Cytosolic and nuclear O-linked N-acetylglucosaminylation has been proposed to be involved in the nuclear transport of cytosolic proteins. We have isolated nuclear and cytosolic N-acetyl-d-glucosamine (GlcNAc)-specific lectins from adult rat liver by affinity chromatography on immobilized GlcNAc and identified these lectins, by a proteomic approach, as belonging to the heat-shock protein (HSP)-70 family (one of them being heat-shock cognate 70 stress protein). Two-dimensional electrophoresis indicated that the HSP-70 fraction contained three equally abundant proteins with molecular masses of 70, 65 and 55kDa. The p70 and p65 proteins are phosphorylated and are themselves O-linked GlcNAc (O-GlcNAc)-modified. The HSP-70 associated into high molecular mass complexes that dissociated in the presence of reductive and chaotropic agents. The lectin(s) present in this complex was (were) able to recognize cytosolic and nuclear ligands, which have been isolated using wheat germ agglutinin affinity chromatography. These ligands are O-GlcNAc glycosylated as demonstrated by [3H]galactose incorporation and analysis of the products released by reductive β-elimination. The isolated lectins specifically recognized ligands present in both the cytosol and the nucleus of human resting lymphocytes. These results demonstrated the existence of endogenous GlcNAc-specific lectins, identified as HSP-70 proteins, which could act as a shuttle for the nucleo-cytoplasmic transport of O-GlcNAc glycoproteins between the cytosol and the nucleus.
Lipomannan (LM) and lipoarabinomannan (LAM) are major glycolipids present in the mycobacterial cell wall that are able to modulate the host immune response. In this study, we have undertaken the structural determination of these important modulins inMycobacterium chelonae, a fast growing pathogenic mycobacterial species. One-dimensional and two-dimensional NMR spectra were used to demonstrate that LM and LAM from M. chelonae, designated CheLM and CheLAM, respectively, possess structures that differ from the ones reported earlier in other mycobacterial species. Analysis by gas chromatography/mass spectrometry of the phosphatidyl-myo-inositol anchor, which is thought to play a role in the biological functions of these lipoglycans, pointed to a high degree of heterogeneity based on numerous combinations of acyl groups on the C-1 and C-2 positions of the glycerol moiety. Characterization of the mannan core of CheLM and CheLAM revealed the presence of novel α1,3-mannopyranosyl side chains. This motif, which reacted specifically with the lectin from Galanthus nivalis, was found to be unique among a panel of nine mycobacterial species. Then, CheLM and CheLAM were found to be devoid of both the mannooligosaccharide cap present in Mycobacterium tuberculosis and the inositol phosphate cap present inMycobacterium smegmatis and other fast growing species. Tumor necrosis factor-α and interleukin-8 production were assessed from human macrophages with LAM preparations from different species. Our results suggest that the inositol phosphate capping may represent the major cytokine-inducing component of LAMs. This work not only underlines the diversity of LAM structures among various mycobacterial species but also provides new structures that could be useful to dissect the structure-function relationships of these complex molecules. Lipomannan (LM) and lipoarabinomannan (LAM) are major glycolipids present in the mycobacterial cell wall that are able to modulate the host immune response. In this study, we have undertaken the structural determination of these important modulins inMycobacterium chelonae, a fast growing pathogenic mycobacterial species. One-dimensional and two-dimensional NMR spectra were used to demonstrate that LM and LAM from M. chelonae, designated CheLM and CheLAM, respectively, possess structures that differ from the ones reported earlier in other mycobacterial species. Analysis by gas chromatography/mass spectrometry of the phosphatidyl-myo-inositol anchor, which is thought to play a role in the biological functions of these lipoglycans, pointed to a high degree of heterogeneity based on numerous combinations of acyl groups on the C-1 and C-2 positions of the glycerol moiety. Characterization of the mannan core of CheLM and CheLAM revealed the presence of novel α1,3-mannopyranosyl side chains. This motif, which reacted specifically with the lectin from Galanthus nivalis, was found to be unique among a panel of nine mycobacterial species. Then, CheLM and CheLAM were found to be devoid of both the mannooligosaccharide cap present in Mycobacterium tuberculosis and the inositol phosphate cap present inMycobacterium smegmatis and other fast growing species. Tumor necrosis factor-α and interleukin-8 production were assessed from human macrophages with LAM preparations from different species. Our results suggest that the inositol phosphate capping may represent the major cytokine-inducing component of LAMs. This work not only underlines the diversity of LAM structures among various mycobacterial species but also provides new structures that could be useful to dissect the structure-function relationships of these complex molecules. phosphatidyl-myo-inositol mannosides arabinofuranosyl lectin from Canavalia ensiformis correlation spectroscopy chemical ionization electron impact gas chromatography/mass spectrometry glycerol lectin from Galanthus nivalis Heteronuclear multiple bound correlation heteronuclear multiple quantum coherence inositol lipomannan homonuclear Hartmann-Hahn spectroscopy lipoarabinomannan matrix-assisted laser-desorption ionization mass spectrometry mannopyranosyl nuclear Overhauser enhancement spectroscopy phosphatidyl-myo-inositol rotating frame Overhauser enhancement spectroscopy total correlation spectroscopy trimethylsilyl digoxigenin interleukin tumor necrosis factor glycosylphosphatidylinositol phosphate-buffered saline Mycobacterium species are responsible for important human diseases including tuberculosis and leprosy. Infection and immunopathogenesis of these diseases widely implicate the mycobacterial cell wall (1Daffé M. Draper P. Adv. Microb. Physiol. 1998; 39: 131-203Crossref PubMed Google Scholar) which is abundantly composed of mannoconjugates, notably polysaccharides and lipoglycans. The latter consist mainly of phosphatidyl-myo-inositol mannosides (PIMs),1 lipomannan (LM), and the structurally related lipoarabinomannan (LAM). LAM is a major cell wall component and is considered as a modulin through its various immunoregulatory and anti-inflammatory effects, which favor the survival of the mycobacteria within the infected host. These effects include suppression of T lymphocyte proliferation through interference with antigen processing (2Moreno C. Mehlert A. Lamb J. Clin. Exp. Immunol. 1988; 74: 206-210PubMed Google Scholar), inhibition of macrophage activation by interferon-γ (3Sibley L.D. Hunter S.W. Brennan P.J. Krahenbuhl J.L. Infect. Immun. 1988; 56: 1232-1236Crossref PubMed Google Scholar, 4Sibley L.D. Adams L.B. Krahenbuhl J.L. Clin. Exp. Immunol. 1990; 80: 141-148Crossref PubMed Scopus (63) Google Scholar), and scavenging of oxygen-derived free radicals (5Chan J. Fan X.D. Hunter S.W. Brennan P.J. Bloom B.R. Infect. Immun. 1991; 59: 1755-1761Crossref PubMed Google Scholar). LAM is not only a virulence factor responsible for macrophage deactivation, but it is also implicated in phagocytosis of mycobacteria into phagocytic cells (6Schlesinger L.S. Hull S.R. Kaufman T.M. J. Immunol. 1994; 152: 4070-4079PubMed Google Scholar). In addition, PIMs that are believed to be precursors of LM and LAM have recently been proposed to recruit NK T cells, which play a primary role in the granulomatous response (7Apostolou I. Takahama Y. Belmant C. Kawano T. Huerre M. Marchal G. Cui J. Taniguchi M. Nakauchi H. Fournie J.J. Kourilsky P. Gachelin G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5141-5146Crossref PubMed Scopus (180) Google Scholar,8Gilleron M. Ronet C. Mempel M. Monsarrat B. Gachelin G. Puzo G. J. Biol. Chem. 2001; 276: 34896-34904Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The biosynthetic relationship of phosphatidylinositol (PI), PIMs, LM, and LAM has recently been supported by biochemical (9Khoo K.H. Dell A. Morris H.R. Brennan P.J. Chatterjee D. Glycobiology. 1995; 5: 117-127Crossref PubMed Scopus (116) Google Scholar, 10Besra G.S. Morehouse C.B. Rittner C.M. Waechter C.J. Brennan P.J. J. Biol. Chem. 1997; 272: 18460-18466Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) and genetic studies (11Schaeffer M.L. Khoo K.H. Besra G.S. Chatterjee D. Brennan P.J. Belisle J.T. Inamine J.M. J. Biol. Chem. 1999; 274: 31625-31631Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 12Kremer L. Gurcha S.S. Bifani P. Hitchen P.G. Baulard A. Morris H.R. Dell A. Brennan P.J. Besra G.S. Biochem. J. 2002; 363: 437-447Crossref PubMed Google Scholar), but the details of this pathway remain highly speculative. However, the structures of LAM from several species including Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium bovis BCG, and Mycobacterium smegmatis have been extensively described during the last decade. LAM is a complex glycolipid composed ofd-mannan and d-arabinan attached to a PI moiety that anchors the glycolipid in the mycobacterial cell wall (13Hunter S.W. Brennan P.J. J. Biol. Chem. 1990; 265: 9272-9279Abstract Full Text PDF PubMed Google Scholar). The biosynthesis of LAM involves the addition of mannopyranosyl (Manp) residues to PI to produce both the short PIMs (2–5 Man residues) and LM, which is further glycosylated with arabinan to form LAM (9Khoo K.H. Dell A. Morris H.R. Brennan P.J. Chatterjee D. Glycobiology. 1995; 5: 117-127Crossref PubMed Scopus (116) Google Scholar, 13Hunter S.W. Brennan P.J. J. Biol. Chem. 1990; 265: 9272-9279Abstract Full Text PDF PubMed Google Scholar, 14Venisse A. Berjeaud J.M. Chaurand P. Gilleron M. Puzo G. J. Biol. Chem. 1993; 268: 12401-12411Abstract Full Text PDF PubMed Google Scholar, 15Chatterjee D. Lowell K. Rivoire B. McNeil M.R. Brennan P.J. J. Biol. Chem. 1992; 267: 6234-6239Abstract Full Text PDF PubMed Google Scholar). In all the species described so far,d-mannan consists of a highly branched structure with an α1,6-linked Manp backbone substituted at C-2 by single Manp units (16Chatterjee D. Hunter S.W. McNeil M. Brennan P.J. J. Biol. Chem. 1992; 267: 6228-6233Abstract Full Text PDF PubMed Google Scholar). The mannan size and the degree of branching can vary depending on the species. The arabinan consists of a linear α1,5-linked arabinofuranosyl (Araf) backbone punctuated by branching produced with 3,5-O-linked α-d-Araf residue. The lateral chains are organized either as linear tetra-arabinofuranosides β-d-Araf-(1,2)-α-d-Araf-(1,5)-α-d-Araf-(1, 5)-α-d-Araf or as a biantennary hexa-arabinofuranosides [β-d-Araf-(1,2)-α-d-Araf-(1-]2-3 and 5)-α-d-Araf-(1,5)-α-d-Araf(17Chatterjee D. Bozic C.M. McNeil M. Brennan P.J. J. Biol. Chem. 1991; 266: 9652-9660Abstract Full Text PDF PubMed Google Scholar, 18Chatterjee D. Khoo K.H. McNeil M. Dell A. Morris H.R. Brennan P.J. Glycobiology. 1993; 3: 497-506Crossref PubMed Scopus (79) Google Scholar). Comparative analyses of LAMs from different mycobacterial species have shown that the non-reducing termini of the arabinosyl side chains are differentially modified. M. tuberculosis and M. leprae modify the termini with Manp residues, thereby yielding “ManLAM,” whereas the rapidly growing speciesM. smegmatis uses inositol phosphate, generating “AraLAM” (19Khoo K.H. Dell A. Morris H.R. Brennan P.J. Chatterjee D. J. Biol. Chem. 1995; 270: 12380-12389Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). It is thought that these modifications are responsible for the marked differences in the biological activities of ManLAM and AraLAM (15Chatterjee D. Lowell K. Rivoire B. McNeil M.R. Brennan P.J. J. Biol. Chem. 1992; 267: 6234-6239Abstract Full Text PDF PubMed Google Scholar, 19Khoo K.H. Dell A. Morris H.R. Brennan P.J. Chatterjee D. J. Biol. Chem. 1995; 270: 12380-12389Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 20Roach T.I. Barton C.H. Chatterjee D. Blackwell J.M. J. Immunol. 1993; 150: 1886-1896PubMed Google Scholar). However, as mentioned above, all available LAM structures are derived from a very limited panel of mycobacterial species, and whether these structures are invariably present in most species remains to be investigated. Awareness that subtle differences among LAMs may affect their biological properties prompted us to establish the structure of LM/LAM fromMycobacterium chelonae, a rapidly growing pathogenic mycobacterium that is found in soil and fresh water throughout the world (21Wolinsky E. Am. Rev. Respir. Dis. 1979; 119: 107-159PubMed Google Scholar). M. chelonae infection typically causes localized skin lesions, often following penetrating trauma or injections. Disseminated disease usually occurs in patients with a significant immune compromised state that is most commonly attributable to exogenous steroid use (22Wallace Jr., R.J. Brown B.A. Onyi G.O. J. Infect. Dis. 1992; 166: 405-412Crossref PubMed Scopus (303) Google Scholar, 23Ingram C.W. Tanner D.C. Durack D.T. Kernodle Jr., G.W. Corey G.R. Clin. Infect. Dis. 1993; 16: 463-471Crossref PubMed Scopus (166) Google Scholar). We report here the detailed structure of LM/LAM from M. chelonae and provide evidence for important differences such as the acylation composition of the PI, branching of the mannan core, and the absence of Manp and inositol phosphate caps. Because these structures were found to be unique among a panel of various mycobacterial species, we propose to designate these components as CheLM and CheLAM. All mycobacterial species used were grown on plates containing Middlebrook 7H11 agar supplemented with 10% oleic acid-albumin-dextrose-catalase enrichment (Difco) or in liquid Sauton medium. Except for M. chelonae (ATCC 19536), which was grown under shaking in Sauton medium at 30 °C for several days, all other species were grown at 37 °C. Confirmation of the identity of the M. chelonae strain was done by analyzing its mycolic acid profile, which is rather unusual because it consists of 60% of α-mycolates and 40% of α′-mycolates (24Kremer L. Dover L. Carrère S. Nampoothiri M. Lesjean S. Brown A. Brennan P.J. Minnikin D.E. Locht C. Besra G.S. Biochem. J. 2002; 364: 423-430Crossref PubMed Scopus (107) Google Scholar, 25Minnikin D.E. Minnikin S.M. Goodfellow M. Stanford J.L. J. Gen. Microbiol. 1982; 128: 817-822PubMed Google Scholar). Extraction of CheLM and CheLAM was adapted from Nigou et al. (26Nigou J. Gilleron M. Cahuzac B. Bounéri J.D. Herold M. Thurnher M. Puzo G. J. Biol. Chem. 1997; 272: 23094-23103Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) based on the Triton X-114 phase partitioning. Briefly, cells were harvested, washed in PBS (20 mm K2HPO4 (pH 7.5), 0.15m NaCl), and resuspended in lysis buffer (8% (v/v) Triton X-114 in PBS, 5 mm EDTA, 10 mmMgCl2). Cells were then heat-inactivated, disrupted using a French pressure cell and stirred overnight at 4 °C. Cellular debris were removed by centrifugation (27,000 × g, 30 min, 4 °C), and phase separation was induced at 37 °C. Lipoglycans present in the lower phase were precipitated by adding 5 volumes of cold ethanol and collected by centrifugation (27,000 ×g, 30 min, 4 °C). The pellet was dissolved in water, and proteinase K was added to a final concentration of 10 μg/ml for 20 min at 55 °C. Proteins were extracted twice by adding saturated phenol. Combined aqueous phases containing lipoglycans were dialyzed for 72 h against water, lyophilized, and resuspended in Tris deoxycholate buffer (10 mm Tris-HCl (pH 8.0), 10 mm EDTA, 0.2 m NaCl, 0.25% deoxycholate). CheLAM and CheLM were then separated by gel filtration on a Sephacryl S-200 (Amersham Biosciences) column (80 × 1.5 cm) in the same buffer. The eluted fractions were monitored by 13% SDS-PAGE stained for carbohydrates according to Tsai and Frasch (27Tsai C.M. Frasch C.E. Anal. Biochem. 1982; 119: 115-119Crossref PubMed Scopus (2315) Google Scholar). The appropriate LAM and LM fractions were pooled and dialyzed for 48 h against 10 mm Tris-HCl (pH 8.0) and then for 48 h against water prior to lyophilization. The endotoxin content of all reagents was measured in a chromogenic Limulus lysate assay (BioWhittaker). The LAM preparations contained insignificant amounts of endotoxin (<25 pg/ml) Prior to NMR spectroscopic analysis, LM (15 mg) and LAM (5 mg) were repeatedly exchanged in2H2O (99.97% purity, Euriso-top, CEA Saclay, France) with intermediate freeze-drying and then dissolved in 250 μl of Me2SO-d6 (Euriso-top). Chemical shifts were expressed in ppm downfield from the signal of the methyl group of Me2SO-d6(δ1H/TMS = 2.52 ppm, δ13C/TMS = 40.98 ppm at 343 K). The samples were analyzed in 200 × 5 mm BMS-005-B Shigemi® tubes on a Bruker ASX-400 spectrometer (Centre d'Analyses RMN, Villeneuve d'Ascq) (1H, 400.33;13C, 100.66, 31P, 162.5 MHz) equipped with a double resonance (1H/X) Broad Band Inverse z-gradient probe head. All NMR data were recorded without sample spinning. The one-dimensional proton 1H spectrum was measured using 90° tipping angle for the pulse and 1.5 s as a recycle delay between each of 32 acquisitions of 2.4 s. The spectral width of 4006 Hz was collected in 16,384 complex data points. The one-dimensional 13C was recorded using a spectral width of 20,161 Hz, and 32,768 data points were collected to obtain a free induction decay resolution of 0.6 Hz per point. The31P spectra of both compounds were acquired with a spectral width of 16,233 Hz collected in 16,384 data points. Both experiments were recorded using a composite pulse decoupling during acquisition using globally optimized alternating phase sequence at the carbon or phosphorus frequency (28Shaka A.J. Barker P.B. Freeman R. J. Magn. Reson. 1985; 64: 547-552Google Scholar). An exponential transformation (line broadening factor = 5 for 13C and 3 Hz for31P) was applied prior to processing data points in the frequency domain. Two-dimensional homonuclear (1H-1H) spectra (COSY-ROESY-TOCSY) were measured using standard Bruker pulse programs. ROESY spectra were acquired with various mixing times (50, 100, 200, and 400 ms) and acquired in States mode according to Bax and Davis (29Bax A.D. Davis J. J. Magn. Reson. 1985; 63: 207-213Google Scholar), whereas both COSY and relayed COSY were acquired in the magnitude calculation mode. Moreover, the two-dimensional TOCSY spectrum was recorded using a MLEV-17 mixing sequence of 120 ms. The spin lock field strength corresponded to a 90° pulse width of 35 μs. The spectral width was 4000 Hz in both dimensions. 512 spectra of 4096 data points with 32 scans per t1 increment were recorded giving a spectral resolution of 0.9 Hz/point in F2 and ∼8 Hz/point in F1. Heteronuclear experiments (1H-13C and1H-31P) were obtained with standard Bruker pulse sequences such as HMQC (inv4tp), HMQC-HOHAHA (inv4mltp), and HMBC (inv4lrnd). HMQC and HMQC-HOHAHA were acquired in the phase-sensitive increment time proportional phase increment (TPPI) method, whereas HMBC was recorded in magnitude mode calculation. All parameters (pulse widths, pulse powers, and delays) were optimized for each experiment. Acquisitions and processing conditions are expressed in the figure legends. Monosaccharides were analyzed as alditol-acetate derivatives. Lipoglycans were hydrolyzed in 4n trifluoroacetic acid for 4 h at 100 °C and reduced with NaBH4 in 0.05 n NH4OH for 4 h. Reduction was stopped by dropwise addition of acetic acid until the pH reached 6, and borate salts were co-distilled by repetitive evaporation in dry methanol. Per-acetylation was performed in acetic anhydride at 100 °C for 2 h, and derivatives were analyzed in GC on a BPX70 12 m × 0.22 mm ID column (Chrompak). For exact quantification of glycerol and myo-inositol, 100 μg of lipoglycans along with 1 μg of scyllo-inositol, as an internal standard, were hydrolyzed with 6 n hydrochloric acid constant boiling (Pierce), at 110 °C for 24 h, and analyzed as TMS derivatives (30Kamerling J.P. Gerwig G.J. Vliegenthart J.F. Clamp J.R. Biochem. J. 1975; 151: 491-495Crossref PubMed Scopus (317) Google Scholar) using GC on a DB-1 60 m ×0.25 mm inner diameter by on-column injection. Linkage analyses of monosaccharides was achieved by two steps of per-methylation according to Ciucanu and Kerek (31Ciucanu I. Kerek F. Carbohydr. Res. 1984; 131: 209-217Crossref Scopus (3205) Google Scholar) and followed by derivatization with acetyl groups with acetic anhydride. Acylglycerols were analyzed according to Nigou et al. (26Nigou J. Gilleron M. Cahuzac B. Bounéri J.D. Herold M. Thurnher M. Puzo G. J. Biol. Chem. 1997; 272: 23094-23103Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) after cleavage of the phosphodiester bond by acetolysis. Briefly, 100 μg of lipoglycans were treated with 400 μl of anhydrous acetic acid/acetic anhydride, 3:2 (v/v), at 110 °C for 12 h. Acetylated acylglycerols were extracted by cyclohexane and analyzed by GC/MS on a WCOT fused silica 30 m × 0.25 mm inner diameter column (Chrompak). Fatty acids were analyzed from intact lipoglycans as well as from extracted acetylated acylglycerol as pyrrolidine derivatives. They were released by methyl esterification with 0.5 m HCl in anhydrous methanol at 80 °C for 20 h, extracted with heptane, and derivatized with 200 μl of pyrrolidine/acetic acid, 9:1 (v/v), at 80 °C for 2 h. Pyrrolidine-derivatized fatty acids were repetitively extracted by CHCl3/H3O, 1:1 (v/v), and analyzed by GC/MS on a WCOT fused silica 30 m × 0.25 mm inner diameter column (Chrompak). The molecular mass of the lipoglycans was measured by matrix-assisted laser desorption ionization on a Vision 2000 time-of-flight instrument (Finnigan Mat) equipped with a 337-nm UV laser. Samples were dissolved in water at a concentration of 100 pmol/μl. One μl of the solution was mixed with an equal volume of 2,5-dihydroxybenzoic acid (10 mg/ml; dissolved in water/methanol, 4:1 (v/v)) matrix solution on the target and then allowed to crystallize at room temperature. Western blot analyses were conducted either on purified LMs and LAMs or on crude mycobacterial lysates. For purified LM and LAM samples, the sugar content was estimated by GC, and the equivalent of 0.5 μg of mannose for each sample was analyzed by 13% SDS-PAGE. For crude lysates, mycobacterial cells were harvested, resuspended in 0.8 ml of PBS, and disrupted for 10 min with a Branson Sonifier 450. Protein concentrations were determined using the BCA Protein Assay Reagent kit (Pierce). Equal amounts of proteins (30 μg) were then separated by 13% SDS-PAGE and then transferred onto a Hybond-C Extra membrane (Amersham Biosciences). Membranes were then saturated with 5% bovine serum albumin in PBS, 0.1% Tween 20, and probed overnight with either ConA-DIG or GNA-DIG (Roche Molecular Biochemicals, dilution 1:1000). After washing, membranes were subsequently incubated with anti-DIG antibodies conjugated to alkaline phosphatase (Roche Molecular Biochemicals, 1:1000 dilution). The human promonocytic THP-1 cell line was grown in RPMI 1640 (Invitrogen) supplemented with 10% fetal calf serum, 2 mm l-glutamine, and 2 10−5m β-mercaptoethanol in an atmosphere of 5% CO2 at 37 °C. THP-1 cells were induced to express CD14 by treatment with 50 nm 1,25-dihydroxyvitamin D3 (Calbiochem) for 48 h. Cells were then washed twice with RPMI 1640 and cultured in 96-well plastic culture plates at a density of 2 × 105 cells/well in RPMI 1640 supplemented with 10% fetal calf serum and glutamine. The purified molecules LAMs from M. Chelonae, M. tuberculosisErdman, and M. smegmatis were added at a final concentration of 10 μg/ml, and triplicates were performed in order to measure cytokine release. Culture supernatants were collected after 6 or 24 h for TNF-α or IL-8 production, respectively. Specific enzyme-linked immunosorbent assays commercial kits were used according the manufacturer's instructions. Human IL-8 and TNF-α kits were purchased from Bender Med systems Diagnostic and R & D Systems, respectively. Cytokine production was quantified with a microtiter plate reader in comparison with a standard curve generated with recombinant human cytokines. The experimental protocols used to extract LM and LAM fromM. chelonae are based on successive detergent and phenol extractions, leading to the recovery of nucleic acid-, protein-, and lipid-free materials. Purity of the preparation was assessed by GC/MS and SDS-PAGE. CheLAM and CheLM were finally resolved on a gel permeation S200 column. Fig.1a represents the elution profile of each compound and shows that CheLM was present in higher amounts than CheLAM. This was confirmed by SDS-PAGE analysis of the total Triton X-114 extract (Fig. 1b). The CheLM/CheLAM ratio (w/w) was also determined by routine monosaccharide analysis and revealed that CheLM was three times as abundant as CheLAM. This result was found for several independent extractions (data not shown). It is noteworthy that the relative abundance of these two compounds largely differs from the ones reported previously for other mycobacterial species, in which LAM represents the major component. For instance, an approximate LM/LAM ratio (w/w) of 1:2.5 was found inM. bovis BCG (26Nigou J. Gilleron M. Cahuzac B. Bounéri J.D. Herold M. Thurnher M. Puzo G. J. Biol. Chem. 1997; 272: 23094-23103Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) compared with the 3:1 ratio observed inM. chelonae. Because LM is thought to be a biosynthetic precursor of LAM, we first undertook the structural elucidation of LM, and we subsequently determined the structure of the more complex LAM molecule. The molecular weight of CheLM was investigated by MALDI analysis. It showed a broad unresolved peak centered at 6900 Da (data not shown). Quantitative analysis of the alditol-acetate derivatives of CheLM led to an average composition of 35 mannose, 1 arabinose, and 1 myo-inositol residues. The exact quantification of TMS derivatives by GC using on-column injection demonstrated the presence of 1.07 unit of glycerol per molecule ofmyo-inositol, which establishes the ratio of mannose/arabinose/inositol/glycerol as 35:1:1:1. Analysis of partially methylated alditol-acetate derivatives revealed the presence of three major components (Table I) identified on the basis of their retention times and fragmentation patterns as t-Manp, 6-Manp, and 3,6-Manp. Another product, different from a mannitol-acetate, was characterized by (M + NH4)+ at m/z 338, indicative of a di-acetylated, tetra-methylated inositol. EI/MS fragments at m/z 200, 191, and 75 identified this compound as a 2,6-Ac2-1,3,4,5-Me4-Ins, which is in agreement with an earlier published report (16Chatterjee D. Hunter S.W. McNeil M. Brennan P.J. J. Biol. Chem. 1992; 267: 6228-6233Abstract Full Text PDF PubMed Google Scholar). An (M + NH4)+ ion at m/z 366 corresponding to a tri-acetylated, tri-methylated inositol was detected as a minor component. However, because its fragmentation pattern was unclear, the detailed structure of this product was not analyzed further.Table IMethylation analysis of CheLM and CheLAMProposed structuremol (%)CheLMCheLAMt-Ara13.52-Ara6.25-Ara45.83,5-Ara14.3t-Man52.79.96-Man16.64.73,6-Man30.75.6 Open table in a new tab In order to establish the main features of both the polysaccharide and the putative GPI anchor of LM from M. chelonae, an exhaustive NMR-based study was conducted. NMR experiments were recorded successively in D2O and in Me2SO. As shown previously (26Nigou J. Gilleron M. Cahuzac B. Bounéri J.D. Herold M. Thurnher M. Puzo G. J. Biol. Chem. 1997; 272: 23094-23103Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), an improved resolution was obtained in Me2SO, which was used for most experiments. 1H and 13C NMR parameters of the polysaccharide moiety from CheLM were assigned using one-dimensional 1H and 13C experiments as well as two-dimensional 1H-1H homonuclear and two-dimensional 1H-13C and1H-31P heteronuclear experiments. Attributed1H and 13C NMR parameters from CheLM are summarized in Table II.Table IICheLM 1H and 13C chemical shifts in Me2SO-d6 at 343 KResiduesChemical shifts1234566′t-α-ManpIV1H4.943.843.663.483.663.693.5513C103.271.0971.5468.3174.2662.176-α-ManpVI1H4.713.683.563.52ND2-aND, not determined.NDND13C100.770.977267NDNDND3,6-α-ManpVIII1H4.713.943.653.643.623.763.6013C100.770.3380.3166.2872.4266.56α-Manp-11H5.153.61NDNDNDNDNDP113C98.7NDNDNDNDNDND1H5.133.58NDNDNDNDNDP313C98.7NDNDNDNDNDNDα-Manp-21H5.193.79NDNDNDNDNDP113C100.9671.2NDNDNDNDND1H5.113.78NDNDNDNDNDP313C101.571.5NDNDNDNDNDIns1H4.034.224.573.63.123.66P113C77.1175.473.15NDND80.31H3.934.193.23.43.053.62P313CNDNDNDNDND80.30Gro11′23.3′1H4.324.095.073.8P113C63.3263.3271.6463.61H4.354.115.093.85P313C63.3263.3271.6463.62-a ND, not determined. Open table in a new tab The anomeric proton region is dominated by two signals at δ 4.94 and δ 4.71 ppm (Fig. 2a). The former signal correlates in the COSY 90 experiment with a single H-2 signal at δ 3.84 ppm, whereas the other correlates with two largely distinct H-2 signals at δ 3.94 ppm and δ 3.68 ppm (Fig.2d), suggesting the presence of two anomeric protons of distinct origins at δ 4.71 ppm. The configuration of these three spin systems was unambiguously attributed to Manp in accordance with TOCSY and NOESY experiments. Moreover, the magnitude of the1JH1,C1 coupling constant, 168–170 Hz, provided evidence for the α-anomeric configuration of these mannose units. This observation was confirmed by the presence of an intra-residual H-1/H-2 NOE contact (data not shown). Based on the complete assignment of its 1H and13C NMR parameters and according to the published literature (14Venisse A. Berjeaud J.M. Chaurand P. Gilleron M. Puzo G. J. Biol. Chem. 1993; 268: 12401-12411Abstract Full Text PDF PubMed Google Scholar, 32Gilleron M. Bala L. Brando T. Vercellone A. Puzo G. J. Biol. Chem. 2000; 275: 677-684Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), the mannose unit deriving from the anomeric proton at δ 4.94 was typified as a terminal α-Manp(IV). The signal at δ 4.71 ppm correlated with a single C-1 signal at δ 100.7 ppm and was tentatively assigned to the anomeric protons of the 6-O-substituted and 3,6-O-substituted mannose units previously observed through methylation analysis. Because of the1H-13C HMQC (Fig. 2b), HMQC-HOHAHA (data not shown), COSY and HMBC experiments (Fig. 2a), all the carbon parameters of these two units could be assigned. In particular, two distinct C-3 signals were identified, one at δ 72 ppm corresponding to an unsubstituted C-3, and one deshielded at δ 80.3 ppm indicative of a substituted C-3. On the other hand, a single deshielded C-6 resonance at δ 66.6 ppm, and a single slightly shielded C-5 resonance at δ 72.42 ppm could also be observed. These parameters confirm that the two mannosyl units are 6-O-substituted and 3,6-O-substituted. Altogether, these NMR data support the fact that the major1H signal at δ 4.71 corresponds to the anomeric proton of both 6- and 3,6-substituted
New results concern Correlation Microwave Thermography, a radiometric process, able to complement Microwave Thermography in biomedical applications. Experimental data point out a spatial resolution which, for steep thermal gradients, can reach about 1 mm for a difference of temperature ΔT ⩽ 1°C. Consequently, Thermal Pattern Recognition can be improved with this method, mainly in the monitoring of Hyperthermia.
The authors have developed a fast MicroWave radiometric Imaging system (MWI) which realizes thermal images with a good spatial resolution. The authors are undertaking at the present time a large scale clinical evaluation of this system for early stage breast tumor characterization. A Receiver Operating Curve (ROC) statistical analsis of an intermediate stage of 60 patients gives a sensibility and a specificity of up to 80%. The authors show here that this technique can sucessfully be used for noninvasive radiological examination in terms of benign-malignant characterization. Moreover, the use of an inverse problem technique in the spatial frequency domain gives a quantitative measurement of cancer temperature. It is now possible to consider a new application such as the following of temperature during a chemotherapy treatment.
The oligosaccharide structures of bovine brain beta-N-acetylhexosaminidases A and B (EC 3.2.1.30) were studied at the glycopeptide level by employing 500 MHz 1H-n.m.r. spectroscopy and methylation analysis involving g.l.c.-m.s. More than 90% of the chains were found to be of the oligomannoside type, containing, on average, five to six mannose residues. Biantennary N-acetyl-lactosamine-type chains terminated in N-acetylneuraminic acid were found to comprise the remaining 5-10% of the total carbohydrate. The isoenzyme forms A and B do not differ from each other in the structure of their carbohydrate moiety, but do deviate in carbohydrate content and, in consequence, in the number of carbohydrate chains per molecule.
The use of radiometers for biomedical applications needs a coherent understanding of thermal signals emitted by the tissues. In this paper, we show first, that some precautions have to be taken when measuring a temperature by the classical radiometric method. For instance, the signal emitted by a lossy material depends on its temperature, permittivity and thickness. This remark allows us to find out a new method for measuring microwave permittivity of liquids when using a Dicke radiometer. We propose a modified radiometric method to determine directly the temperature of the material whatever its reflection coefficient. The applicability of this method is tested with a X band set-up including FET microwave amplifiers. Possibilities of using probes for in situ temperature measurements are discussed.
We describe a new radiometric method capable of reaching a better control of the volume of the tissue under investigation in microwave thermography (this technique is mainly used in biomedical applications). Our process is based on the fact that two (or several) probes with a volume under investigation in common are collecting thermal noise signals which are correlated.
The glycan primary structure of the main glycopeptide fraction obtained by pronase and carboxypeptidase A digestions of porcine pancreatic lipase has been investigated by 500‐MH z 1 H‐NMR spectroscopy and methylation analysis. The results demonstrate that the glycopeptide fraction was a mixture containing the following structures: image
