Active site mapping of Loxosceles phospholipases D: Biochemical and biological features.

Brown spider phospholipases D from Loxosceles venoms are among the most widely studied toxins since they induce dermonecrosis, triggering inflammatory responses, increase vascular permeability, cause hemolysis, and renal failure. The catalytic (H12 and H47) and metal-ion binding (E32 and D34) residues in Loxosceles intermedia phospholipase D (LiRecDT1) were mutated to understand their roles in the observed activities. All mutants were identified using whole venom serum antibodies and a specific antibody to wild-type LiRecDT1, they were also analyzed by circular dichroism (CD) and differential scanning calorimetry (DSC). The phospholipase D activities of H12A, H47A, H12A-H47A, E32, D34 and E32A-D34A, such as vascular permeability, dermonecrosis, and hemolytic effects were inhibited. The mutant Y228A was equally detrimental to biochemical and biological effects of phospholipase D, suggesting an essential role of this residue in substrate recognition and binding. On the other hand, the mutant C53A-C201A reduced the enzyme's ability to hydrolyze phospholipids and promote dermonecrosis, hemolytic, and vascular effects. These results provide the basis understanding the importance of specific residues in the observed activities and contribute to the design of synthetic and specific inhibitors for Brown spider venom phospholipases D.

Characterization of Brown spider (Loxosceles intermedia) hemolymph: cellular and biochemical analyses.

This is the first study on the hemolymph from a spider of the Loxosceles genus. These animals are responsible for a great number of envenomation cases worldwide. Several studies on Loxosceles venoms have been published, and the knowledge about the venom and its toxins is considerable, not only regarding the biological and biochemical characterization, but also regarding structural, genetic and phylogenetic approaches. However, the literature on Loxosceles hemolymph is nonexistent. The main goal of the present study was to characterize biochemically the hemolymph content, and especially, to identify its different hemocytes. Moreover, many papers have already shown molecules whose source is the hemolymph and their very interesting activities and biomedical applications, for example, antifungal and antibacterial activities. A 2D-SDS-PAGE of brown spider hemolymph showed approximately 111 spots for pH 3-10 and 150 spots for pH 4-7. A lectin-blotting assay showed that hemolymph carbohydrate residues were similar to those found in venom. Several types of TAG and DAG phospholipids were found in the hemolymph and characterized by HPTLC and mass spectrometry. Four different hemocytes were characterized in Loxosceles intermedia hemolymph: prohemocyte, plasmatocyte, granulocyte and adipohemocyte. This paper opens new possibilities on toxinology, studying an unknown biological material, and it characterizes a source of molecules with putative biotechnological applications.

Sphingolipids of the mycopathogen Sporothrix schenckii: identification of a glycosylinositol phosphorylceramide with novel core GlcNH(2)alpha1-->2Ins motif.

Acidic glycosphingolipid components were extracted from the yeast form of the dimorphic mycopathogen Sporothrix schenckii. Two minor and the major fraction from the yeast form (Ss-Y1, -Y2, and -Y6, respectively) have been isolated. By a combination of 1- and 2-D 1H-nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and gas chromatography/mass spectrometry (GC/MS), Ss-Y6 was determined to be triglycosylinositol phosphorylceramide with a novel glycan structure, Manalpha1-->3Manalpha1-->6GlcNH(2)alpha1-->2Ins1-P-1Cer (where Ins=myo-inositol, P=phosphodiester). While the GlcNH(2)alpha1-->6Ins1-P- motif is found widely distributed in eukaryotic GPI anchors, the linkage GlcNH(2)alpha1-->2Ins1-P- has not been previously observed in any glycolipid. Ss-Y1 and Ss-Y2 were both found to have the known glycan structure Manalpha1-->3Manalpha1-->2Ins1-P-1Cer. Together with the results of a prior study [Toledo et al. (2001) Biochem. Biophys. Res. Commun. 280, 19-24] which showed that the mycelium form expresses GIPCs with the structures Manalpha1-->6Ins1-P-1Cer and Manalpha1-->3Manalpha1-->6Ins1-P-1Cer, these results demonstrate that S. schenckii can synthesize glycosylinositol phosphorylceramides with at least three different core linkages.

Prevalence of serum antibodies to Helicobacter pylori VacA and CagA and gastric diseases in Chile.

The objective of this study was to evaluate the prevalence of antibodies to Helicobacter pylori CagA and VacA proteins and correlate this prevalence with gastric diseases in colonised Chileans. The study was performed in 418 adults colonised with H. pylori: 316 with gastroduodenal pathology (152 duodenal ulcer, 14 gastric cancer and 150 gastritis patients) and 102 asymptomatic subjects. Serum IgG antibodies to H. pylori were determined by enzyme immunoassay (EIA). Antibodies to VacA and CagA proteins were detected by Western blotting. In a subgroup of the patients, the vacuolating activity was determined by HeLa cell assay and the CagA product was confirmed by PCR assay. IgG antibodies to both VacA and CagA proteins of H. pylori were found in 270 (85%) of 316 colonised gastric patients and in 72 (71%) of 102 asymptomatic subjects. Colonisation with virulent strains was significantly higher among duodenal ulcer and gastric cancer patients than in gastritis patients or asymptomatic subjects. Infections with VacA+/ CagA+ H. pylori strains is common in Chile but, in contrast to some Asian countries, this phenotype was more prevalent in isolates from patients with more severe gastric pathologies.

Biotypes, serogroups and antibiotic susceptibility of Campylobacter jejuni and Campylobacter coli in Chile.

Phenotypic markers were studied in 105 strains of thermophilic campylobacters isolated from human beings, animals and drinking water in Santiago, Chile. The strains were identified as Campylobacter jejuni (n = 49) and Campylobacter coli (n = 56). Biotypes I and II (Lior schema) accounted for 96% C. jejuni isolates, the other 4% being biotype IV but the two biotypes of C. coli were about equally represented. A total of 28 serogroups (Lior's heat-labile antigens) were identified. Lior 13, 9, 79, 2 and 4 were prevalent among the C. jejuni, while Lior 8, 21 and 29/75 were prevalent among the C. coli isolates. These serogroups accounted for 73% all isolates. The distribution of biotypes and serogroups in patients and asymptomatic persons were similar. Human campylobacters were often resistant to ampicillin (31%) but sensitive to erythromycin and furazolidone. Swine C. coli isolates proved resistant to streptomycin (46%), tetracycline (38%) and erythromycin (15%). Determination of phenotypic and serological characters provides valuable epidemiological markers in the study of campylobacter infections.

Identification of GM3 as a marker of therapy-resistant periradicular lesions.

The purpose of this study was to analyze the profile of glycosphingolipids (GSLs) in periradicular lesions refractory to endodontic treatment. Sixteen periapical lesions were removed surgically from patients (experimental group) and compared with 10 samples of periodontal ligament removed from extracted intact third molars (control group). After the GSLs extraction and purification procedures were performed the neutral and acidic GSL fractions were analyzed by high-performance thin-layer chromatography and quantified by densitometry. Data reported herein show that: (i) tissues in the experimental group presented about twice as much GSLs as the control group; (ii) lesion tissues express lactoneotetraosylceramide, and lactofucopentaosyl (IV) ceramide, whereas these neutral GSLs are absent in normal tissues; and (iii) normal tissues express GT1b, whereas lesions cells do not express this ganglioside. In contrast lesion tissues express GM3, which is conspicuously absent in normal tissues.

Immunochemical characterization of carbohydrate antigens from fungi, protozoa and mammals by monoclonal antibodies directed to glycan epitopes.

Cell surface carbohydrates constitute the major antigenic determinants of fungi and protozoa. Glycoconjugates also represent a large variety of antigens or markers present in mammals such as histo-blood groups ABO, differentiation and heterophile antigens, among others. This article focuses on the general properties of glycoconjugate antigens and production and characterization of the anti-carbohydrate monoclonal antibodies (MoAbs). It describes the specificity and some properties of monoclonal antibodies directed against carbohydrate epitopes present in tumor-associated glycoproteins, in glycosaminoglycans of higher eukaryotes and in glycolipid antigens of protozoa and fungi. The epitopes recognized by the anti-carbohydrate MoAbs range from one sugar unit up to ten sugar units. Although most anti-carbohydrate MoAbs are directed predominantly toward terminal sugar residues, a few MoAbs are also reactive with internal sugar residues. The fine structure of the carbohydrate epitopes has been chemically defined by [1H] NMR, GC/MS of alditol acetates of partially permethylated compounds, -FAB/MS, degradation with exoglycosidases and inhibition with different methyl-glycosides and oligosaccharides.

Isolation and possible composition of glucosylceramides from Paracoccidioides brasiliensis.

Twelve different species of neutral monohexosyl ceramides (CMHs) and two species of neutral monohexosyl ceramides were isolated from mycelium and yeast forms of Paracoccidioides brasiliensis, respectively, by a combination of ion-exchange chromatography, HPLC, and HPTLC. The glucosylceramides did not react with sera from patients with paracoccidioidomycosis (PCM). Carbohydrate analysis indicated that CMHs contain glucose. Analysis of 1H-NMR and mass spectrometry data suggest that the structure of the CMHs is Glcp beta 1-->Cer (mycelium forms present 12 different ceramides and yeast forms present 2 different ceramides). The composition of the lipid moieties was analyzed by negative fast atom bombardment mass spectrometry. No glycosphingolipid other than glucosylceramide was detected in P. brasiliensis.

Glycosphingolipid antigens from Leishmania (L.) amazonensis amastigotes. Binding of anti-glycosphingolipid monoclonal antibodies in vitro and in vivo.

Specific glycosphingolipid antigens of Leishmania (L.) amazonensis amastigotes reactive with the monoclonal antibodies (MoAbs) ST-3, ST-4 and ST-5 were isolated, and their structure was partially elucidated by negative ion fast atom bombardment mass spectrometry. The glycan moieties of five antigens presented linear sequences of hexoses and N-acetylhexosamines ranging from four to six sugar residues, and the ceramide moieties were found to be composed by a sphingosine d18:1 and fatty acids 24:1 or 16:0. Affinities of the three monoclonal antibodies to amastigote glycosphingolipid antigens were also analyzed by ELISA. MoAb ST-3 reacted equally well with all glycosphingolipid antigens tested, whereas ST-4 and ST-5 presented higher affinities to glycosphingolipids with longer carbohydrate chains, with five or more sugar units (slow migrating bands on HPTLC). Macrophages isolated from footpad lesions of BALB/c mice infected with Leishmania (L.) amazonensis were incubated with MoAb ST-3 and, by indirect immunofluorescence, labeling was only detected on the parasite, whereas no fluorescence was observed on the surface of the infected macrophages, indicating that these glycosphingolipid antigens are not acquired from the host cell but synthesized by the amastigote. Intravenous administration of 125I-labeled ST-3 antibody to infected BALB/c mice showed that MoAb ST-3 accumulated significantly in the footpad lesions in comparison to blood and other tissues.

Structural properties of lipid reconstructs and lipid composition of normotensive and hypertensive rat vascular smooth muscle cell membranes.

Multiple cell membrane alterations have been reported to be the cause of various forms of hypertension. The present study focuses on the lipid portion of the membranes, characterizing the microviscosity of membranes reconstituted with lipids extracted from the aorta and mesenteric arteries of spontaneously hypertensive (SHR) and normotensive control rat strains (WKY and NWR). Membrane-incorporated phospholipid spin labels were used to monitor the bilayer structure at different depths. The packing of lipids extracted from both aorta and mesenteric arteries of normotensive and hypertensive rats was similar. Lipid extract analysis showed similar phospholipid composition for all membranes. However, cholesterol content was lower in SHR arteries than in normotensive animal arteries. These findings contrast with the fact that the SHR aorta is hyporeactive while the SHR mesenteric artery is hyperreactive to vasopressor agents when compared to the vessels of normotensive animal strains. Hence, factors other than microviscosity of bulk lipids contribute to the vascular smooth muscle reactivity and hypertension of SHR. The excess cholesterol in the arteries of normotensive animal strains apparently is not dissolved in bulk lipids and is not directly related to vascular reactivity since it is present in both the aorta and mesenteric arteries. The lower cholesterol concentrations in SHR arteries may in fact result from metabolic differences due to the hypertensive state or to genes that co-segregate with those that determine hypertension during the process of strain selection.

[Application of serology to confirm the eradication of Helicobacter pylori in peptic ulcer patients]. / Aplicación de la serología para confirmar la erradicación de Helicobacter pylori en pacientes con úlcera péptica.

BACKGROUND: Measurement of changes in serum antibodies is an excellent predictor of Helicobacter pylori eradication after antibiotic treatment. AIM: To measure the changes in serum antibody titers to Helicobacter pylori, before and after treatment. MATERIAL AND METHODS: IgG antibodies to H. pylori were prospectively evaluated in 107 duodenal ulcer patients treated either with antibiotics (amoxicillin, metronidazole and bismuth subsalicylate) plus omeprazole or omeprazole alone. IgG antibody levels were determined using an "in house" ELISA in sera from 49 eradicated patients that received quadruple therapy and 58 non-eradicated patients (12 in whom antibiotic therapy failed and 46 that received omeprazole alone). Endoscopy, urease test, microscopy, and culture of gastric biopsies confirmed H. pylori eradication. RESULTS: Patients in whom H. pylori was eradicated, showed a maintained drop in serum antibody titers that ranged from 15%, 62%, 74% to 76% at 28 days, 4, 8 and 12 months respectively. Such reduction was not observed in patients treated with omeprazole. Patients, in whom quadruple therapy failed to eradicate H. pylori, showed a discrete and transient decrease in antibody titers. By the fourth month, patients in whom eradication with quadruple therapy was not achieved, irrespective of whether they received quadruple therapy or omeprazole alone. CONCLUSIONS: A 45% decrease in IgG titer after 4 months is indicative of therapeutic success in H. pylori eradication. Therefore, serology may be useful to monitor the outcome of antibiotic therapy (Rev Méd Chile 2000; 128: 1119-26).

Helicobacter pylori infection in Chile.

This article summarizes studies designed to evaluate the role of Helicobacter pylori infection in Chile, described in 21 reports from nine centers in various Chilean regions published between 1985 and 1995. According to their data, H. pylori infection is quite frequent among patients with a variety of gastric conditions, including adults (43%-92%) and children (6%-100%). Levels of specific IgG antibodies to H. pylori are also elevated among patients with duodenal ulcers (100%) and gastritis (86%) as well as asymptomatic adults (75%). Combination therapy with three (but not two) drugs has been proved effective, with clinical improvement, ulcer cure, and H. pylori eradication occurring in well-controlled studies. Available evidence suggests that antibiotic resistance is not a major problem in treatment. The H. pylori reinfection rate is low (4.2% per year), suggesting that combination therapy with three drugs constitutes a cost-effective alternative for treating colonized symptomatic patients. Concurrent preliminary studies revealed that antibodies to VacA but not CagA proteins correlate with disease severity in Chilean patients. It can be concluded that local research assists local administrators of health resources to implement adequate policies to prevent, control, and treat H. pylori-related pathologies.

A monoclonal antibody directed to terminal residue of beta-galactofuranose of a glycolipid antigen isolated from Paracoccidioides brasiliensis: cross-reactivity with Leishmania major and Trypanosoma cruzi.

A mouse monoclonal antibody, MEST-1, was produced against Band 1 glycolipid antigen of Paracoccidioides brasiliensis. The glycan structure of Band 1 antigen was recently elucidated and the monosaccharides sequence was defined as: Galf beta 1-->6(Manp alpha 1-->3)Manp beta 1-->2Ins. The reactivity of MEST-1 MAb was determined by solid-phase radioimmunoassay and high performance thin layer chromatography immunostaining. Selective oxidation of galactofuranose residues and inhibition assays with different methyl-glycosides, revealed that MAb MEST-1 is directed against the terminal residue of beta-D-galactofuranose of Band 1, a phosphoglyceroglycolipid antigen of P. brasiliensis. By indirect immunofluorescence, it was observed that the epitope recognized by MEST-1 is accessible to the antibody in yeast forms of this fungus. Reactivity of MEST-1 with parasites known to express galactofuranose containing glycoconjugates was also analyzed by indirect immunofluorescence. A positive fluorescence was observed with promastigotes of Leishmania major and epimastigotes of Trypanosoma cruzi. GIPL-1 was identified as the antigen recognized by MEST-1 in Leishmania major, indicating that the MAb MEST-1 recognizes terminal galactofuranose residue in either beta 1-->6 or beta 1-->3 linkage to the mannose.

[The IgG antibody response in patients colonized by Helicobacter pylori]. / Respuesta de anticuerpos IgG en pacientes colonizados por Helicobacter pylori.

The IgG antibody response specific to Helicobacter pylori was evaluated through ELISA in a group of 92 gastric patients colonized by this bacteria. 74 had gastritis and 19 gastroduodenal ulcer. Three control groups were studied in a similar way: normal adult volunteers (n = 17), adults with E coli or S typhi bacteremia (n = 30) and normal infants (n = 30). IgG antibody response to H pylori was demonstrated in 98% of colonized patients and 0% of infants. Asymptomatic individuals and those with bacteremia had high rates of antibody response (76 and 90% respectively), although this rate and also the titers of antibody response were significantly lower than that of colonized patients (p < 0.05). ELISA reactive sera from colonized patients and asymptomatic individuals evidenced a similar antibody pattern when tested by blotting. This profile was absent in non reactive sera, including those with high antibody titers to C jejuni. The presence of specific IgG antibodies to H pylori in the majority of colonized gastric patients and asymptomatic adults suggests that this infection is very common in our population.

[Prospective study of erradication of Helicobacter pylori in patients with duodenal ulcer: analysis of antral and gastric body biopsies]. / Estudio prospectivo de erradicación de Helicobacter pylori en ulcerosos duodenales: análisis de biopsias de antro y cuerpo gástrico.

Omeprazole may not eradicate Helicobacter pylori from the stomach but rather displace it from the antrum to the stomach body. This fact could interfere with colonization studies in patients receiving the drug. The aim of this study was to assess the presence of Helicobacter pylori, defined as a positive urease test, culture or microscopical examination, in antral and gastric body biopsies in patients receiving treatment with omeprazole. Sixty four paired antral and gastric body biopsies obtained at the end of a 28 day course of omeprazole, 62 obtained four months later, 40 obtained eight months later and 23 obtained 12 months later were analyzed. There was a 92% concordance between antral and gastric body biopsies for the presence of Helicobacter pylori. However, nine of the samples obtained at 28 days (14%) were negative for H pylori in the antrum but positive in the gastric body. It is concluded that for early assessment of Helicobacter pylori eradication after omeprazole treatment, paired biopsies of antrum and gastric body are needed.

Structure elucidation of sphingolipids from the mycopathogen Paracoccidioides brasiliensis: an immunodominant beta-galactofuranose residue is carried by a novel glycosylinositol phosphorylceramide antigen.

Two major acidic glycolipid components (Pb-1 and Pb-2) have been extracted from the mycopathogen Paracoccidioides brasiliensis, a thermally dimorphic fungus endemic to rural areas of South and Central America. Sera of all patients exhibiting paracoccidioidomycosis were found to be reactive with Pb-1, but not with Pb-2; no reactivity was observed with sera of healthy patients or those with histoplasmosis [Toledo, M. S., Suzuki, S., Straus, A. H., and Takahashi, H. K. (1995) J. Med. Vet. Mycol. 33, 247-251]. We report here the complete structure elucidation of both P. brasiliensis glycolipids using monosaccharide, fatty acid, sphingosine, and inositol component analysis by GC-MS; 1H- and 31P NMR spectroscopy; ESI-MS and -MS/CID-MS, linkage analysis, and exoglycosidase digestion. The compounds were found to be glycosylinositol phosphorylceramides (GIPCs) with the following structures: Pb-2, Manpalpha1-->3Manpalpha1-->2Ins1-P-1Cer; and Pb-1, Manpalpha1-->3[Galfbeta1-->6]Manpalpha1-->2Ins1- P-1Cer. The serologically nonreactive Pb-2 appears to be a biosynthetic intermediate between mannosylinositol phosphorylceramide (MIPC), which is widely distributed among fungi, and the antigenic Pb-1. Pb-1 is a novel glycosphingolipid, similar to a triglycosyl IPC (Hc-VI) reported from Histoplasma capsulatum [Barr, K., Laine, R.A, and Lester, R. L. (1984) Biochemistry 23, 5589-5596], but differing in the anomeric configuration of the terminal Galf1-->6 residue, which is immunodominant. The significance of these structures as serological and taxonomic markers, as well as their potential utility as targets for immunodiagnostic agents, is discussed.

[Evaluation of an immunoenzyme technique (ELISA) to diagnose typhoid fever]. / Evaluación de un ensayo inmunoenzimático (ELISA) para el diagnóstico de la fiebre tifoídea.

The efficiency of an ELISA method, designed to detect polyvalent IgG and IgM antibodies to Salmonella typhi polysaccharide was evaluated in patients admitted or convalescing from typhoid fever and in control subjects. Polyvalent antibodies to S typhi were demonstrated in 28/30 (93%) typhoid patients, 0/11 bacteremic patients (E coli or S paratyphi A) and 0/15 asymptomatic individuals. Widal test showed significant anti-0 agglutinin values (> = 1: 160) in only 12/30 (40%), 1/11 (9%) and 0/15 subjects from each group respectively. Typhoid patients tested on admission or at discharge showed similar high reactivity rates to ELISA. On the contrary, the Widal test detected only 2/15 (13%) patients on admission (p < 0.02) and 10/15 (67%) at discharge. These results and additional immunoblotting tests suggest that ELISA developed to detect polyvalent anti-LPS antibodies could represent a highly specific diagnostic tool to confirm typhoid fever in endemic areas.

Dimorphic expression of cerebrosides in the mycopathogen Sporothrix schenckii.

Major neutral glycosphingolipid components were extracted from Sporothrix schenckii, a dimorphic fungus exhibiting a hyphal saprophytic phase and a yeast parasitic phase responsible for chronic mycotic infections in mammalian hosts. These components, one from the mycelial form and two from the yeast form, were purified and their structures were elucidated by (1)H nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and tandem ESI-MS/MS. All three were characterized as cerebrosides (monohexosylceramides) containing (4E, 8E)-9-methyl-4,8-sphingadienine as the long-chain base attached to N-2'-hydroxyoctadecanoate and N-2'-hydroxy-(E)-Delta(3)-octadecenoate as the fatty acyl components. However, while the mycelial form expressed only beta-glucopyranosylceramide, the yeast form expressed both beta-gluco- and beta-galactopyranosylceramides in approximately equal amounts. In addition, while the glucosylceramides of both mycelial and yeast forms had similar proportions of saturated and (E)-Delta(3) unsaturated 2-hydroxy fatty acid, the galactocerebroside of the yeast form had significantly higher levels of (E)-Delta(3) unsaturation. The differences in cerebroside hexose structure represent a novel type of glycosphingolipid dimorphism not previously reported in fungi. Possible implications of these findings with respect to regulation of morphological transitions in S. schenckii and other dimorphic fungi are discussed.

Characterization of monoclonal antibody MEST-2 specific to glucosylceramide of fungi and plants.

An IgG2a monoclonal antibody anti-glucosylceramide was established and termed MEST-2. High performance thin layer chromatography immunostaining, and solid-phase radioimmunoassay showed that MEST-2 reacts with glucosylceramide from yeast and mycelium forms of Paracoccidioides brasiliensis, Histoplasma capsulatum, and Sporothrix schenckii; from hyphae of Aspergillus fumigatus; and from yeast forms of Candida albicans, Cryptococcus neoformans, Cryptococcus laurentii, and Cryptococcus albidus. Studies on the fine specificity of MEST-2 showed that it recognizes the beta-D-glucose residue, and that the 2-hydroxy group present in the fatty acid is an important auxiliary feature for the antibody binding. It was also demonstrated that phosphatidylcholine and ergosterol modulate MEST-2 reactivity to glucosylceramide, by solid-phase radioimmunoassay. Indirect immunofluorescence showed that MEST-2 reacts with the surface of yeast forms of P. brasiliensis, H. capsulatum and S. schenckii. Weak staining of mycelial forms of P. brasiliensis and hyphae of A. fumigatus was also observed. The availability of a monoclonal antibody specific to fungal glucosylceramide, and its potential use in analyzing biological roles attributed to glucosylceramide in fungi are discussed.

Characterization of cerebrosides from the thermally dimorphic mycopathogen Histoplasma capsulatum: expression of 2-hydroxy fatty N-acyl (E)-Delta(3)-unsaturation correlates with the yeast-mycelium phase transition.

Cerebroside (monohexosylceramide) components were identified in neutral lipids extracted from both the yeast and mycelial forms of the thermally dimorphic mycopathogen Histoplasma capsulatum. The components were purified from both forms and their structures elucidated by 1- and 2-dimensional nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and low energy tandem collision-induced dissociation mass spectrometry (ESI-MS/CID-MS). Both components were characterized as beta-glucopyranosylceramides (GlcCers) containing (4E,8E)-9-methyl-4,8-sphingadienine as the long-chain base, attached to 18-carbon 2-hydroxy fatty N-acyl components. However, while the fatty acid of the yeast form GlcCer was virtually all N-2'-hydroxyoctadecanoate, the mycelium form GlcCer was characterized by almost exclusive expression of N-2'-hydroxy-(E)-delta(3)-octadecenoate. These results suggest that the yeast-mycelium transition is accompanied by up-regulation of an as yet uncharacterized ceramide or cerebroside 2-hydroxy fatty N-acyl (E)-delta(3)-desaturase activity. They also constitute further evidence for the existence of two distinct pathways for ceramide biosynthesis in fungi, since glycosylinositol phosphorylceramides (GIPCs), the other major class of fungal glycosphingolipids, are found with ceramides consisting of 4-hydroxysphinganine (phytosphingosine) and longer chain 2-hydroxy fatty acids. In addition to identification of the major glucocerebroside components, minor components (< 5%) detectable by molecular weight differences in the ESI-MS profiles were also characterized by tandem ESI-MS/CID-MS analysis. These minor components were identified as variants differing in fatty acyl chain length, or the absence of the sphingoid 9-methyl group or (E)-delta(8)-unsaturation, and are hypothesized to be either biosynthetic intermediates or the result of imperfect chemical transformation by the enzymes responsible for these features. Possible implications of these findings with respect to chemotaxonomy, compartmentalization of fungal glycosphingolipid biosynthetic pathways, and regulation of morphological transitions in H.capsulatum and other dimorphic fungi are discussed.