Plasma desorption mass spectrometry of peptides adsorbed on nitrocellulose from a glutathione matrix.
Explore the source record for details and available documents.
Collodion↗
Biomedical subjects
I Jardine
Publications and source records attributed to I Jardine.
Trehalose-containing lipooligosaccharide antigens of Mycobacterium sp.: presence of a mono-O-methyltri-O-acyltrehalose "core".
We have described the surface antigens of Mycobacterium kansasii as trehalose-containing lipooligosaccharides (LOS) which at the nonreducing "epitope" end bear a unique amino sugar containing diglycosyl unit, whereas the putative reducing end consists of an acylated alpha, alpha-trehalose-containing tetraglucosyl "core" [Hunter, S. W., Jardine, I., Yanagihara, D. L., & Brennan, P. J. (1985) Biochemistry 24, 2798-2805]. The presence of a new variation on this core, in Mycobacterium szulgai, is now reported, ----3)beta-D-Glcp-(1----6)alpha-D-Glcp(1----1)3,4,6-tri-O-acyl-2-O- Me-alpha-D-Glcp, representing the first example of an O-methyltrehalose unit in nature. The simplest of the LOS class of glycolipids in M. szulgai was defined as alpha-L-2-O-Me-Fucp(1----3)alpha-L-Rhap(1----3)alpha-L-Rh ap(1----3) beta-D-Glcp(1----6)alpha-D-Glcp(1----1)3,4,6-tri-O-acyl-2-O-Me-alpha-D-G lcp. Further glycosylation of this nonantigen, by an incompletely defined 6-deoxyhexosyl residue, confers specific antigenicity on the organism. Thus, these extraordinary structures, in a manner analogous to the better known lipopolysaccharides from rough variants of Enterobactericiae, are highly amphipathic and display variability not only in the immunogenic, distal region but also in the "invariant" lipophilic core. The contribution of these glycolipids to the hydrophobic barrier, the pseudo outer membrane of mycobacteria, is discussed.
Acylation↗
Location of acyl groups of trehalose-containing lipooligosaccharides of mycobacteria.
A variant of a Mycobacterium sp. originating in a patient with Crohn's disease, but not necessarily implicated in the disease, provided a simple version of a newer class of species-specific surface glycolipids, the trehalose-containing lipooligosaccharides. A combination of high-resolution 1H nuclear magnetic resonance, methylation, ethylation, and absolute configurational analysis established the structure of the oligosaccharide unit as beta-D-Glcp(1----3)-alpha-L-Rhap(1----3)-alpha-D-Glcp(1----1)-alph a-D-Glcp (where Glc is glucose, Rha is rhamnose, and p is pyranosyl), and gas chromatography-electron impact mass spectrometry allowed identification of the fatty acyl esters as primarily 2,4-dimethyltetradecanoate. The relative simplicity of the glycolipid combined with the application of a mild methylation procedure and californium-252 plasma desorption mass spectrometry allowed recognition of three such acyl residues on the 3-, 4-, and 6-hydroxyl positions of the terminal glucosyl residue of the trehalose unit. Thus, the glycolipid is decidedly amphipathic yet is clearly not membranous. This observation leads to speculation about the role of these novel lipooligosaccharides in contributing to the outer segment of the hydrophobic barrier of the cell wall of certain mycobacteria.
Chemical Phenomena↗
Definition of the surface antigens of Mycobacterium malmoense and use in studying the etiology of a form of mycobacteriosis.
Mycobacterium malmoense is the latest of a roster of atypical mycobacteria implicated in pulmonary infections. Yet it lacks recognizable phenotypic features to allow its ready identification. Some 23 clinical isolates of M. malmoense were examined for homologous seroagglutination reactions and characteristic surface antigens. One group showed concordant agglutination interreactions and an identical spectrum of glycolipids and are regarded as M. malmoense sensu stricto. The glycolipids are of the newly found, trehalose-containing lipooligosaccharide class. De-O-acylation followed by high-pressure liquid chromatography revealed one major and several minor oligosaccharides. Partial acidic cleavage to release glycosidically linked trehalose, alpha-mannosidase digestion to demonstrate the presence of a non-reducing-end mannobiose, perdeuteriomethylation, partial acid hydrolysis, reduction, and O ethylation, combined with 1H nuclear magnetic resonance and electron impact and fast-atom bombardment mass spectrometry revealed the structure of the major oligosaccharide as alpha-D-Manp-(1----3) -alpha-D-Manp-(1----[2-alpha-L-Rhap-(1--]4--3)-alpha-L-Rh ap- (1----3)-alpha-D-Glcp-(1----1)-alpha-D-Glcp, in which two of the 2-alpha-L-Rhap residues are O methylated at C-3. (Man, mannose; Rha, rhamnose; Glc, glucose; p, pyranosyl). The structures of the minor oligosaccharides were also determined; they differ at the distal nonreducing end. The dominant oligosaccharide was acylated by octanoate, 2-methyleicosanoate, and 2,4-dimethylpentacosanoate to yield the major species-specific surface antigen of M. malmoense, which we regard as the most characteristic feature of the pathogen.
Agglutinins↗
Chemical characterization and regulation of endogenous morphine and codeine in the rat.
Data on the tissue distribution of morphine and codeine in rat are presented. The concentration of these two opiate alkaloids seems to be distributed uniformly in the cortex, midbrain, pons/medulla and cerebellum. The spinal cord and the adrenal gland have high levels of morphine and codeine and the adrenal has more codeine than morphine. The major fraction of the alkaloids reside in a synaptosomal fraction and are present in tissues as the sulfate conjugate. The levels of morphine in the spinal cord and the urinary excretion of morphine are elevated in the arthritic rat model. We used extracted alkaloid samples from arthritic rats spinal cord for analysis by mass spectrometry and found molecular ions identical with morphine and codeine. The results are discussed in the light of possible physiological roles of endogenous morphine and codeine.
Animals↗
A novel mass spectrometric procedure to rapidly determine the partial structure of heparin fragments.
The molecular weight and degree of sulfation has been obtained for di-, tetra- and hexasaccharide fragments of heparin obtained by enzymatic depolymerization of porcine mucosal heparin. The sodium salt form of the sulfated oligosaccharide is adsorbed onto an immobilized cationic surfactant film which is inserted directly into the mass spectrometer. Analyses are routinely obtained on 25-50 microgram samples in less than an hour. This approach provides rapid confirmatory structural information that is complementary to existing methodologies.
Heparin↗
Heterogeneity of bacterial antigenic lipooligosaccharides determined by californium-252 plasma desorption mass spectrometry.
Californium-252 plasma desorption mass spectrometric analysis of complex lipooligosaccharide antigens from Mycobacterium kansasii has provided molecular weight information. From the spectra obtained and from other data it can be concluded that these lipooligosaccharides generally contain three 2,4-dimethyltetradecanoyl groups, and considerable heterogeneity in the attached acyl functions is apparent.
Antigens, Bacterial↗
Implementation of the gas chromatographic/mass spectrometric peptide sequencing program PEPALG on a personal computer for off-line analysis.
The gas chromatographic/mass spectrometric peptide sequencing program PEPALG has been implemented in both FORTRAN and PASCAL on a personal computer for convenient off-line analysis. The availability of PEPALG in a microcomputer-based version of both FORTRAN and PASCAL should now allow ready access to this powerful protein sequencing technique by any interested laboratory.
Amino Acid Sequence↗
Mass spectrometric analysis of cyclosporine metabolites.
Cyclosporine (CsA, mol. wt 1202) is a cyclic undecapeptide with potent immunosuppressive properties which have made it extremely useful for preventing the rejection of transplanted organs. The drug has severe side-effects, however, which may be related to its metabolism. Sensitive methods to determine the structure of metabolites of CsA have, therefore, been developed using mass spectrometric techniques. First, the molecular weight of the metabolite is determined using fast atom bombardment mass spectrometry or thermospray liquid chromatography/mass spectrometry. The metabolite is then hydrolysed to its component amino acids, which are esterified and acylated and identified by gas chromatography/mass spectrometry. To distinguish metabolism at the four identical N-methyl leucines, the metabolite is partially hydrolysed, the resulting peptides are derivatized to the trimethylsilyl-polyamino alcohols, and these in turn are analysed by gas chromatography/mass spectrometry. These procedures have been used to determine the structure of metabolites of CsA isolated from rabbit bile. The determination of the structure of one metabolite carboxylated on the eta-carbon of amino acid 1, and of one metabolite hydroxylated on the eta-carbon of amino acid 1 and on the gamma-carbon of N-methyl leucine 9 is presented. These procedures should be generally useful for the structural analysis of microgram amounts of CsA metabolites and analogs.
Amino Acids↗
A novel cyclosporine binding assay.
The binding of [3H]cyclosporine A (CsA) to BALB/c mouse spleen cells was examined with a novel centrifugation assay which rapidly removes free [3H]CsA from cell surfaces with a minimal loss of low affinity specifically bound [3H]CsA. A single class of specific and saturable CsA binding sites was found under equilibrium binding conditions. Scatchard analysis of the data resulted in a straight line with KD and Bmax values of 95 nM and 2.4 million binding sites/cell, respectively. Kinetic studies conducted with a wider range of [3H]CsA concentrations revealed two distinct binding sites, with KD's of 290 nM and 9.6 microM, respectively. [3H]CsA bound only nonspecifically to phosphatidylcholine:cholesterol liposomes. Specific [3H]CsA binding sites were found in murine WEHI-5 B-lymphoma cells, murine N1E-115 neuroblastoma cells and human A204 rhabdosarcoma cells. We conclude from these results that there are at least two nonlipid CsA binding sites in BALB/c mouse spleen cells and that CsA binding sites are present in both lymphoid and nonlymphoid tissue.
Animals↗
An acid metabolite of cyclosporine.
The primary biliary metabolite of cyclosporine has been isolated from rabbit and human bile. The material has been identified by mass spectrometry and by nuclear magnetic resonance spectrometry as an acidic metabolite of cyclosporine in which the eta-methyl group of the cyclosporine-specific nine carbon amino acid #1 has been oxidized to an alpha, beta unsaturated carboxylic acid functionality. This major cyclosporine metabolite is inactive in a phytohemagglutinin stimulated lymphocyte proliferation assay.
Animals↗
Trehalose-containing lipooligosaccharides from mycobacteria: structures of the oligosaccharide segments and recognition of a unique N-acylkanosamine-containing epitope.
The structures of the oligosaccharide segments of nine trehalose-containing lipooligosaccharides (LOS) of Mycobacterium kansasii have been established by positive and negative fast-atom bombardment mass spectrometry, acetolysis, partial acid hydrolysis, methylation analyses, and nuclear magnetic resonance. Upon acetolysis, all produce the alpha,alpha-trehalose-containing tetraglucose (Glc4) "core" -beta-D-Glcp(1----3)-beta-D-Glcp(1----4)-alpha-D-Glcp(1----1)-alpha-D-Gl cp. The simplest (LOS I') contains an additional alpha 1----3-linked 3-O-methyl-L-rhamnopyranose (3-O-Me-L-Rhap) unit; those of intermediate complexity (LOS I-III) contain an additional D-xylopyranose (D-Xylp) residue or xylobiose in beta 1----4 linkage; and those of ultimate complexity (LOS IV-VIII) contain further D-Xylp residues and the distal N-acylkanosamine- (KanNAcyl) and fucopyranosyl- (Fucp) containing disaccharide KanNAcyl(1----3)Fucp. Thus, the structure of the oligosaccharide from LOS VII is KanNAcyl(1----3)Fucp(1----4) [-beta-D-Xylp(1----4)]6-alpha-L-3-O-Me-Rhap(1----3)Glc4++ +. Polyclonal rabbit and murine monoclonal antibodies react only with the more complex KanNAcyl-Fucp-containing lipooligosaccharides, indicating that the KanNAcyl distal end, not the trehalose end, contains the antibody binding site unique to M. kansasii and is responsible for the serological distinctiveness of M. kansasii among mycobacterial species.
Carbohydrate Conformation↗
Influence of hepatic oxidative metabolism on the immunosuppressive activities of 6-thiopurines.
It has been suggested that in vitro and in vivo hepatic metabolism of 6-thiopurine and its prodrug azathioprine generates immunosuppressive metabolites with far greater activity than the parent drugs. To examine this possibility, in vitro and in vivo drug-metabolizing systems were interfaced with in vitro immune response assays for the detection of active 6-thiopurine metabolites. In this way, it was demonstrated that preincubation of 6-thiopurine with hepatic microsomes from Balb/c mice does not enhance the immunosuppressive activity of the parent drug in in vitro lymphocyte proliferation assays performed with Balb/c splenocytes stimulated by T and B cell mitogenic lectins. In fact, prior microsomal metabolism generally decreased the inhibitory effects of 6-thiopurine on lymphocyte mitogenic responses, indicating that any metabolites formed under these conditions were relatively inactive. In contrast, the immunosuppressive effects of cyclophosphamide, which was used as a positive control in this system, were profoundly increased by microsomal metabolism. It was shown, moreover, that in vivo metabolism of 6-thiopurine and azathiopurine did not lead to the generation of metabolites with detectably greater immunosuppressive activity than the parent drugs. The circulating immunosuppressive activity present in the serum of drug-treated Balb/c mice, when quantitated in vitro with a sensitive murine mixed lymphocyte culture system, was found to correlate strongly with the serum levels of the unmetabolized thiopurines, which were measured specifically by high-pressure liquid chromatography assay. Taken together, these results fail to support earlier hypotheses that blood-borne active metabolites contribute significantly to the immunosuppressive actions of 6-thiopurines.
Animals↗
Electron ionization mass spectra of reduced and permethylated urinary oligosaccharides from patients with mannosidosis.
The electron ionization mass spectra of reduced and permethylated isomeric mixtures of the major urinary tri- to deca-oligosaccharides of patients with mannosidosis are reported. Many of the oligosaccharide isomers can be differentiated in the mixtures on the basis of their distinct fragmentation patterns.
Humans↗
Characterization of cholecystokinin from the human brain.
Human forms of cholecystokinin have not previously been characterized chemically. In this study, we have extracted and purified the predominant molecular form of cholecystokinin present in human cerebral cortex. The peptide was characterized by amino acid analysis, automated peptide sequencing, and fast atom bombardment mass spectrometry. It appears to be identical to porcine cholecystokinin-octapeptide, with the sequence of Asp-Tyr(SO3)-Met-Gly-Trp-Met-Asp-Phe(NH2). This structural identity is consistent with the observations that the peptide in human brain and porcine cholecystokinin-octapeptide are recognized similarly by a battery of antisera to porcine cholecystokinin; that they coelute from several chromatographic systems, including gel filtration, ion exchange, and reversed-phase; and that they possess similar biological activities.
Brain Chemistry↗
Synthesis of mono- and diglucosiduronates of metabolites of deoxycorticosterone and corticosterone and analysis by a new mass spectrometric technique.
By condensing 3 alpha,21-dihydroxy-5 beta-pregnan-20-one, or its appropriate monoacetate, with methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-alpha-D-glucuronate in the Koenigs-Knorr reaction beta-D-glucosiduronates 10, 4, and 7 were obtained as polyacetate methyl esters. Alkaline hydrolysis of these substances cleaved the ester groups and gave the corresponding steroidal glucosiduronic acids 12, 6 and 8. Upon treatment with diazomethane, these acids produced the equivalent methyl esters. The C-3, the C-21 and the C-3,21 glucosiduronates of 3 alpha,21-dihydroxy-5 beta-pregnan-11,20-dione were prepared by previously reported methods and converted into the corresponding C-20 semicarbazones (14, 20 and 26). With C-20 stabilized by the semicarbazone group against reduction, it was possible to reduce the 11-oxo function in these substances to an 11 beta-hydroxyl group; after removal of the semi-carbazone moiety from these products at pH 2.0, glucosiduronic acids 18, 22 and 28 were obtained. The mass spectra of a representative group of the mono- and diglucosiduronic acids and esters were determined by utilizing fast atom bombardment and monitoring ions in both positive and negative modes of operation.
Chemical Phenomena↗
Synthesis and pH-dependent stability of purine-6-sulfenic acid, a putative reactive metabolite of 6-thiopurine.
Previous studies have shown that 6-thiopurine is metabolically activated by hepatic cytochrome P-450 to an intermediate capable of binding to proteins by a mixed disulfide linkage. The identity of the active metabolite was postulated to be purine-6-sulfenic acid. In the present report, we describe the synthesis of the sulfenic acid derivatives of 6-thiopurine and two structurally similar compounds, 9-methyl-6-thiopurine and 4-mercapto-1H-pyrazolo[3,4-d]-pyrimidine. The unusual pH-dependent stability profiles of these compounds in buffered aqueous media are presented and explained on the basis of a disproportionation mechanism of sulfenic acid decomposition. Studies with radiolabeled purine-6-sulfenic acid demonstrate that this species binds directly to hepatic microsomal protein. These results support the proposed involvement of purine-6-sulfenic acid in the metabolic activation and tissue binding of 6-thiopurine.
Animals↗
In vitro alteration of canine renal allografts with cyclophosphamide metabolites.
4-Hydroxycyclophosphamide (4-HCY) is generated spontaneously in cryoprecipitated plasma from synthesized 4-hydroperoxycyclophosphamide (4-HPCY); its immunosuppressive and cytotoxic effects seem to depend on milieu, temperature, time, and dosage. Our preliminary results suggest that prolonged canine renal allograft survival can be achieved with grafts exposed to CY metabolites (4-HPCY and PM) in vitro for 24 hr. Further studies with larger dosages (greater than or equal to 100 micrograms/ml of 4-HPCY) are indicated to test these suggestions conclusively.
Animals↗