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Biomedical subjects

B Lanne

Publications and source records attributed to B Lanne.

At least 19 recordsLinked to original sources

Antifibrinolytic proCPU is present in the peritoneal cavity during surgery.

The fibrinolytic capacity of the peritoneum plays a pivotal role in peritoneal wound healing. During surgery the balance between fibrin deposition and degradation is tilted towards deposition, leading to the formation of adhesions. In blood, carboxypeptidase U (CPU) stabilizes clots by retarding fibrinolysis. The purpose of this study was to investigate whether the more stable zymogen, proCPU, is also present in the peritoneal cavity and, if so, to examine its origin. Levels of proCPU were measured in plasma and serosal peritoneal fluid collected during surgery. Peritoneal biopsies were stained for proCPU. Two-dimensional gel electrophoresis was performed to study the protein composition of the serosal fluid compared to plasma and Western blotting to identify differences in glycosylation of proCPU, indicating possible different cellular origin. Cultured human mesothelial cells were examined for proCPU production under normal conditions and conditions mimicking surgery. We found comparable and correlating levels of proCPU in serosal fluid and plasma. ProCPU was also found where fibrin covered the injured peritoneal surface. A protein composition very similar in serosal fluid and plasma was shown by two-dimensional gel electrophoresis, and the proCPU pattern did not indicate a different origin. No proCPU production was found in cultured mesothelial cells. This is the first study to report on the presence of proCPU in the peritoneal cavity, which seems to be the result of plasma oozing out during the inflammatory reaction to the surgical trauma. This is likely to be important for the balance between fibrin deposition and degradation and thereby in the formation of postoperative adhesions.

Antifibrinolytic Agents↗

Thiourea enhances mapping of the proteome from murine white adipose tissue.

Adipose tissue imposes problems in two-dimensional (2-D) analysis due to its extremely high content of fat. To improve protein separation detergents and chaotropes were varied in the IEF step. The most important factor for obtaining distinct spots in the 2-D gel was whether thiourea was included or not. Many high molecular weight spots became resolved by using thiourea, while no spots disappeared or showed inferior characteristics, thus approximately twice as many spots were possible to quantify. Hydrophobic indices were compared for a set of proteins that gave rise to sharper spots with proteins that were not improved on the use of thiourea. The comparison did not give any statistically significant difference between the two groups of proteins. One of the effects obtained by inclusion of thiourea was that the dominating protein, serum albumin, appeared as more condensed spots allowing other minor proteins to be detected. This work resulted in a protocol which greatly enhances the resolution of proteins in adipose tissue. A 2-D map of mouse white adipose tissue from epididymal fat pads was constructed in which 140 spots were identified by mass spectrometry. This work lays the ground for our further studies on white adipose tissue in metabolic diseases such as obesity and dyslipidemia.

Adipose Tissue↗

Mass spectrometric analysis of ceramide composition in mono-, di-, tri-, and tetraglycosylceramides from mouse kidney: an experimental model for uropathogenic Escherichia coli.

Many bacteria have been shown to bind to the carbohydrate part of glycosphingolipids, but also the lipid moieties of receptor-active glycolipids are of importance. To investigate the chemistry of the ceramides of kidney glycolipids to which the uropathogenic Escherichia coli bind, different mass spectrometric techniques were utilized. First, a mixture of glycolipids isolated from man and mice kidney was separated by thin-layer chromatography (TLC) and scanned by direct desorption from the plate by fast atom bombardment mass spectrometry (TLC/FAB-MS). Second, the glycolipids were purified by preparative TLC and analyzed by negative-ion FAB-MS. After methylation, further analyses were made with positive-ion FAB-MS, positive-ion electron ionization (EI)-MS, high-temperature capillary gas chromatography (GC/EI-MS) and positive-ion matrix-assisted laser desorption/ionization (MALDI)-MS. The ceramide compositions of the four glycolipids were determined using all these MS techniques and the reliability of the different methods for this type of analyses is discussed. Comparison of the mouse kidney glycolipids with the corresponding glycolipids from human kidney showed the same degree of hydroxylation of ceramides among mono- and disaccharide glycolipids, but a significantly higher degree of hydroxylation among mouse kidney glycolipids with three and four sugar residues. This result might be of relevance for the binding of P-fimbriated E. coli to the urinary tract tissues.

Animals↗

Binding of cholera toxin B-subunits to derivatives of the natural ganglioside receptor, GM1.

In a previous paper we showed that the B-pentamer of cholera toxin (CT-B) binds with reduced binding strength to different C(1) derivatives of N-acetylneuraminic acid (NeuAc) of the natural receptor ganglioside, GM1. We have now extended these results to encompass two large amide derivatives, butylamide and cyclohexylmethylamide, using an assay in which the glycosphingolipids are adsorbed on hydrophobic PVDF membranes. The latter derivative showed an affinity approximately equal to that earlier found for benzylamide ( approximately 0.01 relative to native GM1) whereas the former revealed a approximately tenfold further reduction in affinity. Another derivative with a charged C(1)-amide group, aminopropylamide, was not bound by the toxin. Toxin binding to C(7) derivatives was reduced by about 50% compared with the native ganglioside. Molecular modeling of C(1) and C(7) derivatives in complex with CT-B gave a structural rationale for the observed differences in the relative affinities of the various derivatives. Loss of or altered hydrogen bond interactions involving the water molecules bridging the sialic acid to the protein was found to be the major cause for the observed drop in CT-B affinity in the smaller derivatives, while in the bulkier derivatives, hydrophobic interactions with the protein were found to partly compensate for these losses.

Biochemistry↗

Binding of N-trifluoroacetyl-derivatized natural glycosphingolipids by uropathogenic Escherichia coli and Neisseria subflava.

Several neutral glycosphingolipids were hydrogenated and subjected to trifluoroacetylation in trifluoroacetic acid/trifluoroacetic acid anhydride under conditions leading to complete exchange of the N-acetyl groups of GalNAc for N-trifluoroacetyl. The derivatized glycosphingolipids were analyzed for binding by P-fimbriated uropathogenic Escherichia coli, recognizing the globo series of glycolipids (carrying Galalpha1-4Gal). Using E. coli it was shown that a GalNCO-CF3 next to the minimum binding epitope Galalpha1-4Gal did not substantially influence the binding, as did not a trifluoro acetyl group on the ceramide. Exchange of N-acetyl of GalNAc in the receptor active gangliotetraosylceramide, Galbeta1-3GalNAcbeta1-4Galbeta1-4Glcbeta1-1Cer, for N-trifluoroacetyl, did not change the binding of two out of the three strains tested of the bacterium Neisseria subflava. Discussion concerning the binding epitopes of the bacterial adhesins to carbohydrates is based on these results.

Bacterial Adhesion↗

Delineation of the epitope recognized by an antibody specific for N-glycolylneuraminic acid-containing gangliosides.

P3 is a mouse monoclonal antibody (mAb) that binds to several NeuGc-containing gangliosides. It also reacts with antigens expressed in human breast tumors (Vázquez et al. (1995) Hybridoma , 14, 551-556). In this work, the binding specificity of P3 has been characterized in more detail using a panel of glycolipids that included several disialylated gangliosides and several chemical derivatives of NeuGc-GM3. The carboxyl group and the nitrogen function of sialic acid were found to play important roles in the antibody binding, whereas the glycerol tail appears to be nonrelevant. Molecular modeling was used to analyze the binding data, including the finding that P3 selectively recognizes the internal NeuGc in GD3. For this purpose, conformational studies of GD3 were performed using molecular dynamics. It was concluded that sialic acid binds the P3 antibody through its upper face (the one on which the carboxyl group is exposed) and the C4-C5 side of the sugar ring, whereas none or very little contact between the galactose residue and the protein is evident. Conformational analysis of GD3 revealed that, despite the large flexibility of the NeuGcalpha8NeuGc linkage, the P3 binding epitope on the external sialic acid is not well exposed for any of the possible conformations this linkage can adopt, whereas the internal sialic acid presents the epitope in a proper way for several of these conformations. As a final result, a coherent picture of the epitope that fits the wide binding data was obtained.

Animals↗

Gal alpha 4Gal-binding antibodies: specificity and use for the mapping of glycolipids of Burkitt lymphoma and other human tumors.

The binding of four different Gal alpha 4Gal-specific antibodies was examined using several natural and synthetic Gal alpha 4Gal-containing glycolipids on thin-layer chromatograms. One of the antibodies, MC2102, bound much better to galabiosylceramide (Gal alpha 4Gal beta Cer) than to the elongated structure globotriaosylceramide (Gal alpha 4Gal beta-4Glc beta Cer, CD77). Two antibodies, 38.13 and Pk002, bound best to globotriaosylceramide but cross-reacted with the P1 antigen (Gal alpha 4Gal beta 3GlcNAc beta 3Gal beta 4Glc beta Cer). The fourth antibody tested, P001, reacted most strongly with the P1 antigen but also to some extent with globotriaosylceramide. None of the antibodies bound to glycolipids with Gal alpha 4Gal placed internally in the carbohydrate chains. The synthetic Gal alpha 4Gal beta-bissulfone (3-hexadecylsulfonyl-2-hexadecylsulfonylmethylprop-1-yl) was bound by MC2102, Pk002, and 38.13 with a strength comparable to Gal alpha 4Gal beta Cer. A large number of glycolipids lacking terminal Gal alpha 4Gal were also tested, but none of the antibodies bound to any of these structures. The specificity of the studied antibodies was used to investigate the presence of Gal alpha 4Gal beta-containing glycolipids in several human and animal tissues and cells and in an experimental Burkitt lymphoma cell line grown in nude mice. Glycolipids were also isolated from a series of human tumors and several of them were, by antibody binding, found to contain Gal alpha 4Gal beta Cer.

Antibodies↗

Glycoconjugate receptors for P-fimbriated Escherichia coli in the mouse. An animal model of urinary tract infection.

Glycosphingolipids were isolated from kidneys, urethers, and bladders (including urethrae) of C3H/HeN mice. Binding was studied of a clinical isolate and recombinant strains of uropathogenic P-fimbriated Escherichia coli to these glycolipids. A series of receptor-active glycolipids with Gal alpha 4Gal in common, previously shown to be recognized by these bacteria, was identified by use of specific monoclonal antibodies, fast-atom bombardment and electron-impact mass spectrometry, and proton nuclear magnetic resonance spectroscopy: galabiosylceramide (Gal alpha 4Gal beta Cer), globotriaosylceramide (Gal alpha 4Gal beta 4Glc beta Cer), globoside (GalNAc beta 3Gal alpha 4Gal beta 4Glc beta Cer), the Forssman glycolipid (GalNAc alpha 3GalNAc beta 3Gal alpha 4Gal beta 4Glc beta Cer), Gal beta 4GlcNAc beta 6(Gal beta 3)GalNAc beta 3Gal alpha 4Gal beta 4Glc beta Cer, and Gal beta 4(Fuc alpha 3)GlcNAc beta 6(Gal beta 3)GalNAc beta 3Gal alpha 4Gal beta 4Glc beta Cer. The binding pattern for mouse kidney glycolipids differed from that for kidney glycolipids of man and monkey. In particular, the dominant 8-sugar glycolipid in the mouse was not detected in the primates. A second difference was found in the binding of E. coli to kidney glycoproteins on blots after electrophoresis; the mouse showed distinct receptor-active bands while human and monkey did not. These differences may be of relevance when using the mouse as a model for clinical urinary tract infection of man.

Animals↗

Enhanced binding of enterotoxigenic Escherichia coli K99 to amide derivatives of the receptor ganglioside NeuGc-GM3.

A natural receptor in pig small intestine [Teneberg, S., Willemsen, P., de Graaf, F. K., & Karlsson, K.-A. (1990) FEBS Lett. 263, 10-14] for the enterotoxigenic bacteria Escherichia coli K99 is the ganglioside NeuGc-GM3 (NeuGc alpha 3Gal beta 4Glc beta Cer) [e.g., H. Smit, W. Gaastra, J. P. Kamerling, J. F. G. Vliegenthart, & F. K. de Graaf (1984) Infect. Immun. 46, 578-584]. Chemical modifications of the carboxyl group of this ganglioside were performed, giving five different amides, the methyl ester, and the primary alcohol. The products were purified, and their structures were investigated by negative FAB mass spectrometry. Binding of E. coli K99 was tested by incubating 35S-labeled bacteria with derivatized compounds separated on thin-layer chromatograms. Modification of the carboxyl group to a primary amide strengthened the binding at least 5-fold, as estimated from autoradiography of dilutions on thin-layer plates. Some strengthening of the binding was also obtained with the methylamide as well as with the carboxyl group reduced to the alcohol. The ethylamide bound equally well as the underivatized NeuGc-GM3. Amide substituents as large as propyl amide and benzyl amide were still recognized by the bacteria, although they bound weaker. The methyl ester was not stable in the chromatogram-binding assay with silica gel and water present, and it reverted to the acid.

Amides↗

Generation of a murine monoclonal antibody specific for N-glycolylneuraminic acid-containing gangliosides that also recognizes sulfated glycolipids.

The P3 murine monoclonal antibody (MAb) was generated by immunizing BALB/c mice with NeuGcGM3 included into liposomes. The specificity of this MAb was defined by an enzyme-linked immunosorbent assay and immunostaining on thin-layer chromatograms. P3 MAb binds to NeuGc-containing gangliosides and was shown also to react with sulfated glycolipids. A preliminary immunohistochemical study showed that the P3 MAb was able to recognize antigens expressed in human breast tumors.

Animals↗

Antigen-specific primary immune response of human B-lymphocytes after in vitro immunization with GM3 ganglioside.

In vitro immunization of human B-lymphocytes was performed with liposomes containing the monosialoganglioside GM3, with or without either complete tetanus toxoid or a synthetic T helper epitope derived from tetanus toxin (determinant 830-843). The immunized B-cells were Epstein-Barr virus transformed and the human anti-ganglioside antibody response was evaluated using an indirect ELISA against different mono- and disialogangliosides. Clones producing antigen-specific human antibodies of the IgM isotype against the ganglioside GM3 used as the immunogen were selected and one clone, IM-11, was further characterized. In addition, a method of positive selection using GM3-coated magnetic beads has been developed which allowed us to rescue unstable clones. The binding of the human antibody IM-11 to a large panel of glycosphingolipids separated on thin-layer plates was studied. The human MAb IM-11 was found to bind strongly to NeuAcGM3, IV3 NeuAcnLc4 and sulfate containing glycosphingolipids and weakly to NeuGcGM3. Immunohistological staining of melanoma and breast cancer biopsy sections showed a selective reactivity of IM-11 with tumor cells which varied among different tumors.

Amino Acid Sequence↗

Binding of the galactose-specific Pseudomonas aeruginosa lectin, PA-I, to glycosphingolipids and other glycoconjugates.

The carbohydrate-binding specificity of Pseudomonas aeruginosa lectin I (PA-I) in iodinated or biotinylated form was studied. A large number of glycosphingolipids, as well as some glycoproteins and neoglycoproteins were used as ligands. Also, inhibition by free saccharides of PA-I binding to glycosphingolipids was tested. It was found that the lectin binds most strongly to terminal and nonsubstituted Gal alpha 3Gal- or Gal alpha 4Gal-structures.

Adhesins, Bacterial↗

On the role of the carboxyl group of sialic acid in binding of cholera toxin to the receptor glycosphingolipid, GM1.

The carboxyl group of the natural cholera toxin receptor, the ganglioside GM1, Gal beta 1-3GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-4Glc beta 1-Cer, has been converted to a number of C(1)-amides of NeuAc. The binding of cholera toxin B-subunit to these derivatives was monitored by exposing the modified glycolipids, on solid phases, to radiolabeled toxin. Binding was obtained, although substantially reduced, with the amide and to a lesser extent with the benzylamide and also the C(1)-alcohol. In the assay system used, the methyl-, ethyl-, or propylamides did not bind. It was concluded that the hydrogen bonding capacity of a carboxyl or amide group is needed for strong binding. This is in agreement with the recently published crystal structure of the B-subunit in complex with the GM1 pentasaccharide.

Binding Sites↗

Non-acid glycosphingolipid expression in plasma of an A1 Le(a-b+) secretor human individual: identification of an ALeb heptaglycosylceramide as major blood group component.

Total non-acid glycosphingolipids were isolated from plasma of an A1 Le(a-b+) secretor individual with Refsum's disease (phytanic acid storage disease). The glycolipids were separated into 11 fractions by open column chromatography and by HPLC. The fractions were analyzed by thin-layer chromatography and tested for different blood group A activities as well as blood group Le(a )and Leb activity. The fractions were structurally characterized by proton NMR spectroscopy and FAB mass spectrometry and in selected cases by EI mass spectrometry of the permethylated and permethylated-reduced derivatives. Degradation analysis was performed on partially permethylated or permethylated-reduced alditol acetates. The dominating blood group compound was found to be a blood group A active type 1 chain difucosylheptaglycosylceramide. Other blood group compounds were identified as a blood group A active type 1 chain monofucosylhexaglycosylceramide, a blood group Leb hexaglycosylceramide, a blood group H active type 1 chain pentaglycosylceramide, and a globotetraosylceramide (the P-antigen). The presence of a Le(a) glycosphingolipid and blood group A type 3/4 chain structures were also found by immunostaining. Glucosyl-, lactosyl-, and globotriaosylceramides were the dominating short chain compounds. The amount of phytanic acid incorporated into the monoglycosylceramide fraction was found to be less than 5% of the fatty acids.

Animals↗

Microbial interaction with animal cell surface carbohydrates.

Microbes have selected primarily carbohydrates for attachment to host animal cells. Recent studies have revealed essential characteristics in the recognition of receptor carbohydrates. Of importance is the property of recognizing also sequences placed inside an oligosaccharide chain, which differs from most animal antibodies. This is the basis for series of isoreceptors with the minimum receptor sequence in common but with separate neighbouring groups. There are families of microbial ligands that show different preferences for members within one series of isoreceptors, indicating only slight differences in the complementary binding sites of the proteins. Such differences may explain shifts in the selectivity of separate host tissues for infection. A second characteristic is the low affinity interaction often found where simple receptor-containing saccharides are unable to inhibit attachment. Technical possibilities are rapidly developing for the design of synthetic receptor analogues to be used in the therapy of clinical infections. This is urgently needed in cases where no rational therapy exists today.

Adhesins, Escherichia coli↗

The resolution into molecular species on desorption of glycolipids from thin-layer chromatograms, using combined thin-layer chromatography and fast-atom-bombardment mass spectrometry.

Using a specially designed, motorised t.l.c.-f.a.b.-m.s. probe with continuous desorption and scanning over a moving t.l.c. plate, it was shown that glycolipids with identical carbohydrate sequences were well resolved into molecular species with differences in long-chain base and fatty acid. There was no serious diffusion of the glycolipids into the matrix. The technique is demonstrated for sulphatides (one and two sugar residues) isolated from human kidney, GM3 ganglioside isolated from human malignant melanoma, and chemically modified gangliotetraosylceramide from mouse intestine. T.l.c.-f.a.b.m.s. is convenient for sequencing and composition analysis of receptor-active glycolipids, the biological activity of which can be monitored in parallel by overlay on the t.l.c. plate with proteins, viruses, bacteria, or animal cells.

Carbohydrate Sequence↗

Detection of endothelial-derived relaxing factor in human plasma in the basal state and following ischemia using electron paramagnetic resonance spectrometry.

The endothelial-derived relaxing factor is a vasodilator agent that is formed in the vascular endothelium in response to various stimuli. It has been identified as nitric oxide (NO). Due to its short half-life the endothelial-derived relaxing factor offers certain analytical problems. We present here a method for quantitative analysis of nitrite, the oxidation product of NO, in human plasma. NO binds strongly to hemoglobin. If the resulting NO-hemoglobin (Hb) complex is subjected to a magnetic field and microwave radiation, a characteristic electron paramagnetic resonance spectrum is obtained. This spectrum is highly specific and its amplitude can be used for quantitative determination of NO in the nanomolar range. Columns of bovine Hb covalently bound to agarose were prepared, and an excess amount of dithionite was used to ensure that the Hb was reduced to a ferrous, nonoxygenated state. Samples of human plasma were treated with dithionite to convert nitrite to nitric oxide. They were then passed over the columns, which were subsequently analyzed at 77 degrees K in an electron paramagnetic resonance spectrometer. As an external standard nitrite was used. The amplitude of the spectrum was linear in the range 1-100 nmol. In healthy subjects the venous plasma level of nitrite ranged from 0 to 0.6 microM. Following forearm or leg ischemia the plasma level of nitrite increased substantially. These data are the first to demonstrate circulating levels of an index of the endothelial-derived relaxing factor in human plasma.

Electron Spin Resonance Spectroscopy↗