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Serological specificity of phenolic glycolipid I from Mycobacterium leprae and use in serodiagnosis of leprosy.

The serological activities of the specific phenolic glycolipid I from Mycobacterium leprae, its dissected parts, and related glycolipids from other mycobacteria were examined by enzyme-linked immunosorbent assay against hyperimmune anti-M. leprae rabbit antiserum and sera from patients with leprosy and other mycobacterial diseases. High anti-phenolic glycolipid I immunoglobulin M antibodies were found in 23 of 24 (96%) of lepromatous leprosy patients on short term chemotherapy and in 8 of 13 tuberculoid leprosy patients (62%). Sera from patients with tuberculosis or atypical mycobacterial infections were devoid of anti-phenolic glycolipid I activity. The structurally related phenolic glycolipids from Mycobacterium kansasii and Mycobacterium bovis and the aglycone segments of the M. leprae product showed no significant activity. Thus, the trisaccharide determinant of phenolic glycolipid I is specific in its structure, serological activity, and, to a lesser extent, the antibody class it evokes.

Animals↗

Porcine 987P glycolipid receptors on intestinal brush borders and their cognate bacterial ligands.

Certain strains of enterotoxigenic Escherichia coli adhere to piglet intestinal epithelial cells by means of the 987P fimbriae. The 987P fimbrial structure consists of a helical arrangement of three fimbrial proteins, namely, the major subunit FasA and two minor subunits, FasF and FasG. FasG, which is located at the fimbrial tip and at various positions along the fimbriae, mediates 987P binding to glycoprotein receptors. In this study, we isolated and analyzed the structure of piglet glycolipid brush border receptors and characterized their cognate ligands on the 987P fimbriae. Two major glycolipid bands recognized by 987P fimbrial probes in thin-layer chromatography overlay assays were further purified by high-performance thin-layer chromatography and shown to comigrate with control galactosylceramide containing hydroxylated fatty acids and with sulfatide. Their structures were confirmed by fast atom bombardment mass spectrometry, which detected homologous series of ceramide monohexoside and sulfatide with hydroxylated fatty acyl chains ranging from h16:0 to h24:0. Assembled 987P fimbriae, pre- and postassembly dissociated fimbrial subunits, and Fab fragments of specific anti-FasG, -FasF, and -FasA were used to inhibit 987P-mediated bacterial binding to the two identified piglet glycolipids and corresponding isoreceptor controls. Only assembled fimbriae and anti-FasG Fab fragments were significantly able to inhibit bacterial binding to sulfatide, indicating that in addition to glycoproteins, FasG recognizes a specific glycolipid of piglet brush borders. In contrast, only anti-FasA Fab fragments were significantly able to inhibit bacterial binding to galactosylceramide with hydroxylated fatty acids and piglet hydroxylated ceramide monohexoside, indicating that FasA may determine a third type of ligand-receptor interaction in the piglet intestines. Since these bacterial adhesins recognize their respective glycolipid receptors only after being assembled in their final fimbrial quaternary structure, adhesin binding may involve cooperative interactions and the subunits by themselves may have very low binding affinities. Alternatively, conformation-sensitive domains of these subunits present in the assembled fimbriae may be required for glycolipid binding.

Adhesins, Escherichia coli↗

Myelin palingenesis. 2. Immunocytochemical localization of myelin/oligodendrocyte glycolipids in multilamellar structures.

Myelin is a membrane with unique characteristics that set it apart from any other membrane. It has a very high lipid:protein ratio (approximately 75:25) not found in other multilamellar membranes; it has a high content of two glycolipids--galactocerebrosides and sulfatides--which account for 26.5% of its lipids. The physiological role of these glycolipids in myelin and/or oligodendrocytes is unknown, but evidence that Abs directed against them interfere with myelination has been presented. Moreover, one of the early events in the process of oligodendrocyte differentiation prior to myelination is the acquisition of galactocerebroside on their surface. In earlier work, we have demonstrated that adhesion of oligodendrocytes to a positively charged substratum signals the commencement of myelinogenesis. Among the events that take place following adhesion are the rapid synthesis of glycolipids. Since over time in culture, oligodendrocytes elaborate multilamellar membranes that contain all the myelin characteristic proteins, we undertook to investigate whether these structures also accumulate myelin characteristic glycolipids. We have used three monoclonal antibodies--two are directed against galactocerebroside, the third one is an antisulfatide--in conjunction with the immunogold method, at the electron microscopic level, to examine the distribution of these glycolipids in the multilammelar structures that accumulate in long-term oligodendrocyte cultures. Our data show that galactocerebrosides and sulfatides are present in the multilamellar structures. Staining is easily demonstrated on the outermost membrane. However, as was the situation with myelin proteins, detection of these glycolipids on the inner lamellae is only achieved at the expense of destroying the ultrastructure. This is so, independent of whether the structures are compact or not.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Functional role of antibody against "core" glycolipid of Enterobacteriaceae.

Antibodies against the "core" glycolipid of Enterobacteriaceae (2-keto, 3-deoxyoctonate-Lipid A) have been associated with protection against the sequelae of gram-negative rod bacteremia. To investigate the nature of this protection, dogs and rabbits were immunized with purified glycolipid prepared by phenol-chloroform-petroleum ether extraction of the "Re" mutant of Salmonella minnesota 595 and opsonophagocytic and bactericidal tests were carried out using lapine peritoneal granulocytes and serum factors. Whereas 1-4 mug/kg of glycolipid i.v. produced hypotension in dogs and 8 mug/kg i.v. was lethal, a rising dosage schedule of immunization with an average total dose of 1 mg/kg produced striking protection against shock and death against challenge with heterologous organisms. 20 control dogs, given approximately 10(10) live, serum-resistant Escherichia coli 0.85:H9 or Serratia marcescens 03 during a continuous intra-arterial pressure transducer recording, showed a drop in mean systolic pressure from 186 (+/- 6 SE) to 101 (+/- 12 SE) MM Hg and a fall in mean diastolic pressure from 118 (+/- 3 SE) to 64 (+/- 8 SE) mm Hg within 60-120 min. Minor pressure changes (average less than 12% of prechallenge levels) were seen in the same number of immunized dogs. In contrast, no significant difference was noted in the bloodstream clearance of these serum-resistant organisms over a period of 4-6 h between immunized and control dogs. Intravascular clearance was greater in animals immunized with the challenged strain or in glycolipid-immunized animals challenged with highly serum-sensitive E. coli 0.14:K7. Antibody against core glycolipid protected against the hemodynamic sequelae of bacillemia, augmented intravascular clearance of serum-sensitive organisms, and abrogated the pyrogenic response to enteric bacilli, but did not enhance clearance of serum-resistant organisms. Although canine and lapine antiserum against core glycolipid passively protected mice against a heterologous challenge, opsonophagocytic and bactericidal activity was at least 100-fold less than type-specific antiserum.

Animals↗

Isolation of mycolic acid-containing glycolipids in Nocardia rubra and their granuloma forming activity in mice.

Three classes of glycolipids (TMM (trehalose monomycolate), TDM (trehalose dimycolate) and GM (glucose mycolate] containing mycolic acids as hydrophobic components were isolated from a strain of Nocardia rubra (Rhodococcus rubrum) and their structures have been partially characterized using infrared spectrometry, gas-liquid chromatography and gas chromatography-mass spectrometry. Acid or alkaline hydrolysis of isolated glycolipids revealed that trehalose was the sole water soluble component in TMM and TDM, while glucose was the hydrophilic component in GM. On the other hand, saturated, monoenoic and dienoic mycolic acids with carbon atoms ranging from C36 to C50 contained constituents of fatty acid moiety at C44. From the analytical results, TMM, TDM and GM were tentatively identified as trehalose monomycolate, trehalose dimycolate and glucose monomycolate, respectively. The mycolic acid composition differed significantly by the glycolipid classes: the highest amount of saturated mycolic acids were detected in TMM and GM, while a significant amount of dienoic mycolic acids have been found in TDM and the cell wall bound lipid fraction (BL). All these three classes of glycolipids containing mycolic acids showed strong granuloma forming activity in lungs and spleen of ICR mice 1 week after intravenous injection of 100 to 500 micrograms glycolipid in W/O/W micelles containing Freund's incomplete adjuvant. These results indicated that glycolipids containing shorter carbon chain mycolic acids ranging C40-50, corresponding to less acyl numbers or monosaccharides such as glucose, can also produce foreign body-type granuloma in mice without protein antigens.

Animals↗

[Granuloma formation in mice by liposomes of new glycolipid containing mycolic acids, "trehalose 2,3,6'-trimycolate"].

After intravenous administration of liposomes containing a novel glycolipid, trehalose 2,3,6'-trimycolate, isolated from Rhodococcus aurantiacus, the granuloma formation was observed distinctively in the lungs, spleen and liver of ICR mice. The concentration of the glycolipid in liposomes influenced profoundly on the inducibility for granulomas and the liposomes containing 10 mol% of trehalose 2,3,6'-trimycolate showed the highest activity for the granuloma formation in mice without a significant loss of body weight of mice with a less toxicity. EggPC liposomes are of more highly inducible for the granuloma formation in mice than eggPC:PS (7:3, mol ratio) liposomes or eggPC:Chol (9:1, mol ratio) liposomes, suggesting that cholesterol in liposomes was not necessarily essential for inducing granulomas in mice. Among the various PC liposomes, synthetic dimyristoyl-phosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC) liposomes were of highly inducible, although a high responsiveness was also observed with natural lecithines such as egg or soy PC. However, distearoylphosphatidylcholine (DSPC) liposomes did not show any activity for the granuloma formation in any organs of mice. Organ responsiveness differed significantly in the micelle forms injected. Glycolipid entrapped with eggPC-Chol liposomes showed a lower lung index below 1.0, in contrast to w/o/w micelles containing Freunds' incomplete adjuvant, which showed a higher granuloma formation in the lungs. It was also noted that the toxicity of glycolipid containing liposomes was comparatively lower than the toxicity of glycolipid containing w/o/w micelles, indicating that the liposomes appeared to be more suitable for the induction of the immunomodifying activities of mycolic acid-containing glycolipids than w/o/w micelles.

Animals↗

Ethanol-induced changes in glycolipids of Saccharomyces cerevisiae.

Total glycolipid content of Saccharomyces cerevisiae cells increased in ethanol-treated yeast cells. Sialic acid and hexosamine contents of glycolipids from ethanol-treated cells decreased, whereas those of hexoses increased. Increased sialidase activity in the presence of ethanol may be responsible for the decrease in sialic acid content of glycolipids. The saccharide moieties of glycolipids of S. cerevisiae consisted of fucose, mannose, galactose, and glucose. Ethanol treatment of yeast cells caused an increase in glucose and a decrease in galactose content of glycolipids. The changes in glucose content can be related to changes in beta-glucosidase activity under alcohol stress. The content of cerebrosides, sulfatides, and monoglucosyldiglycerides was enhanced following ethanol treatment. An increase in cerebroside as well as in sulfatide content during alcohol stress might play an important role in stabilizing the membrane both physically and structurally. Such variations in glycolipid content and composition of S. cerevisiae cells may represent an adaptive response to ethanol stress.

Cerebrosides↗

The carbohydrate- and lipid-containing cell wall of mycobacteria, phenolic glycolipids: structure and immunological properties.

Phenolic glycolipids were first discovered as cell-wall constituents of M. bovis, M. bovis BCG, M. marinum, and M. kansasii. Recently, such compounds were also isolated from M. leprae and have been shown to be specific-species serological markers. Moreover, they seem to be involved, in the case of lepromatous leprosy, in the stimulation of the suppressor T-cells. The functional activities of these phenolic glycolipids over the immune cells stimulation emphasized the role played by these molecules in the mycobacteria pathogenicity. Phenolic glycolipids have also been found in M. gastri and M. tuberculosis strain Canetti. From a structural point of view, these glycolipids contain the same aglycon moiety mainly assigned to phenolphthiocerol diester while the sugar part structure confers to some of these glycolipids their antigenic specificity. The search of immunoreactive glycolipids and their function analysis remain a challenge for chemists and immunologists for the understanding of the mycobacteria pathogenicity.

Animals↗

The fatty acid and monosaccharide compositions of three neutral and three phosphorylated glycolipids isolated from Leishmania donovani promastigotes grown in a chemically defined medium.

Several lipids and macromolecular lipoconjugates of Leishmania spp. have now been well characterized; however, the glycolipids of L. donovani have not been thoroughly examined. In the present study, 3 neutral and 3 phosphorylated glycolipids were detected in promastigote forms of the organism grown in a chemically defined medium. The fatty acid and sugar compositions of these glycolipids, isolated and purified by adsorption column chromatography and thin-layer chromatographic procedures, were identified and quantified by gas-liquid chromatography and mass spectrometry. Myristate (14:0), palmitate (16:0), palmitoleate (16:1), stearate (18:0), oleate (18:1), and linoleate (18:2) were the major fatty acids in all 6 glycolipids. Arabinose, mannose, glucose, and galactose were detected in the glycolipids. The biochemical nature of these lipids suggested that the major components in the isolated preparations of the 6 glycolipids are diacylglycerophospholipids, distinct from the major precursors of macromolecular lipoconjugates such as the lipid anchors of cell surface antigens that have been reported. These appear to be terminal products of lipid biosynthesis in this parasite.

Animals↗

Brefeldin A induced inhibition of de novo globo- and neolacto-series glycolipid core chain biosynthesis in human cells. Evidence for an effect on beta 1-->4galactosyltransferase activity.

De novo synthesis of neolacto-series glycolipids has been studied in human cell lines via metabolic labeling of ceramide with [3H]serine. Intense labeling of ceramide mono- and dihexoside glycolipids occurred with labeling of progressively longer chain derivatives with increasing time. Most of the label was recovered in neutral glycolipids with about 5% of the total labeling in the ganglioside fraction. Experiments done using cell treatment with 2.5 micrograms/ml brefeldin A resulted in a stimulation in the total amount of labeling, accumulation of a neutral glycolipid identified as Lc3 due to inhibited transfer of the neolacto-series core chain terminal beta-Gal residue, and a corresponding inhibition of labeling of longer chain neutral glycolipids in all cell lines. Brefeldin A also blocked synthesis of the globo-series precursor, Gb3, longer chain sialylated structures such as IV3NeuAcnLc4, but not de novo GM3 synthesis. Brefeldin A treatment had no effect on cellular beta 1-->3N-acetylglucosaminyl-, beta 1-->4galactosyl-, or alpha 1-->3fucosyltransferase specific activities, nor was it inhibitory in beta 1-->4galactosyltransferase assays in vitro. The results describe brefeldin A-induced blocks in globo- and neolacto-series glycolipid biosynthesis, consistent with differential localization of enzymes in intracellular membranes. In particular, the results suggest that the beta 1-->4galactosyltransferase in these cells is either not redistributed by brefeldin A or is otherwise rendered nonfunctional.

Anti-Bacterial Agents↗

[Screening for anti-glycolipid antibody profiles from patients with immune-mediated peripheral neuropathies by Dotzen Ganglio Profile Antibodies].

The presence of anti-glycolipid specific antibodies have been found to be associated with acute and chronic immune-mediated peripheral neuropathies. Recently a number of anti-glycolipid antibody assays have became commercially available. In this study we established specific anti-glycolipid antibody profiles in a series of sera by the Dotzen Ganglio Profile antibodies. This kit screens for the simultaneous detection of ten anti-glycolipid antibodies against GM3, GM2, GM1, GD3, GD1a, GD1b, GT1a, GT1b, GQ1b gangliosides and sulfatides of the IgM and IgG classes. Sera from 89 patients with acute and chronic neuropathies were selected in a well-characterized cohort of banked sera with anti-glycolipid antibody profiles identified by in-house immunodot assay. Serum from 52 clinical variants of Guillain-Barré syndrome with IgG autoantibody profiles and 37 chronic acquired peripheral neuropathy with IgM autoantibody profiles were tested. The assay correctly identified with good agreement 50 of 52 IgG antibody profiles and 32 of 37 IgM antibody profiles. The assay compared well with in-house immunodot assay. It is easy to screen 10 crossreacting glycolipid antibodies to establish specific antibody profiles to define different subgroups of immune-mediated peripheral neuropathies for classification and immune management.

Autoantibodies↗

[The glycolipids of the causative agent of tularemia].

Up to 10 glycolipids were detected in F. tularensis with the use of thin-layer chromatographic techniques. These glycolipids were slime antigens of F. tularensis membrane. Attenuated F. tularensis strains were found to have defects in their glycolipid composition: in the vaccine strain glycolipid 8 was replaced by more polar lipid 8-a; the avirulent strain had only two glycolipids, and one of them was not typical for virulent strains. Considering that glycolipids differed from entero-bacterial Vi-antigen in their physical-chemical and biological properties, the suggestion was made that the use of the symbol "Vi" to denote the surface substances of F. tularensis should be abolished.

Bacterial Proteins↗

Had-1, a uridine 5'-diphosphogalactose transport-defective mutant of mouse mammary tumor cell FM3A: composition of glycolipids, cell growth inhibition by lactosylceramide, and loss of tumorigenicity.

Glycolipid compositions of mouse mammary tumor cell FM3A and its Newcastle disease virus-resistant mutant cell, Had-1, which was also characterized as a defective mutant of UDP-galactose transport to Golgi apparatus, have been studied. The major neutral glycolipid in FM3A was Gal beta 1-4Glc beta 1-1Cer (LacCer) (95%) and the rest was Glc beta 1-1Cer. The concentration of neutral glycolipids in Had-1 was only about one-fifth of that in FM3A. GlcB1-1Cer in Had-1 accounted for 79% of neutral glycolipids and the rest was LacCer, the content of which was decreased to 4% of that in FM3A. Ganglioside patterns of the two cell lines were similar, although gangliosides with N-glycolylneuraminic acid were increased in Had-1 cells compared with that in FM3A cells. The presence of NeuAc alpha 2-3-Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer, NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-2Cer, GM3, and GD3 was demonstrated by thin-layer chromatography immunostaining. 125I-Labeled Newcastle disease virus bound only poorly to gangliosides extracted from either FM3A or Had-1 cells on a high performance thin-layer chromatography plate. The effects of glycolipids on the growth of the two cell lines were also studied. Had-1 cells were more sensitive to glycolipids added exogenously than FM3A cells. Addition of GM3 had a stimulative effect on cell growth of Had-1. LacCer, Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 4-1Cer, and Glc beta 1-1Cer inhibited the growth of Had-1 cells. LacCer was the most potent inhibitor. LacCer immobilized on the culture plate also inhibited the growth of Had-1 cells. The inhibitory effect was recovered completely overcome by transferring the cells to LacCer-free medium. Had-1 cells were not tumorigenic in C3H/He mice, and furthermore the tumorigenic activity of FM3A cells was suppressed by the prior administration of Had-1 cells.

Animals↗

Characterization of sulfated glucuronic acid containing glycolipids reacting with IgM M-proteins in patients with neuropathy.

In some patients with neuropathy and plasma cell dyscrasia, the serum IgM M-proteins are known to bind to the myelin associated glycoprotein and to peripheral nerve glycolipids. We have isolated two acidic glycolipids which bind to the M-protein from human cauda equina by DEAE-Sephadex, Iatrobeads, and high performance liquid column chromatographies. The major acidic glycolipid migrated between GM1 and GD1a and the minor acidic glycolipid migrated between GD1a and GD1b. Their structures were elucidated by sugar analysis, enzymatic digestion, mild acid hydrolysis, permethylation, fast atom bombardment mass spectrometry, and NMR studies. Their core structure was confirmed to be paragloboside by high performance thin-layer chromatography-immunostaining using anti-paragloboside monoclonal antibody. Both acidic glycolipids lacked sialic acid but contained sulfated glucuronic acid as their acidic moiety. The sulfate group in the glucuronic acid was established by periodate oxidation and permethylation studies to be attached to the 3 position. The structures of the two acidic glycolipids are therefore consistent with the following: IV3GlcUA(3-sulfate)nLcOse4Cer and VI3GlcUA(3-sulfate)nLcOse6Cer. Additionally, the free carboxyl group on the glucuronic acid residue was shown to be necessary to bind the IgM M-proteins from neuropathy patients.

Antibodies, Monoclonal↗

Adhesion of Mycoplasma pneumoniae to sulfated glycolipids and inhibition by dextran sulfate.

A virulent strain of Mycoplasma pneumoniae was metabolically labeled with [3H]palmitate and studied for binding to glycolipids and to WiDr human colon adenocarcinoma cells. The organism binds strongly to sulfatide and other sulfated glycolipids, such as seminolipid and lactosylsulfatide which all contain terminal Gal(3SO4) beta 1-residues and weakly to some neolactoseries neutral glycolipids. M. pneumoniae do not bind gangliosides including the sialylneolacto-series and other neutral glycolipids that were tested. Only metabolically active M. pneumoniae cells bind to sulfatide, as binding is maximal in RPMI medium at 37 degrees C and almost completely abolished in nutrient-deficient medium or by keeping the cells at 4 degrees C. Dextran sulfate but not other sulfated or anionic polysaccharides at 10 micrograms/ml completely inhibits binding of M. pneumoniae to purified sulfatide. Dextran sulfate does not inhibit binding to the neolacto-series neutral glycolipids. Dextran sulfate partially inhibits adhesion of M. pneumoniae to cultured human colon adenocarcinoma cells (WiDr). The biological relevance of these data is suggested by our finding that sulfatide occurs in large amounts in human trachea, lung, and WiDr cells. Thus, there are at least two distinct receptors that mediate binding of M. pneumoniae to cells: glycolipids containing terminal Gal(3SO4) beta 1-residues as reported here, and glycoproteins containing terminal NeuAc alpha 2-3Gal beta 1-4GlcNAc sequences (Roberts, D. D., Olson, L. D., Barile, M. F., Ginsburg, V., and Krivan, H. C. (1989) J. Biol. Chem. 264, 9289-9293).

Animals↗

Comparative studies of antigenic glycolipids of mycobacteria related to the leprosy bacillus.

The leprosy bacillus, Mycobacterium leprae, is a member of a small group of mycobacteria comprising the species Mycobacterium bovis, Mycobacterium marinum, Mycobacterium kansasii, Mycobacterium tuberculosis, Mycobacterium ulcerans and related taxa. This relationship is based on the similarity of the characteristic lipid types in the cell envelope. Mycobacterium leprae produces a phenolic glycolipid antigen which is species specific. This communication reports a comparison of the specificity of the lipid antigens of other members of this group of mycobacteria. Mycobacterium kansasii, in accordance with previous studies, produces phenolic glycolipid and trehalose-based lipooligosaccharide antigens which do not cross react with antisera raised against other mycobacteria. The phenolic glycolipid and an uncharacterised polar glycolipid, with the properties of a lipooligosaccharide, from Mycobacterium marinum are also shown to be specific antigens. An acylated trehalose glycolipid antigen from Mycobacterium tuberculosis H37Rv reacts strongly with antisera raised against the same strain and sera from eight out of ten tuberculosis patients. The phenolic glycolipid antigen, isolated only from Mycobacterium tuberculosis "Canetti" variants, did not react with antisera raised against the type strain, Mycobacterium tuberculosis H37Rv, although it had been shown previously to react with sera from tuberculosis patients. It is apparent that there are populations of the tubercle bacillus which differ in the lipid antigens expressed on their cell surface.

Antigens, Bacterial↗

[Functional significance of glycolipid as an important plasma membrane constituent].

Glycolipids are ubiquitous membrane components in various mammalian cells and participate in several functions through membrane. Until now, about 180 glycolipids with different carbohydrate structures have been characterized. Since alteration in glycolipids during cellular proliferation, differentiation and transformation is dramatic in general, several approaches have been attempted to clarify the meaning. Our findings that ganglioside composition in human milk is characteristically changed during the periods of lactation and that N-(O-linoleoyl-omega-hydroxylignoceroyl) sphingosyl glucose (ester CMH) in mammalian skin is characteristically synthesized during differentiation of epidermis are the examples to indicate the close association of glycolipids in cellular functions. GM3 ganglioside appeared in the later period of lactation has an activity to suppress cell growth of human breast cancer and ester CMH has an activity to promote the keratinization of epidermal cells, respectively. The observations indicate that glycolipids synthesized during cellular differentiation function as an signal transducer through plasma membrane. Structural bases of glycolipids to give such physiological activities are summarized and discussed in this communication.

Animals↗

The generation of infective stage Leishmania major promastigotes is associated with the cell-surface expression and release of a developmentally regulated glycolipid.

A monoclonal antibody, 3F12, was generated which reacted specifically against infective or metacyclic stage Leishmania major promastigotes, but not with noninfective promastigotes obtained from log phase cultures. The antibody recognized a cell surface and released molecule that could be metabolically labeled with [14C]glucose, [3H]mannose, [3H]galactose, and [3H]palmitic acid, but not with [35S]methionine or [3H]leucine. The molecule was the major species surface-labeled by [3H]sodium borohydride after periodate treatment. The glycolipid appeared to be shed primarily as free carbohydrate because 70% of the released material partitioned in the aqueous fraction after phase separation in TX-114. The molecule could be distinguished from the L. major glycolipid which has already been extensively described because its migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was of higher relative m.w. However, a close relationship between the two molecules was indicated by the finding that another monoclonal antibody, WIC-79.3, recognized both forms of the glycolipid; one produced and released only by log phase promastigotes, and one produced and released only by metacyclic promastigotes. The loss of agglutination with peanut agglutinin which has been shown to accompany metacyclogenesis was found to be caused by the loss of expression of the log form of the glycolipid which in most cases appeared to be the result of the developmental modification of this molecule. A survey of a number of virulent and avirulent. L. major strains and clones reinforced an absolute association between the ability of these promastigotes to initiate infection in BALB/c mice and their expression and release of the 3F12-binding, developmentally regulated form of the glycolipid. Not only does this glycolipid serve as the first well defined molecular marker for infective stage metacyclic promastigotes, but its unique structure is very likely to contribute to the adaptive changes that allow these parasites to survive within the vertebrate host.

Animals↗