated GLUT-4 from the LM to the PM fraction, but was without effect on the distribution of the 60-kDa protein labelled by 2F11. Digestion with endoproteinase lys-C produced the same pattern of immunoreactive fragments of the protein in the vesicular PM and LM fractions, indicating similar membrane topology of the 2F11-reactive, 60-kDa protein in vesicles of PM and LM fractions.


Subject(s)
Adipocytes/chemistry , Caveolins/analysis , Muscle Proteins , Adipocytes/drug effects , Adipocytes/ultrastructure , Animals , Antibodies, Monoclonal , Blotting, Western , Caveolin 1 , Caveolins/immunology , Cell Fractionation , Cell Membrane/chemistry , Glucose Transporter Type 4 , Hybridomas , Hypoglycemic Agents/pharmacology , Insulin/pharmacology , Mice , Microscopy, Electron , Microscopy, Immunoelectron , Microsomes/chemistry , Monosaccharide Transport Proteins/analysis , Monosaccharide Transport Proteins/immunology , Rats , Rats, Wistar
7.
Glycobiology ; 11(6): 473-9, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11445552

ABSTRACT

Sulfatide is a glycolipid that has been associated with insulin-dependent diabetes mellitus. It is present in the islets of Langerhans and follows the same intracellular route as insulin. However, the role of sulfatide in the beta cell has been unclear. Here we present evidence suggesting that sulfatide promotes the folding of reduced proinsulin, indicating that sulfatide possesses molecular chaperone activity. Sulfatide associates with insulin by binding to the insulin domain A8--A10 and most likely by interacting with the hydrophobic side chains of the dimer-forming part of the insulin B-chain. Sulfatide has a dual effect on insulin. It substantially reduces deterioration of insulin hexamer crystals at pH 5.5, conferring stability comparable to those in beta cell granules. Sulfatide also mediates the conversion of insulin hexamers to the biological active monomers at neutral pH, the pH at the beta-cell surface. Finally, we report that inhibition of sulfatide synthesis with chloroquine and fumonisine B1 leads to inhibition of insulin granule formation in vivo. Our observations suggest that sulfatide plays a key role in the folding of proinsulin, in the maintenance of insulin structure, and in the monomerization process.


Subject(s)
Insulin/chemistry , Proinsulin/metabolism , Protein Folding , Sulfoglycosphingolipids/pharmacology , Animals , Crystallography , Humans , Insulin/biosynthesis , Models, Molecular , Proinsulin/drug effects , Protein Conformation , Swine
8.
Eur J Ultrasound ; 8(2): 129-33, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9845795

ABSTRACT

OBJECTIVE: The purpose of this study was to investigate the identification of the anterior glenoid labrum with ultrasonography. METHODS: Twelve cadaveric shoulders from subjects 74 to 85 years of age were examined by ultrasonography. Under ultrasonographic guidance, a small amount of dye was injected on the structure believed to be the anterior glenoid labrum, whereafter the shoulders were dissected. RESULTS: In 11 shoulders the dye was found in close relation to the glenoid labrum. Comparing the labrum to a watch face numbered clockwise in the left shoulder, and counter clockwise in the right, the part seen was located between 1 and 5 o'clock. Signs of degeneration were found in six labra by ultrasonography and verified by blindfolded dissection. CONCLUSIONS: Ultrasonography can identify the anterior glenoid labrum and distinguish degenerated from normal labra.


Subject(s)
Joint Capsule/diagnostic imaging , Shoulder Joint/diagnostic imaging , Aged , Aged, 80 and over , Arthroscopy , Cadaver , Female , Humans , Joint Capsule/pathology , Joint Instability/diagnostic imaging , Joint Instability/pathology , Male , Shoulder Joint/pathology , Ultrasonography
9.
EMBO J ; 17(6): 1598-605, 1998 Mar 16.
Article in English | MEDLINE | ID: mdl-9501081

ABSTRACT

Streptolysin O (SLO) is a bacterial exotoxin that binds to cell membranes containing cholesterol and then oligomerizes to form large pores. Along with rings, arc-shaped oligomers form on membranes. It has been suggested that each arc represents an incompletely assembled oligomer and constitutes a functional pore, faced on the opposite side by a free edge of the lipid membrane. We sought functional evidence in support of this idea by using an oligomerization-deficient, non-lytic mutant of SLO. This protein, which was created by chemical modification of a single mutant cysteine (T250C) with N-(iodoacetaminoethyl)-1-naphthylamine-5-sulfonic acid, formed hybrid oligomers with active SLO on membranes. However, incorporation of the modified T250C mutant inhibited subsequent oligomerization, so that the hybrid oligomers were reduced in size. These appeared as typical arc lesions in the electron microscope. They formed pores that permitted passage of NaCl and calcein but restricted permeation of large dextran molecules. The data indicate that the SLO pore is formed gradually during oligomerization, implying that pores lined by protein on one side and an edge of free lipid on the other may be created in the plasma membrane. Intentional manipulation of the pore size may extend the utility of SLO as a tool in cell biological experiments.


Subject(s)
Erythrocyte Membrane/chemistry , Streptolysins/chemistry , Animals , Bacterial Proteins , Cell Membrane Permeability , Complement Hemolytic Activity Assay , Cysteine/chemistry , Erythrocyte Membrane/metabolism , Erythrocyte Membrane/ultrastructure , Membrane Lipids/chemistry , Mutation , Naphthalenesulfonates , Protein Conformation , Rabbits , Streptolysins/genetics
10.
Eur J Biochem ; 247(1): 209-16, 1997 Jul 01.
Article in English | MEDLINE | ID: mdl-9249028

ABSTRACT

Vibrio cholerae cytolysin (VCC) is produced by many non-choleratoxigenic strains of V. cholerae, and possibly represents a relevant pathogenicity determinant of these bacteria. The protein is secreted as a pro-toxin that is proteolytically cleaved to yield the active toxin with a molecular mass of approximately 63 kDa. We here describe a simple procedure for preparative isolation of mature VCC from bacterial culture supernatants, and present information on its mode of binding and pore formation in biological membranes. At low concentrations, toxin monomers interact with a high-affinity binding site on highly susceptible rabbit erythrocytes. This as yet unidentified binding site is absent on human erythrocytes, which are less susceptible to the toxin action. At higher concentrations, binding of the toxin occurs to both rabbit and human erythrocytes in a non-saturable manner. Cell-bound toxin monomers oligomerize to form supramolecular structures that are seen in the electron microscope as apparently hollow funnels, and oligomerization correlates functionally with the appearance of small transmembrane pores. Osmotic protection experiments indicate that the toxin channels are of finite size with a diameter of 1-2 nm. The mode of action of VCC closely resembles that of classical pore-forming toxins such as staphylococcal alpha-toxin and the aerolysin of Aeromonas hydrophila.


Subject(s)
Cytotoxins/metabolism , Vibrio cholerae/pathogenicity , Amino Acid Sequence , Animals , Binding Sites , Cytotoxins/chemistry , Cytotoxins/pharmacology , Erythrocyte Membrane/drug effects , Humans , Molecular Sequence Data , Molecular Weight , Rabbits
11.
Eur J Biochem ; 236(1): 34-9, 1996 Feb 15.
Article in English | MEDLINE | ID: mdl-8617283

ABSTRACT

Streptolysin 0 (SLO) is the prototype of a family of cytolysins that consists of proteins which bind to cholesterol and form very large transmembrane pores. Structure/function studies on the pore-forming cytolysin SLO have been complicated by the proteolytic inactivation of a substantial portion of recombinant SLO (rSLO) expressed in Escherichia coli. To overcome this problem, translational fusions between the E. coli maltose-binding protein (MBP) gene and SLO were constructed, using the vectors pMAL-p2 and pMAL-c2. MBP-SLO fusion proteins were degraded if secreted into the E. coli periplasm, but intact, soluble MBP-SLO fusion proteins were produced at high levels in the cytoplasm. Active SLO with the expected N-terminus was separated from the MBP carrier by cleavage with factor Xa. Cleavage with plasmin or trypsin also yielded active, but slightly smaller forms of SLO. Surprisingly, uncleaved MBP-SLO was also hemolytic and cytotoxic to human fibroblasts and keratinocytes. The MBP-SLO fusion protein displayed equal activities to SLO. Sucrose density gradient analyses showed that the fusion protein assembled into polymers, and no difference in structure was discerned compared with polymers formed by native SLO. These studies show that the N-terminal 70 residues of mature (secreted) SLO are not required for pore formation and that the N-terminus of the molecule is probably not inserted into the bilayer. In addition, they provide a simple means for producing mutants for structure/function studies and highly purified SLO for use as a permeabilising reagent in cell biology research.


Subject(s)
ATP-Binding Cassette Transporters , Bacterial Proteins/biosynthesis , Escherichia coli Proteins , Hemolysis , Monosaccharide Transport Proteins , Streptococcus pyogenes/genetics , Streptolysins/biosynthesis , Bacterial Proteins/genetics , Bacterial Proteins/pharmacology , Base Sequence , Carrier Proteins/biosynthesis , Carrier Proteins/genetics , Cloning, Molecular , Erythrocytes/drug effects , Escherichia coli/genetics , Fluoresceins/metabolism , Humans , Liposomes/metabolism , Maltose-Binding Proteins , Molecular Sequence Data , Protein Conformation , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/pharmacology , Sequence Analysis , Sequence Deletion , Streptolysins/genetics , Streptolysins/pharmacology , Structure-Activity Relationship
12.
J Exp Med ; 182(6): 1959-71, 1995 Dec 01.
Article in English | MEDLINE | ID: mdl-7500042

ABSTRACT

Combined treatment with trypsin, cholesterol esterase, and neuraminidase transforms LDL, but not HDL or VLDL, to particles with properties akin to those of lipid extracted from atherosclerotic lesions. Single or double enzyme modifications, or treatment with phospholipase C, or simple vortexing are ineffective. Triple enzyme treatment disrupts the ordered and uniform structure of LDL particles, and gives rise to the formation of inhomogeneous lipid droplets 10-200 nm in diameter with a pronounced net negative charge, but lacking significant amounts of oxidized lipid. Enzymatically modified LDL (E-LDL), but not oxidatively modified LDL (ox-LDL), is endowed with potent complement-activating capacity. As previously found for lipid isolated from atherosclerotic lesions, complement activation occurs to completion via the alternative pathway and is independent of antibody. E-LDL is rapidly taken up by human macrophages to an extent exceeding the uptake of acetylated LDL (ac-LDL) or oxidatively modified LDL. After 16 h, cholesteryl oleate ester formation induced by E-LDL (50 micrograms/ml cholesterol) was in the range of 6-10 nmol/mg protein compared with 3-6 nmol/mg induced by an equivalent amount of acetylated LDL. At this concentration, E-LDL was essentially devoid of direct cytotoxic effects. Competition experiments indicated that uptake of E-LDL was mediated in part by ox-LDL receptor(s). Thus, approximately 90% of 125I-ox-LDL degradation was inhibited by a 2-fold excess of unlabeled E-LDL. Uptake of 125I-LDL was not inhibited by E-LDL. We hypothesize that extracellular enzymatic modification may represent an important step linking subendothelial deposition of LDL to the initiation of atherosclerosis.


Subject(s)
Arteriosclerosis/etiology , Complement Activation , Lipoproteins, LDL/metabolism , Membrane Proteins , Receptors, Lipoprotein , Complement C3/metabolism , Complement Membrane Attack Complex , Complement System Proteins/metabolism , Foam Cells/metabolism , Glycoproteins/metabolism , Humans , Lipid Metabolism , Lipoproteins, LDL/chemistry , Macrophages/metabolism , Neuraminidase/metabolism , Receptors, Immunologic/metabolism , Receptors, Scavenger , Scavenger Receptors, Class B , Sterol Esterase/metabolism , Trypsin/metabolism
13.
Eur J Endocrinol ; 133(5): 626-34, 1995 Nov.
Article in English | MEDLINE | ID: mdl-7581995

ABSTRACT

The expression of sodium-potassium pumps and glucose transporters in pure adipocyte plasma membranes from a hyperthyroid animal model was studied. Hyperthyroidism was induced by enteral administration of five doses of 90 micrograms of triiodothyronine every second day to 8-week-old rats. Following isolation of epididymal adipocytes, 3-O-methylglucose transport was measured and the number of Na/K-ATPase-(alpha 1- and alpha 2-isoforms) and glucose transporter (GLUT1 and GLUT4) molecules in sheets of adipocyte plasma membrane were determined by quantitative immunoelectron microscopy, using gold labelling. Maximal in vitro insulin stimulation of adipocytes increased the glucose transport rate and the amount of GLUT4 in the plasma membrane 15-fold, whereas the amount of alpha 2 was unaffected. In adipocytes from hyperthyroid rats, mean adipocyte volume was decreased by 18% and the quantities of GLUT4 per unit area of plasma membrane (maximal insulin stimulation) and of alpha 2 were decreased by 19% and 15%, respectively. Thus, hypotrophia of fat tissue in the hyperthyroid state is associated with a decreased expression in the plasma membrane of the glucose transporter GLUT4 and the alpha 2-isoform of Na/K-ATPase.


Subject(s)
Adipocytes/chemistry , Monosaccharide Transport Proteins/analysis , Muscle Proteins , Sodium-Potassium-Exchanging ATPase/analysis , Triiodothyronine/pharmacology , 3-O-Methylglucose , Adipocytes/metabolism , Adipocytes/ultrastructure , Animals , Biological Transport/physiology , Blood Glucose/analysis , Cell Membrane/chemistry , Cell Membrane/drug effects , Cell Membrane/metabolism , Disease Models, Animal , Dose-Response Relationship, Drug , Glucose Transporter Type 1 , Glucose Transporter Type 4 , Hyperthyroidism/enzymology , Hyperthyroidism/metabolism , Insulin/pharmacology , Isomerism , Male , Methylglucosides/metabolism , Methylglucosides/pharmacokinetics , Microscopy, Immunoelectron , Monosaccharide Transport Proteins/metabolism , Rats , Rats, Wistar , Sodium-Potassium-Exchanging ATPase/metabolism
14.
J Membr Biol ; 140(3): 215-23, 1994 Jun.
Article in English | MEDLINE | ID: mdl-7932656

ABSTRACT

Insulin receptors were incorporated into liposomes by two different procedures, one using dialysis and one using detergent removal by Bio-Beads. Receptor incorporation was analyzed by gradient centrifugation and electron microscopy. Reconstituted receptors projected up to 12 nm above the membrane and exhibited a T-shaped structure compatible with that previously described for the solubilized receptor. Insulin binding and autophosphorylation experiments indicated that approx. 50% of the receptors were incorporated right-side out. Such random orientation was confirmed by immunogold labeling of the alpha- and the beta-subunit of the receptor. Immunogold labeling of the C-terminus of the beta-subunit indicates that it resides about 6 nm off the membrane, while two alpha-subunit epitopes were labeled at about twice this distance, confirming that the alpha-subunit is harbored in the cross-bar of the T-structure.


Subject(s)
Membranes, Artificial , Receptor, Insulin/chemistry , Humans , Insulin/metabolism , Liposomes , Microscopy, Electron , Models, Biological , Receptor, Insulin/metabolism , Receptor, Insulin/ultrastructure
15.
Infect Immun ; 62(6): 2249-56, 1994 Jun.
Article in English | MEDLINE | ID: mdl-8188346

ABSTRACT

Chemical modification of histidine residues in staphylococcal alpha-toxin leads to loss of functional activity. Site-directed mutants of the toxin in which each of the four histidine residues was replaced by several amino acids were therefore produced. The mutant proteins were purified and characterized. Exchange of H-259 or H-144 was sometimes tolerated without reduction in hemolytic activity. These histidine residues are thus not essential for toxin function. Exchange of H-35 and H-48, however, had marked effects. H-35 mutant toxins bound with high affinity to rabbit erythrocytes but displayed faulty oligomerization and were unable to form pores. H-48 mutant toxins also had severely impaired hemolytic activity due probably to faulty hexamerization. We interpret these results to indicate that the N-terminal domain of alpha-toxin in the region of H-35 and H-48 is involved in protomer-protomer interactions that underlie the hexamerization and pore-forming process.


Subject(s)
Bacterial Toxins/chemistry , Hemolysin Proteins/chemistry , Animals , Bacterial Toxins/metabolism , Erythrocyte Membrane/ultrastructure , Hemolysin Proteins/metabolism , Hemolysis , Histidine , Mutagenesis, Site-Directed , Rabbits , Structure-Activity Relationship
16.
Liver ; 13(6): 305-10, 1993 Dec.
Article in English | MEDLINE | ID: mdl-7507546

ABSTRACT

Characteristics of primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) are bile duct destruction and portal inflammation. Increased levels of circulating complement activation products are also present. This raises the possibility of involvement of complement-dependent cytotoxic mechanisms in the pathogenesis. Therefore, we investigated liver biopsy specimens from 21 patients with PBC, six patients with PSC and six controls for complement deposits by immunohistochemistry using polyclonal and monoclonal antibodies against C3d, the terminal complement complex (TCC) and vitronectin (S-protein). We found C3d, TCC and vitronectin deposits only in the portal tracts. C3d and TCC were present in the walls of the hepatic arteries and in the connective tissue stroma but never around the bile ducts. We found vitronectin deposits throughout the connective tissue, often independent of the TCC deposits. When vitronectin and TCC were co-localized, the staining patterns were inverse; that is, intense staining for TCC accompanied weak staining for vitronectin and vice versa. Occasionally complete dissociation between TCC and vitronectin staining was observed. Deposits of TCC and vitronectin showed a focal distribution leaving many portal tracts free of TCC. Our results question whether complement-dependent cytotoxic mechanisms take part in the bile duct destruction in PBC and PSC.


Subject(s)
Blood Proteins/analysis , Cholangitis, Sclerosing/immunology , Complement C3d/analysis , Complement Membrane Attack Complex/analysis , Glycoproteins/analysis , Liver Cirrhosis, Biliary/immunology , Liver/immunology , Adult , Aged , Humans , Immunohistochemistry , Vitronectin
17.
J Membr Biol ; 136(1): 63-73, 1993 Oct.
Article in English | MEDLINE | ID: mdl-8271273

ABSTRACT

We have quantitated and studied the topology of isoforms of the Na+/K(+)-ATPase and of the glucose transporter in rat adipocyte plasma membranes. Adipocytes were incubated with or without insulin for 15 min. Sheets of native plasma membrane, with the cytoplasmic face exposed, were prepared by adsorption to EM grids. Grids were incubated in parallel with monoclonal antibodies against the Na+/K(+)-ATPase isoforms alpha 1 and alpha 2, and the glucose transporter isoforms GLUT1 and GLUT4, followed by immunogold labeling, negative staining and quantitation by counting of the gold particles in electron micrographs. In addition, the distribution of glucose transporters and Na+/K(+)-ATPase isoforms in subcellular membrane fractions prepared by an established fractionation procedure was monitored by Western blotting. We found that the Na+/K(+)-ATPases and the glucose transporters were confined to the planar part of the plasma membrane, without association to caveolar invaginations. The vast majority of the Na+/K(+)-ATPase molecules in the adipocyte plasma membrane were of the alpha 2 isoform; GLUT4 was the dominating glucose transporter isoform. The total number of Na+/K(+)-ATPase molecules labeled in the plasma membrane was 3.5 x 10(5) per cell, independent of insulin stimulation. Concomitantly, insulin increased GLUT4 labeling sevenfold to a value of 3.5 x 10(5) per cell.


Subject(s)
Adipose Tissue/chemistry , Adipose Tissue/cytology , Monosaccharide Transport Proteins/analysis , Muscle Proteins , Sodium-Potassium-Exchanging ATPase/analysis , Adipose Tissue/ultrastructure , Animals , Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/immunology , Blotting, Western , Cell Fractionation , Cell Membrane/chemistry , Cell Membrane/enzymology , Cell Membrane/ultrastructure , Cells, Cultured , Glucose Transporter Type 1 , Glucose Transporter Type 4 , Immunohistochemistry , Insulin/pharmacology , Isomerism , Male , Microscopy, Electron , Microscopy, Immunoelectron , Monosaccharide Transport Proteins/immunology , Monosaccharide Transport Proteins/physiology , Rats , Rats, Wistar , Sodium-Potassium-Exchanging ATPase/immunology
18.
Biosci Rep ; 12(6): 471-82, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1298437

ABSTRACT

Human transferrin receptor was isolated from Triton X-100 solubilized placental plasma membranes by a rapid one-step chromatographic procedure based on immunoadsorption of the receptor-transferrin complex on anti-transferrin Sepharose and lectin-affinity on wheat germ agglutinin. Following exchange of Triton X-100 with CHAPS or n-octylglucoside, the purified receptor was incorporated into egg phosphatidylcholine liposomes upon detergent removal by dialysis (lipid/protein ratio 15:1 to 45:1 (w/w)). Reconstitution of the receptor was confirmed by trypsin cleavage to dissociate the large extracellular receptor domain from the liposomal membranes. Electron micrographs of the receptor-lipid recombinants negatively stained with sodium sillicotungstate, showed that the receptor molecules distributed very inhomogeneously on the liposomes, most receptors being clustered. Single copies of the receptor were seen as elongate structures (5 x 10 nm) oriented with their long axis parallel to the liposome surface and separated from this by a 2-3 nm gap. This result provides evidence for a narrow connecting link between the globular extracellular receptor domain and the membrane spanning segment.


Subject(s)
Placenta/metabolism , Receptors, Transferrin/isolation & purification , Cell Membrane/metabolism , Chromatography, Affinity , Female , Humans , In Vitro Techniques , Liposomes , Microscopy, Electron , Octoxynol , Polyethylene Glycols , Pregnancy , Protein Conformation , Receptors, Transferrin/chemistry , Receptors, Transferrin/ultrastructure , Trypsin
19.
Am J Physiol ; 262(5 Pt 1): E700-11, 1992 May.
Article in English | MEDLINE | ID: mdl-1590380

ABSTRACT

To study the mechanism of acceleration of glucose transport in skeletal muscle after stimulation with insulin and contractions, we isolated a subcellular vesicular membrane fraction, highly enriched in the plasma membrane enzyme K(+)-stimulated p-nitrophenylphosphatase and also enriched in some intracellular membranes. Protein recovery, morphology, lipid content, marker enzyme activities, total intravesicular volume, Western blot quantitation of GLUT-1, and glucose-inhibitable cytochalasin B binding were identical in membrane fractions from control, insulin-stimulated, contraction-stimulated, and insulin- and contraction-stimulated muscle. Time course of D-[3H]glucose entry in membrane vesicles at equilibrium exchange conditions showed that initial rate of transport at 30 mM of glucose was increased 19-fold and that equilibrium distribution space was increased 4-fold in vesicles from maximum stimulated muscle. The effects of insulin and contractions on initial rate of transport as well as on equilibrium distribution space were additive, and stimulation increased the substrate saturability of glucose transport. Furthermore, cytochalasin B binding to membranes prepared by using less centrifugation time than usual showed that, after stimulation with insulin and contractions, at least 35% of the total number of glucose transporters were redistributed from one kind of vesicles to a more slowly sedimenting kind of vesicles, probably reflecting translocation within the membrane preparation from intracellular vesicles to the plasma membrane upon stimulation. In the present membrane preparation the effects of insulin and/or contractions on glucose transport resemble those seen in intact muscle, and the effects are thus not dependent on cellular integrity.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Glucose/pharmacokinetics , Insulin/pharmacology , Muscle Contraction , Muscles/metabolism , Animals , Biological Transport , Male , Microscopy, Electron , Monosaccharide Transport Proteins/metabolism , Muscles/ultrastructure , Osmolar Concentration , Rats , Rats, Inbred Strains
20.
Microbiol Rev ; 55(4): 733-51, 1991 Dec.
Article in English | MEDLINE | ID: mdl-1779933

ABSTRACT

Alpha-toxin, the major cytotoxic agent elaborated by Staphylococcus aureus, was the first bacterial exotoxin to be identified as a pore former. The protein is secreted as a single-chain, water-soluble molecule of Mr 33,000. At low concentrations (less than 100 nM), the toxin binds to as yet unidentified, high-affinity acceptor sites that have been detected on a variety of cells including rabbit erythrocytes, human platelets, monocytes and endothelial cells. At high concentrations, the toxin additionally binds via nonspecific absorption to lipid bilayers; it can thus damage both cells lacking significant numbers of the acceptor and protein-free artificial lipid bilayers. Membrane damage occurs in both cases after membrane-bound toxin molecules collide via lateral diffusion to form ring-structured hexamers. The latter insert spontaneously into the lipid bilayer to form discrete transmembrane pores of effective diameter 1 to 2 nm. A hypothetical model is advanced in which the pore is lined by amphiphilic beta-sheets, one surface of which interacts with lipids whereas the other repels apolar membrane constitutents to force open an aqueous passage. The detrimental effects of alpha-toxin are due not only to the death of susceptible targets, but also to the presence of secondary cellular reactions that can be triggered via Ca2+ influx through the pores. Well-studied phenomena include the stimulation of arachidonic acid metabolism, triggering of granule exocytosis, and contractile dysfunction. Such processes cause profound long-range disturbances such as development of pulmonary edema and promotion of blood coagulation.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Bacterial Toxins/pharmacology , Cell Membrane/drug effects , Hemolysin Proteins/chemistry , Staphylococcus aureus , Animals , Bacterial Toxins/chemistry , Cell Membrane Permeability/drug effects , Humans , Structure-Activity Relationship
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