Biomedical subjects

N Qureshi

Publications and source records attributed to N Qureshi.

At least 91 records · Page 5Linked to original sources

PubMed1987

Substitution of phosphatidylserine by lipid A in the activation of purified rabbit brain protein kinase C.

Three lipid A derivatives (hexaacyl monophosphoryl lipid A, hexaacyl diphosphoryl lipid A, and disaccharide precursor IVA) were shown to activate protein kinase C from rabbit brain. These derivatives substituted for phosphatidylserine in a concentration-dependent manner and did not compete for binding of [3H]phorbol dibutyrate to its receptor site. Instead, phorbol dibutyrate binding was increased on raising the concentration of the derivatives in a similar manner to phosphatidylserine. The phorbol ester 12-0-tetra-decanol 13-acetate augmented the activation of protein kinase C by the lipid A derivatives.

Animals↗

PubMed1987

Structural determination of lipid A from gram negative bacteria using laser desorption mass spectrometry.

Laser desorption mass spectrometry has been employed for the structural determination of lipid A components derived from the lipopolysaccharides (LPS) of gram-negative bacteria. Mass spectra were obtained for methylated monophosphoryl lipid A from Neisseria gonorrhoeae and Rhodopseudomonas sphaeroides, for diphosphoryl lipid A from Escherichia coli and for the intact LPS from the Re Mutant of E. coli consisting of triphosphoryl lipid A and two KDO (2-keto-3-deoxyoctonate) units. Fragmentation of the phosphate (or pyrophosphate) on the reducing glucosamine is followed by fragmentation of acyl-linked fatty acids. Also observed are fragment ions which correspond to the distal portion of the molecule.

Escherichia coli↗

PubMed1986

Characterization of a structural series of lipid A obtained from the lipopolysaccharides of Neisseria gonorrhoeae. Combined laser desorption and fast atom bombardment mass spectral analysis of high performance liquid chromatography-purified dimethyl derivatives.

Monophosphoryl lipid A (MLA) obtained from the lipopolysaccharides of serum-sensitive strains of Neisseria gonorrhoeae was fractionated on a silicic acid column to yield the hexaacyl and pentaacyl MLAs. The dimethyl derivative of the hexaacyl MLA was analyzed by proton nuclear magnetic resonance spectroscopy. The dimethyl esters of hexaacyl and pentaacyl MLAs were further purified by reverse-phase high performance liquid chromatography, and all of the peaks were analyzed by laser desorption mass spectrometry. Considerable structural information was obtained by laser desorption mass spectrometry due to three kinds of specific fragmentations of the sugar at the reducing end. Two major fractions were also analyzed by positive ion fast atom bombardment mass spectrometry. High performance liquid chromatography was able to separate the dimethyl MLA according to number, nature, and position of the fatty acyl groups. Since almost no structural information is available, the mass spectra of the samples were interpreted on the basis of the established structure of a model lipid A (hexaacyl MLA derived from Salmonella minnesota). Thirteen different structures of dimethyl MLA were identified. The four prominent dimethyl MLAs found in the fractionated samples were M1 (Mr = 1463), M2 (Mr = 1479), M3 (Mr = 1661), and M4 (Mr = 1677). These MLAs appear to have a 1'----6 linked glucosamine disaccharide backbone. The most prominent hexaacyl MLA was M3. We propose that it contains hydroxylaurate at the 3- and 3'-positions in ester linkage and lauroxymyristate at the 2- and 2'-positions in amide linkage of the glucosamine disaccharide. The most abundant pentacyl MLA was M2. We propose that it contains hydroxylaurate at the 3- and 3'-positions in ester linkage, lauroxymyristate at the 2'-position in amide linkage, and hydroxymyristate at the 2-position in amide linkage of the disaccharide. The lipid A of N. gonorrhoeae appeared to differ from that of the Salmonella strains by the presence of shorter-chain fatty acids and by the normal fatty acid distribution in the reducing and distal subunits.

Chromatography, High Pressure Liquid↗

PubMed1985

Isolation and characterization of eight lipid A precursors from a 3-deoxy-D-manno-octylosonic acid-deficient mutant of Salmonella typhimurium.

Temperature-sensitive mutants of Salmonella typhimurium that are defective in the biosynthesis of 3-deoxy-D-manno-octulosonate are known to accumulate disaccharide precursor(s) of lipid A at 42 degrees C (Rick, P. D., Fung, L. W.-M., Ho, C., and Osborn, M. J. (1977) J. Biol. Chem. 252, 4904-4912). We have devised new methods for purifying this material by chromatography on DEAE-cellulose and silicic acid columns and have fractionated it into eight related anionic components that fall into four sets, as judged by their charge. Substances IA and IB have an apparent net charge of -1, IIA and IIB of -2, IIIA and IIIB of -3, and IVA and IVB of -4. Negative ion fast atom bombardment mass spectrometry reveals that the simplest component is IVA [( M - H]- at m/z 1404). Compound IVA is also the most abundant, representing 30-50% of the accumulated lipids after 3 h at 42 degrees C. Structural studies of IVA, including NMR spectroscopy described in the accompanying paper, reveal that it consists of O-(2-amino-2-deoxy-beta-D-glucopyranosyl)-(1----6)-2-amino-2-deoxy-alpha - D-glucose, acylated at positions 2, 3, 2', and 3' with beta-hydroxymyristoyl moieties and bearing phosphate groups at positions 1 and 4'. Compound IIIA ([M - H]- at m/z 1527) contains an additional phosphoethanolamine residue, while IIA ([M - H]- m/z 1535) bears an aminodeoxypentose substituent, presumably 4-amino-4-deoxy-L-arabinose. Compound IA ([M - H]- at m/z 1658) bears both a phosphoethanolamine and an aminodeoxypentose. The compounds of the less abundant B series are further derivatized with an ester-linked palmitoyl moiety. Our results demonstrate that these precursors are far more heterogeneous than previously suspected.

Glycolipids↗

PubMed1985

Location of polar substituents and fatty acyl chains on lipid A precursors from a 3-deoxy-D-manno-octulosonic acid-deficient mutant of Salmonella typhimurium. Studies by 1H, 13C, and 31P nuclear magnetic resonance.

Eight anionic disaccharide precursors of lipid A accumulate at 42 degrees C in 3-deoxy-D-manno-octulosonic acid-deficient temperature-sensitive mutants of Salmonella typhimurium. These compounds comprise a series of lipids based on the minimal structure, O-[2-amino-2-deoxy-N2,O3-bis(3-hydroxytetradecanoyl)-beta-D-glucopyranos yl] -(1----6)-2-amino-2-deoxy-N2, O3-bis(3-hydroxytetradecanoyl)-alpha-D-glucopyranose 1,4'- bisphosphate (designated lipid IVA) that differ from each other by the presence of an additional phosphoethanolamine moiety (IIIA), or an aminodeoxypentose moiety (IIA), or both (IA). A homologous set of metabolites is further derivatized with a palmitoyl function; these are designated IVB, IIIB, IIB, and IB (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16088). The attachment of the palmitoyl moiety, known to be on the reducing terminal GlcN residue by mass spectrometry, was determined to be O-beta of the N2-linked beta-hydroxymyristoyl group of that residue of IVB by 13C NMR and two-dimensional 1H chemical shift correlation spectroscopy experiments. 31P NMR indicated the presence of diphosphodiester moieties in IIIA, IIIB, and IA and monophosphodiester moieties in IIA and IA. Selective 1H decoupling of the 31P spectrum of IIIA demonstrated that the O-diphosphoethanolamine moiety is attached to the O4' position in IIIA. On the basis of the observed 31P chemical shifts it was concluded that the aminodeoxypentose is located at position 1 in IIA and IA, while diphosphoethanolamine is most likely located at O-4' in IA and IIIB, as in IIIA.

Acylation↗

PubMed1985

Monophosphoryl lipid A obtained from lipopolysaccharides of Salmonella minnesota R595. Purification of the dimethyl derivative by high performance liquid chromatography and complete structural determination.

The monophosphoryl lipid A (MLA) obtained from the lipopolysaccharides of Salmonella minnesota R595 was fractionated on a silicic acid column to yield the heptaacyl, hexaacyl, and pentaacyl MLA. Each of these MLAs was methylated with diazomethane to yield the dimethyl derivative and purified to homogeneity by reverse-phase high performance liquid chromatography. The molecular ions obtained by positive ion fast atom bombardment mass spectrometry of purified dimethyl heptaacyl MLA allowed us to establish the molecular formula and Mr of C112H211N2O23P and 1983.3, respectively. Cleavage at the glycosidic linkage yielded an oxonium ion of mass 1115, which showed that the distal sugar unit contained one phosphate (dimethyl), two hydroxymyristates, one laurate, and one myristate, while the reducing sugar unit contained two hydroxymyristates and one palmitate. By utilizing two-dimensional NMR spectroscopy, we were able to assign all of the protons of dimethyl heptaacyl MLA. This assignment included the beta protons of the three acyloxyacyl groups. A substantial downfield shift of the protons at the 3- and 3' -carbons was observed, which indicated that these two positions are occupied by ester groups. Fast atom bombardment mass spectral analysis of the hexaacyl and pentaacyl MLAs showed that these structures were identical to the previously designated TLC-3 and TLC-5 fractions, respectively, from Salmonella typhimurium. From this study, the complete structures of the MLA series found in the LPS of S. minnesota can now be described.

Chemical Phenomena↗

PubMed1985

Purification and characterization of C28-55 fatty acids from Mycobacterium smegmatis.

The nonmycolic C16 to C55 fatty acids obtained from Mycobacterium smegmatis ATCC 356 by saponification were enriched with respect to the C28 to C55 acids by successive chromatography on silicic acid and Sephadex LH-20 columns. These partially purified fatty acids were then derivatized to the p-bromophenacyl ester and further fractionated by argentation thin-layer chromatography and reverse-phase high-performance liquid chromatography into their individual components. The esters were characterized by electron impact mass spectrometry. Two structural series of C28:1 to C42:1 and C45:2 to C55:2 fatty acids were identified as possible precursors of the monoenyl and dienyl mycolic acids, respectively. These acids were structurally related to the alpha-alkylhydroxyl group of the corresponding mycolic acid. The results suggest that these C28 to C55 fatty acids (meromycolic acids) of M. smegmatis might be precursors of mycolic acids.

Chromatography, High Pressure Liquid↗

PubMed1984

Influence of fine structure of lipid A on Limulus amebocyte lysate clotting and toxic activities.

We examined the relationship between the fine structure of lipid A and the toxicity of endotoxin or lipopolysaccharides as measured by the Limulus amebocyte lysate (LAL), rabbit pyrogenicity, chicken embryo lethal dose, and dermal Shwartzman reaction tests. Lipid A and lipid A-like compounds obtained from deep-rough mutants of Salmonella spp. and Escherichia coli had a wide range of structural variations. These compounds included native lipopolysaccharides, diphosphoryl and monophosphoryl lipid A's, and lipid X (a monosaccharide). The LAL test was positive for all lipids tested with lysates from Travenol Laboratories and from Associates of Cape Cod (2.9 X 10(3) to 2.6 X 10(7) endotoxin units per mg), except for O-deacylated and dephosphorylated lipid X, which were negative. The Mallinckrodt lysate gave negative tests for lipid X. In the rabbit pyrogenicity and chicken embryo lethal dose tests, only native lipopolysaccharide and diphosphoryl lipid A's were judged toxic. The Shwartzman reaction was positive for a specific purified diphosphoryl lipid A (thin-layer chromatography-3 fraction) but negative for the purified monophosphoryl lipid A (also a thin-layer chromatography-3 fraction). These results show that the LAL test is not a valid measure of all parameters of toxicity of a lipid A or lipid A-like compound and can yield false-positive results. However, these findings are not in conflict with the widespread use of the LAL assay for pyrogens in the pharmaceutical industry since a good correlation exists between LAL results and pyrogenicity when undegraded endotoxin is evaluated in parallel assays.

Animals↗

PubMed1984

Biosynthesis of C30 to C56 fatty acids by an extract of Mycobacterium tuberculosis H37Ra.

A 10,800 X g supernatant fraction from disrupted cells of Mycobacterium tuberculosis H37Ra was incubated with [2-14C]malonate to produce labeled long-chain fatty acids upon saponification. These acids were derivatized to the p-bromophenacyl ester and separated into the nonmycolic saturated, monounsaturated, and multiunsaturated esters by argentation thin-layer chromatography. Each of these fractions was then analyzed by high-performance liquid chromatography by using a C18-bonded silica cartridge and a mobile phase of a linear gradient of 0 to 70% p-dioxane in acetonitrile. The results showed that the cell-free system is able to synthesize both saturated and monounsaturated fatty acids of the sizes C30 to C40 and C48 to C56. This latter series was strikingly similar to meromycolic acid, a putative precursor of mycolic acid. When acetate or oleate was used as the labeled substrate, the major products were no longer than C32. When palmitate was used as the labeled substrate, the saturated acids ranged in size from C18 to C32, whereas the monounsaturated products contained C18, C26 to C30 and C40 fatty acids. Fatty acids greater than C40 were also detected. When methyl-labeled S-adenosyl-L-methionine was used as the substrate, the methyl group was incorporated into short-chain and C48 to C56 fatty acids. Unlabeled malonyl-coenzyme A was included in all of these reactions. This cell-free system was not able to synthesize mycolic acid (final product) or its keto derivative (intermediate product). However, since the meromycolate-like C48 to C56 fatty acids were synthesized, we suggest that the present system is able to take the synthesis to a point before the alpha-alkyl condensation reaction.

Chromatography, High Pressure Liquid↗

PubMed1983

Glucosamine-derived phospholipids in Escherichia coli. Structure and chemical modification of a triacyl glucosamine 1-phosphate found in a phosphatidylglycerol-deficient mutant.

Certain Escherichia coli mutants defective in phosphatidylglycerol biosynthesis accumulate novel glucosamine-derived phospholipids. We previously demonstrated that the simplest of these substance (lipid X) is a diacylglucosamine 1-phosphate bearing beta-hydroxymyristoyl groups at positions 2 and 3 (Takayama, K., Qureshi, N., Mascagni, P., Nashed, M. A., Anderson, L., and Raetz, C. R. H. (1983) J. Biol. Chem. 258, 7379-7385). We now report the structural characterization of a triacylglucosamine 1-phosphate (designated lipid Y) that is also found in these mutants. Hydrolyzates of Y contain 2 mol of beta-hydroxymyristate and 1 mol of palmitate/mol of glucosamine. In the lipid, one of the beta-hydroxymyristates is amide-linked at position 2, while the two other fatty acyl groups are ester-linked. Fast atom bombardment mass spectrometry is used to confirm that Y is a monosaccharide derivative and that the molecular weight of Y as the free acid (C50H96NO13P) is 950.29. Analysis of Y by proton NMR spectroscopy at 200 MHz reveals that the anomeric configuration is alpha. Further, one of the esterified fatty acid residues is attached to the 3 OH of the sugar, while the second is linked to an OH moiety of a hydroxymyristate. The 4 and 6 OH groups of the sugar are unsubstituted, as in E. coli lipid X. To establish the precise location of each esterified fatty acyl residue, we subjected Y to a very mild alkaline hydrolysis in the presence of triethylamine. This resulted in the selective removal of a single hydroxymyristoyl group. The triethylamine-treated derivative (lipid Y) has a molecular weight of 723. NMR spectroscopy of Y shows that the 3 OH of the sugar is no longer substituted, while the beta OH of the remaining amide-linked hydroxymyristate is still esterified with palmitate. On the basis of these findings, we propose that lipid Y has the same fundamental structure as lipid X, except for the additional presence of a palmitoyl moiety on the N-linked hydroxymyristate. Presumably, lipid Y is synthesized from X by a selective acylation reaction.

Endotoxins↗

PubMed1983

Complete structure of lipid A obtained from the lipopolysaccharides of the heptoseless mutant of Salmonella typhimurium.

A highly purified monophosphoryl lipid A, TLC-3 fraction obtained from the lipopolysaccharides of the heptoseless mutant Salmonella typhimurium G30/C21 was converted to the dimethyl pentatrimethylsilyl derivative and analyzed by proton NMR spectroscopy at 400 MHz. Substantial downfield shifts of the resonances for protons at the 3- and 3'-carbons of the glucosamine disaccharide to 5.06 and 5.15 ppm, respectively, occurred from the normal range of 3.5-4.1 ppm, indicating that these two positions on the sugar rings were acylated. Significant downfield shift of the resonances for protons at the 4- and 6'-carbons did not occur, indicating the absence of acyl groups at these two positions. Since positive ion fast atom bombardment mass spectrometry previously established the presence of hydroxymyristoyl and myristoxymyristoyl esters at the reducing end and distal subunits, respectively, these acyl groups must be attached to the oxygen of the corresponding 3- and 3'-carbons of lipid A. With these results, we can now describe the complete structure of the monophosphoryl lipid A, TLC-3 from S. typhimurium.

Heptoses↗

PubMed1983

Position of ester groups in the lipid A backbone of lipopolysaccharides obtained from Salmonella typhimurium.

Lipopolysaccharides extracted from the heptoseless mutant of Salmonella typhimurium G30/C21 were hydrolyzed with either 0.1 N HCl at 100 degrees C or treated twice with 20 mM sodium acetate, pH 4.5, at 100 degrees C for 45 min and finally purified by preparative thin layer chromatography to yield a structural series of mono- and diphosphoryl lipid A, respectively. Positive ion fast atom bombardment mass spectrometry of the diphosphoryl lipid A TLC-3 (a highly acylated major band) showed a major component with (M + H)+ ion of mass 1798, which fragmented to yield a (M - H2PO4)+ ion of mass 1700. Cleavage at the glycosidic bond gave rise to an oxonium ion fragment of mass 1087. In conjunction with other studies, this establishes the molecular formula and Mr of the major component to be C94H178N2O25P2 and 1797.2 (as the free acid), respectively. Similar analysis of monophosphoryl lipid A TLC-3 produced an (M + H)+ peak at m/z 1718, (M + Na)+ adduct peak at m/z 1740, and a fragment of mass 1087. The spectrum of monophosphoryl lipid A TLC-5 was devoid of the m/z 1087 peak and instead contained the phosphorylated oxonium ion of mass 876. This fragment ion is assigned as the distal subunit, and these results show that the distal subunit of the major lipid A TLC-3 contains two hydroxymyristoyl, one myristoyl, and one lauroyl residues, whereas the reducing end subunit contains two hydroxymyristoyl groups. A revised structure of the lipid A backbone in lipopolysaccharides of S. typhimurium is proposed.

Chromatography, Thin Layer↗

PubMed1983

Fatty acyl derivatives of glucosamine 1-phosphate in Escherichia coli and their relation to lipid A. Complete structure of A diacyl GlcN-1-P found in a phosphatidylglycerol-deficient mutant.

We have determined the complete structure of a glycolipid (designated lipid X) previously found to accumulate in certain Escherichia coli mutants defective in phosphatidylglycerol synthesis (Nishijima, M., and Raetz, C.R.H. (1979) J. Biol. Chem. 254, 7837-7844). Based on fast atom bombardment mass spectrometry and proton nuclear magnetic resonance studies, this substance is an acylated metabolite of glucosamine 1-phosphate. Lipid X of E. coli has a Mr = 711.87 as the free acid (C34H66NO12P) and contains two beta-hydroxymyristate moieties, one attached as an amide at the 2 position and the other as an ester at the 3 position of the sugar. It has free hydroxyl groups at the 4 and 6 positions, and the anomeric configuration is alpha. The structure of lipid X from E. coli closely resembles the reducing end subunit of lipid A, and it might represent a very early precursor in the biosynthesis of lipid A. To our knowledge, fatty acyl derivatives of glucosamine 1-phosphate have not been reported previously.

Escherichia coli↗

PubMed1982

Purification and structural determination of nontoxic lipid A obtained from the lipopolysaccharide of Salmonella typhimurium.

Endotoxin extracted from the heptose-less mutant of Salmonella typhimurium was hydrolyzed in 0.1 N HCl in methanol/water (1:1, v/v) at 100 degrees C to yield lipid A, which was then fractionated on a Sephadex LH-20 column to yield a major monophosphoryl lipid A fraction. The monophosphoryl lipid A was further fractionated by preparative thin layer chromatography. This process yielded three major bands (TLC-1, -3, and -5) and two minor bands (TLC-7 and -9). The purity of these fractions was established by ion exchange and reverse phase high performance liquid chromatography. The thin layer fractions were analyzed by fast atom bombardment mass spectrometry. TLC-1 and -3 gave molecular ions (M-H)- at m/e 1730 and 1716, respectively. Both of these fractions contained beta-hydroxymyristic, lauric, and 3-myristoxymyristic acids in O-acyl linkages. The molecular formula and Mr of TLC-1 are C95H179O22N2P and 1731.16; those of TLC-3 are C94H177O22N2P and 1717.15. TLC-1 was a methyl homolog of TLC-3. The major component of TLC-5 (C80H151O22N2P and Mr = 1506.99) gave a molecular ion at m/e 1506 and contained two beta-hydroxymyristic acids and a lauric acid in the O-acyl linkages. The major component of TLC-7 (C66H125O19N2P and Mr = 1280.83) and the single component of TLC-9 gave molecular ions at m/e 1280 and 1098, respectively. TLC-7 contained lauric and beta-hydroxymyristic acids in the O-acyl linkages. TLC-9 (C54H103O18N2P and Mr = 1098.69) contained a single O-acylated beta-hydroxymyristate group. TLC-1 and -3 were nontoxic in the chick embryo lethality test and regressed established tumors in the syngeneic guinea pigs.

Chromatography, High Pressure Liquid↗

PubMed1981