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Control of plastidic glycolipid synthesis and its relation to chlorophyll formation.

Mechanisms restricting the accumulation of chloroplast glycolipids in achlorophyllous etiolated or heat-treated 70S ribosome-deficient rye leaves (Secale cereale L. cv "Halo") and thereby coupling glycolipid formation to the availability of chlorophyll, were investigated by comparing [(14)C]acetate incorporation by leaf segments of different age and subsequent chase experiments. In green leaves [(14)C]acetate incorporation into all major glycerolipids increased with age. In etiolated leaves glycerolipid synthesis developed much more slowly. In light-grown, heat-bleached leaves [(14)C]acetate incorporation into glycolipids was high at the youngest stage but declined with age. In green leaves [(14)C]acetate incorporation into unesterified fatty acids and all major glycerolipids was immediately and strongly diminished after application of an inhibitor of chlorophyll synthesis, 4,6-dioxoheptanoic acid. The turnover of glyco- or phospholipids did not differ markedly in green, etiolated, or heat-bleached leaves. The total capacity of isolated ribosome-deficient plastids for fatty acid synthesis was not much lower than that of isolated chloroplasts. However, the main products synthesized from [(14)C]acetate by chloroplasts were unesterified fatty acids, phosphatidic acid, and diacylglycerol, while those produced by ribosome-deficient plastids were unesterified fatty acids, phosphatidic acid, and phosphatidylglycerol. Isolated heat-bleached plastids exhibited a strikingly lower galactosyltransferase activity than chloroplasts, suggesting that this reaction was rate-limiting, and lacked phosphatidate phosphatase activity.

Journal Article↗

Preparation of monoclonal antibodies against a glycolipid asialo GM1.

Five IgM monoclonal antibodies (MAbs), MW-1, MW-2, MW-3, MW-4, and MW-5, against a glycolipid asialo GM1 were prepared from hybridoma clones obtained by the fusion of mouse NS-1 myeloma cells with spleen cells from a mouse immunized with asialo GM1 adsorbed to naked Salmonella. All the MAbs reacted only with asialo GM1 when their reactivities were examined by enzyme-linked immunosorbent assay (ELISA) and thin-layer chromatography (TLC)-immunostaining using structurally related glycolipids. The MAbs showed a complement-dependent lysis of mouse natural killer (NK) cells, but the lytic activities were weaker than that of a rabbit polyclonal anti-asialo GM1 antibody. When they were mixed, the anti-NK activity was increased to a level almost comparable to that of the polyclonal antibody. These results suggest that all the MAbs obtained are specific for asialo GM1 and that they may be different in fine specificity for the glycolipid. Significance of the MAbs in immunological and neurochemical studies is discussed.

Animals↗

Structural requirements for the glycolipid receptor of human uropathogenic Escherichia coli.

The binding of uropathogenic Escherichia coli to the globo series of glycolipids via P pili is a critical step in the infectious process that is mediated by a human-specific PapG adhesin. Three classes of PapG adhesins exist with different binding specificities to Gal alpha 4Gal-containing glycolipids. The structural basis for PapG recognition of the human glycolipid receptor globoside was investigated by using soluble saccharide analogues as inhibitors of bacterial haemagglutination. The minimum binding epitope was confirmed as the Gal alpha 4Gal moiety, but parts of the GalNAc beta and glucose residues, which flank the Gal alpha 4Gal in globoside (GbO4), were also shown to be important for strong binding. Furthermore, the same five hydroxyl groups of Gal alpha 4Gal in globotriasyl ceramide that were recognized by a previously characterized PapG variant were also recognized by the human-specific PapG in binding the GbO4 that dominates in the human kidney. Saccharide analogues that blocked haemagglutination also blocked the adherence of human uropathogenic E. coli to human kidney sections. Knowledge of the molecular details of the PapG-GbO4 interaction will make it possible to design antiadherence therapeutics.

Adhesins, Escherichia coli↗

Piglet ileal mucus contains protein and glycolipid (galactosylceramide) receptors specific for Escherichia coli K88 fimbriae.

The aim of this study was to characterize the Escherichia coli K88-specific receptors in mucus from the small intestines of 35-day-old piglets with the isogenic strains E. coli K-12(pMK005) (K88+) and E. coli K-12(pMK002) (K88-). These strains differed only in that the latter one cannot produce intact K88 fimbriae because of a deletion in the gene coding for the major fimbrial subunit. Adhesion was studied by incubating 3H-labeled bacteria with crude mucus, pronase-treated whole mucus, mucus fractionated by gel filtration, delipidated mucus, or extracted lipids immobilized in microtiter wells. In addition, E. coli strains were tested for adhesion to glycolipids extracted from mucus by overlaying glycolipid chromatograms with 125I-labeled bacteria. The recently reported finding that K88 fimbriae bind to glycoproteins in mucus from the piglet small intestine was confirmed in two ways. Pronase treatment of immobilized mucus reduced adhesion by 82%, and adhesion to delipidated mucus was 14 times greater for the K88+ than for the K88- strain. E. coli K88+ adhered to several of the fractions collected after gel filtration of crude mucus, including the void volume (M(r), > 250,000). Receptor activity specific for the K88 fimbriae was demonstrated in the lipids extracted from mucus, as the neutral lipids contained six times as much receptor activity as the acidic lipid fraction. Specificity was confirmed by demonstrating that adhesion to the total lipids could be inhibited by pretreatment of the immobilized lipids with K88 fimbriae. Relative to K-12 (K88-), the K-12 (K88+) bacterial cells bound more avidly to galactosylceramide when the neutral lipids were separated on thin-layer chromatography plates. No adhesion to lipids in the acidic fraction separated on thin-layer plates was detected. Relative to adhesion of K-12 (K88-), adhesion of K-12 (K88+) to commercially available galactosylceramide immobilized in microtiter wells confirmed the results with the thin-layer plates. It can be concluded that 35-day-old piglet mucus contains both protein and glycolipid receptors specific for K88 fimbriae, the latter being galactosylceramide.

Animals↗

Glycolipid receptors for attachment of Mycoplasma hyopneumoniae to porcine respiratory ciliated cells.

Glycolipid receptors for Mycoplasma hyopneumoniae attachment were analyzed by using a thin-layer chromatography (TLC) overlay assay. M. hyopneumoniae bound specifically to sulfatide, globoside, and monosialoganglioside GM3. No binding to sphingomyelin, cerebroside, lactosyl ceramide, ceramide trihexoside, monosialogangliosides GM1 and GM2, disialogangliosides (GD1a, GD1b, and GD3), trisialoganglioside (GT1b), cholesterol, cholesterol sulfate, palmitic acid, tripalmitin, or cholesteryl palmitate was detected. Total lipids extracted from cilia of the swine respiratory epithelium, the natural targets of M. hyopneumoniae infection, were also separated on TLC plates and overlaid with mycoplasmas. M. hyopneumoniae bound specifically to three ciliary glycolipids identified as La, Lb, and Lc. Binding to Lc was stronger than to La and Lb. All three lipids were believed to be sulfated glycolipids, as determined by laminin binding and staining with azure A. Lc was identified as a putative sulfatide because it has a mobility similar to that of authentic sulfatide and comigrated with sulfatide on TLC plates. Laminin bound to La, Lb, and Lc and produced dose-dependent inhibition of adherence of the mycoplasma to the three ciliary receptors. Binding of the mycoplasma to sulfatide, La, Lb, and Lc was partially inhibited by dextran sulfate, heparin, fucoidan, mucin, and chondroitin sulfate B. These substances blocked the adherence of M. hyopneumoniae to cilia and ciliated cells as shown in a previous study (Q. Zhang, T. F. Young, and R. F. Ross, Infect. Immun. 62:1616-1622, 1994). These results indicate that La, Lb, and Lc are the major native receptors for M. hyopneumoniae adherence to ciliated cells.

Animals↗

Brief heat shock treatment induces a long-lasting alteration in the glycolipid receptor binding specificity and growth rate of Haemophilus influenzae.

After brief heat shock treatment, clinical strains of nontypeable Haemophilus influenzae show a long-lasting change in the binding specificity for glycolipids and a markedly increased growth rate in vitro. Non-heat-shocked H. influenzae specifically binds to phosphatidylethanolamine (PE), gangliotetraosylceramide (Gg4), and gangliotriosylceramide (Gg3) and binds minimally to sulfatoxygalactosylceramide (SGC; also called sulfatide). After a 5-min heat shock at 42 degrees C, strains of H. influenzae showed a marked increase in binding to SGC and acquired the ability to bind to sulfatoxygalactosylglycerol (SGG) in thin-layer chromatography overlays. Additionally, heat-shocked H. influenzae cells showed an increased growth rate (twofold). Increased sulfatide binding and growth rate were retained for approximately 60 generations, after which the heat-shocked organisms reverted to their original glycolipid binding pattern (i.e., PE, Gg3, and Gg4) and growth rate. Such organisms could then be reexposed to heat, and the heat shock phenotype would be reestablished. After exposure of the organisms to brief heat shock, Western blotting of a surface extract of H. influenzae with anti-bovine-brain hsp-70 monoclonal antibody showed an increase in two protein bands at 82 and 60 kDa. This antibody was a potent inhibitor of the binding of heat-shocked H. influenzae to SGC and SGG but had no effect on PE, Gg3, or Gg4 binding in vitro. In contrast, an antibody against an H. influenzae PE-Gg3-Gg4-binding adhesin that was recently identified (J. Busse, E. Hartmann, and C. A. Lingwood, J. Infect. Dis. 175:77-83, 1996) selectively inhibited the organism's binding to PE and Gg3. This indicates that cell surface hsp-70-related heat shock proteins can mediate H. influenzae attachment to sulfoglycolipids following heat shock. We suggest that such increased binding to sulfated glycolipids may be a response to fever following H. influenzae infection in humans.

Antibodies, Blocking↗

Mycoside G, a Specific Glycolipid in Mycobacterium marinum (Balnei).

Navalkar, R. G. (University of Wisconsin, Madison), E. Wiegeshaus, E. Kondo, H. K. Kim, and D. W. Smith. Mycoside G, a specific glycolipid in Mycobacterium marinum (Balnei). J. Bacteriol. 90:262-265. 1965.-A new specific glycolipid in extracts prepared from strains designated Mycobacterium marinum and M. balnei has been demonstrated by use of the techniques of column chromatography and infrared spectroscopy. Since there is now agreement among many workers that M. marinum and M. balnei are identical, the demonstration of the same specific glycolipid in both species is not surprising. This substance, which we have designated mycoside G, is chemically similar to mycosides A and B, and apparently differs only in the sugar moiety. In addition, the lipids extracted from these cultures contain phthiocerol dimycocerosate, a wax component found also in M. tuberculosis and M. bovis.

Journal Article↗

Receptor-specific agglutination tests for detection of bacteria that bind globoseries glycolipids.

Specific binding to the globoseries of glycolipid receptors explains the adherence of uropathogenic Escherichia coli to host cells. The minimal receptor disaccharide Gal alpha 1----4Gal beta [galactose alpha (1----4)galactose beta] is recognized by most attaching clinical isolates. However, wild-type isolates can express adhesins with several different receptor specificities. Bioassays do not permit separate analysis of each receptor specificity, since the target cells contain multiple potentially receptor-active molecules. In this study, bacterial adhesins were analyzed by using receptors immobilized into latex beads in one of two ways. In one way, di- and trisaccharides were covalently linked via a spacer arm to latex beads coupled with bovine serum albumin. In the other way, receptor-active glycolipids were coated onto the bovine serum albumin-latex beads. The latex beads were subsequently used for agglutination by using type strains with known receptor specificity. The composition was optimized regarding receptor structure and size of latex beads. Gal alpha 1----4Gal beta was as active as the trisaccharide derivative Gal alpha 1----4Gal beta 1----3glucose or Gal alpha 1----4Gal beta 1----3glucosamine. Among the natural glycolipids tested, globotetraosylceramide was the most active. Subsequently, the sensitivity and specificity of the Gal alpha 1----4Gal beta-latex and globotetraosylceramide-latex reagents were compared for 733 E. coli urinary isolates. Hemagglutination of human erythrocytes was used as the positive standard. No significant difference in the specificity or sensitivity of the latex reagents was found; the sensitivity ranged from 86%, when isolates agglutinating human erythrocytes of blood groups P1 and p were included, to 93%, when those isolates agglutinating erythrocytes of blood group p were excluded. These reagents provide tools for bacterial identification in patients with urinary tract infection.

Adhesins, Escherichia coli↗

Glycolipid antigen induces long-term natural killer T cell anergy in mice.

Natural killer T (NKT) cells recognize glycolipid antigens presented by the MHC class I-related glycoprotein CD1d. The in vivo dynamics of the NKT cell population in response to glycolipid activation remain poorly understood. Here, we show that a single administration of the synthetic glycolipid alpha-galactosylceramide (alpha-GalCer) induces long-term NKT cell unresponsiveness in mice. NKT cells failed to proliferate and produce IFN-gamma upon alpha-GalCer restimulation but retained the capacity to produce IL-4. Consequently, we found that activation of anergic NKT cells with alpha-GalCer exacerbated, rather than prevented, B16 metastasis formation, but that these cells retained their capacity to protect mice against experimental autoimmune encephalomyelitis. NKT cell anergy was induced in a thymus-independent manner and maintained in an NKT cell-autonomous manner. The anergic state could be broken by IL-2 and by stimuli that bypass proximal TCR signaling events. Collectively, the kinetics of initial NKT cell activation, expansion, and induction of anergy in response to alpha-GalCer administration resemble the responses of conventional T cells to strong stimuli such as superantigens. Our findings have important implications for the development of NKT cell-based vaccines and immunotherapies.

Animals↗

Functions and potential applications of glycolipid biosurfactants--from energy-saving materials to gene delivery carriers.

Biosurfactants (BS) produced by various microorganisms show unique properties (e.g., mild production conditions, lower toxicity, higher biodegradability and environmental compatibility) compared to their chemical counterparts. The numerous advantages of BS have prompted applications not only in the food, cosmetic, and pharmaceutical industries but in environmental protection and energy-saving technology as well. Glycolipid BS are the most promising, due to high productivity from renewable resources and versatile biochemical properties. Mannosylerythritol lipids (MEL), which are glycolipid BS produced by a yeast Candida antarctrica, exhibit not only excellent interfacial properties but also remarkable differentiation-inducing activities against human leukemia cells. MEL also show a potential anti-agglomeration effect on ice particles in ice slurry used for cold thermal storage. Recently, the cationic liposome bearing MEL has been demonstrated to increase dramatically the efficiency of gene transfection into mammalian cells. These features of BS should broaden its applications in new advanced technologies. The current status of research and development on glycolipid BS, especially their function and potential applications, is discussed.

Journal Article↗

Alpha 2-adrenoceptor activity induces the antidepressant-like glycolipid in mouse forced swimming.

Antidepressants show an antidepressive-like behavioral effect on mouse forced swimming via induction of GalNAc alpha 1-3GalNAc-lipid. In order to detect the mechanisms of the neuron receptor activities on the effect, the glycolipid reactivities in the serum induced by tandospirone, a specific 5-HT1A receptor agonist, and clonidine, a specific alpha 2-adrenoceptor agonist, were investigated. Both of these agonists showed dose-dependent behavioral effects which were decreased by pretreatment with yohimbine, a specific alpha 2-adrenoceptor antagonist. The glycolipid reactivities corresponded with the effects. These present findings strongly suggest that alpha 2-adrenoceptor activity in the brain mediates the induction of the antidepressant glycolipid in the forced swimming test.

Adrenergic alpha-Agonists↗

Transfer of glycolipid between membranes of tissue culture cells, using dansylcerebroside as a model.

Donor cells, which had incorporated dansylcerebroside in their membranes, could further transfer this glycolipid to monolayers of acceptor cells. The case of transfer varied among acceptor cells, BHK cells being the best and MDBK cells the poorest acceptors of the cells tested. The process of transfer seemed to be mediated by lipids rather than by proteins of the membranes. The mode of attachment between donors and acceptors, such as classified as loose contact, tight adhesion or binding by lectin, did not significantly influence the extent of glycolipid transfer. However, modification of plasma membranes by infection of acceptor cells with myxoviruses resulted in enhancement of glycolipid transfer in some cases. Various factors have been evaluated with respect to dynamics of cellular membranes.

Animals↗

Immunochemical relationship between Forssman and globoside glycolipid antigens.

The rabbit antibody response to the human blood group P glycolipid antigen, globoside, GalNAc(beta 1-3)Gal(alpha 1-4)Gal(beta 1-4)Glc-Cer, has been examined with respect to cross-reactions with the structurally related Forssman glycolipid GalNAc(alpha 1-3)GalNAc(beta 1-3)Gal(alpha 1-4)Gal(beta 1-4)Glc-Cer. Immunoadsorbent columns were used to isolate three purified antibody populations from the anti-globoside sera: (1) fraction A, antibodies that cross-react with both glycolipids; (2) fraction B, antibodies that react with Forssman antigen but not with globoside; and (3) fraction C, antibodies that are specific for globoside. The proportion of each fraction in the total antibody response to globoside appears to be related to preexisting immunity to these antigens. A rabbit with a high preimmune titer to Forssman antigen produced a large amount of Forssman-specific antibody, whereas a rabbit with a low or nonexistent preimmune titer of anti-Forssman antibody produced large amounts of globoside-specific antibody. The presence of Forssman-specific antibody in an immune response to globoside is an example of a heteroclitic type of immune response.

Animals↗

"Core" glycolipid of enterobacteriaceae: immunofluorescent detection of antigen and antibody.

The Re chemotype mutant of Salmonella minnesota R595 has a cell-wall glycolipid composed principally of 2-keto, 3 deoxyoctonate and Lipid A, which is an antigen widely shared by Enterobacteriaceae. High-titered antiserum against this antigen can be conjugated with fluorescein isothiocyanate for direct detection of this antigen in heterologous bacteria and staining of bacteria in tissue. Alternatively, the indirect immunofluorescence technique can be used for antigen detection on bacterial surfaces and in tissues, and this method can quantitate glycolipid antibody in mammalian sera. The latter may be particularly useful in serologic studies because, although the glycolipid antigen is a surface antigen and purified extracts can be used to coat latex particles, high-titered antisera will not agglutinate bacteria or coated latex particles.

Animals↗

Another view of T cell antigen recognition: cooperative engagement of glycolipid antigens by Va14Ja18 natural T(iNKT) cell receptor [corrected].

Va14Ja18 natural T (iNKT) cells rapidly elicit a robust effector response to different glycolipid Ags, with distinct functional outcomes. Biochemical parameters controlling iNKT cell function are partly defined. However, the impact of iNKT cell receptor beta-chain repertoire and how alpha-galactosylceramide (alpha-GalCer) analogues induce distinct functional responses have remained elusive. Using altered glycolipid ligands, we discovered that the Vb repertoire of iNKT cells impacts recognition and Ag avidity, and that stimulation with suboptimal avidity Ag results in preferential expansion of high-affinity iNKT cells. iNKT cell proliferation and cytokine secretion, which correlate with iNKT cell receptor down-regulation, are induced within narrow biochemical thresholds. Multimers of CD1d1-alphaGalCer- and alphaGalCer analogue-loaded complexes demonstrate cooperative engagement of the Va14Ja18 iNKT cell receptor whose structure and/or organization appear distinct from conventional alphabeta TCR. Our findings demonstrate that iNKT cell functions are controlled by affinity thresholds for glycolipid Ags and reveal a novel property of their Ag receptor apparatus that may have an important role in iNKT cell activation.

Animals↗

[Characterization of glycolipid sulfotransferase fractionated from rat kidney and involvement of the enzyme in human cancer].

3'-Phosphoadenosine 5'-phosphosulphate: galactosylceramide sulfotransferase which catalyzes the sulfation of galactosylceramide (GalCer) was partially purified from a rat kidney light membrane fraction, and the properties were studied with special reference to substrate specificity. In order to study minimum molecular requirement of the acceptor substrate for the sulfotransferase, analogues of galactosylceramide where omega-amiocaproic or omega-aminododecanoic acid is substituted for the acyl moiety of the native glycolipid were chemically synthesized by improved procedures. The artificial glycolipids were sulfated effectively by the kidney enzyme, suggesting that the synthetic compounds will serve for affinity ligands for the purification of the enzyme. The extent of sulfation in the synthetic compounds was comparable with that of galactosylceramide containing normal acids and higher than that of galactosylsphingonine in which one of hydrocarbon chains is deleted from the native glycolipid. Through substrate specificity experiments, the sulfotransferase has relatively broad specificity acting on beta-linked galactosides at nonreducing ends of mono- and disaccharides which bind, at least, one hydrocarbon chain. Enzyme kinetic analysis by competition assay using mixed acceptors demonstrated that the same, single sulfotransferase catalyzes sulfation of galactosylceramide, lactosylceramide and galactosylsphingosine. As to human cancer, the sulfotransferase activity was hardly detectable in Wilms' tumor tissues that contrasts with renal cell carcinoma tissues where the markedly elevated level was previously demonstrated. When sera from patients with various cancers were examined for the enzyme level, many cases of hepatocellular carcinoma showed significantly increased activity, whereas the hepatoma tissues had hardly detectable level of the enzyme. These observations suggest that a humoral factor derived from the hepatoma induces the sulfotransferase.

Animals↗

Specificity of the glycolipid transfer protein from pig brain.

Lipid specificity has been studied in the lipid transfer reaction facilitated by the glycolipid transfer protein from pig brain. The lipid transfer was measured by determining the transfer of a radioisotopically labeled lipid from donor liposomes to either acceptor liposomes or mitochondria. Whenever possible, the liposomes contained 1 mol % of the lipid whose transfer was under study. The transfer protein accelerates the transfer of glucosylceramide, galactosylceramide (GalCer), lactosylceramide (LacCer), galactosylceramide 3-sulfate, globotriaosylceramide, LacCer sulfate, sialosyl-LacCer, globotetraosylceramide, and globopentaosylceramide. An inverse relationship is found between the length of sugar chains in glycosphingolipids and the transfer rates. In addition to the glycosphingolipids, the transfer protein facilitates the transfer of galactosyldiacylglycerol, digalactosyldiacylglycerol, glucosyldiacylglycerol, and diglucosyldiacylglycerol. The protein does not facilitate the transfer of dimannosyldiacylglycerol. The transfer of periodate-oxidized and subsequently reduced derivatives of GalCer and LacCer is facilitated by the transfer protein. The derivatives of GalCer are transferred at lower rates than GalCer, whereas the derivatives of LacCer are transferred at higher rates than LacCer. The transfer protein does not facilitate the transfer of phosphatidylcholine, phosphatidylinositol, cholesterol, or cholesteryloleate. These results suggest that the glycolipid transfer protein from pig brain has specificity to hydroxyl groups present in the sugar residue directly linked to either ceramide or diacylglycerol. The presence of glucose or galactose linked to these hydrophobic moieties makes the glycolipid transferable by the protein.

Animals↗

Glycolipids of murine lymphocyte subpopulations. Structural characterization of thymus gangliosides.

Several sialic acid-containing glycolipids have been isolated and purified from murine thymus. Eleven of these acidic glycolipids (gangliosides) have been analyzed by several techniques, permitting us to propose a tentative structure for each. Sugar analysis reveals that all 11 gangliosides contain the same base structure, Gal(beta 1-3)GalNAc(beta 1-4)Gal(beta 1-4)Glc(beta 1-1) ceramide, which is gangliotetraosylceramide. No glucosamine or fucose is present in glycolipids from this tissue. There appears to be considerable heterogeneity in the sialic acid moiety of these gangliosides with respect to their structures and linkages. One ganglioside contains only N-glycolylneuraminic acid. Four of the gangliosides are common brain-type structures and contain sialic acid linked to internal galactose which is insusceptible to Vibrio cholerae neuraminidase. In addition, four of the gangliosides are susceptible to base hydrolysis and are believed to contain O-acetyl derivatives of sialic acid. Two gangliosides have also been shown to contain both N-acetyl- and N-glycolylneuraminic acid. There is also heterogeneity within the ceramide portion of some gangliosides which may be responsible for the appearance of an additional three ganglioside bands on thin layer chromatography.

Animals↗