Browsing by Author "Smith, David F."
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- Affinity purification of blood group A-active glycolipids on immobilized Helix pomatia lectinTorres-López, Beatriz Virginia (Virginia Polytechnic Institute and State University, 1988)Lectin affinity chromatography has proven to be a powerful method to separate oligosaccharides based on their stereochemical structures. This technique has not been used for the separation of glycolipids since mixtures of these compounds form micelles in aqueous solution. Since N-acetylgalactosamine (GalNAc) is commonly found in glycolipids, three GalNAc-specific lectins were selected to develop a lectin affinity chromatographic method for glycolipids. To circumvent the difficulty of working with micelles, the autoradiographic detection of ¹²⁵l-labeled lectins binding to glycolipids on thin-layer chromatograms was used to study the glycolipid-binding specificity of the lectins from Helix pomatia, Wisteria floribunda and Dolichos biflorus. All three lectins detected the Forssman glycolipid which has a terminal GalNAcα1-3 residue. The Helix pomatia and Wisteria floribunda lectins are also bound to glycolipids with GalNAcβ-linked residues. The interactions of these lectins with glycolipid derived, ³H-labeled oligosaccharides were also analyzed by affinity chromatography on agarose-immobilized lectins. Only the immobilized Helix pomatia lectin was able to specifically bind oligosaccharides with α-linked GalNAc residues. The Helix pomatia lectin was selected to develop an affinity chromatography system for the purification of intact glycolipids having terminal GalNAcα1-3 residues. This technique relies on the ability of the immobilized lectin to bind its oligosaccharide ligands in aqueous solutions of tetrahydrofuran (THF) which inhibits micelle formation and permits the separation of non-specifically bound glycolipids. Forssman glycolipid and a human blood group A-active hexaosylceramide were bound to the Helix pomatia column equilibrated in water/THF (5:95). After applying a step gradient of increasing water content (to 50% water), the specifically bound glycolipids were eluted when GalANc was included in the mobile phase. Using these chromatographic conditions, the Forssman glycolipid from the neutral lipid fraction of sheep erythrocyte stroma and the A-active glycolipids from a total extract of type A human erythrocytes were purified in the Helix pomatia column. The ability to purify human A-active glycolipids from total lipid extracts in a single chromatographic step with the Helix pomatia column was used to isolate A-active glycolipids present in erythrocytes from donors from a rare blood group B(A). The erythrocytes from B(A) subgroup of blood group B individuals, are weakly hemagglutinated by a murine monoclonal anti-A antibody although these erythrocytes should not express blood group A antigens. The Helix pomatia lectin was used to determine the presence and isolate A-active glycolipids from the neutral lipid fraction of erythrocytes from two blood group B(A) donors. However, A-active glycolipids were absent in the glycolipid extracts from erythrocytes from a third B(A) donor and plasma of all three B(A) donors as well as erythrocytes of blood group B and O donors. Based on the fact that only glycolipids and oligosaccharides with GalNAcα1-3 residues specifically bind to the Helix pomatia column, this lectin column was used to isolate the 'terminal products' of the biosynthetic pathway of the human blood group A glycolipids and glycopeptides from the human epidermoid carcinoma cell line A-431. The metabolically active A-431 cells were grown in the presence of ³H-labeled monosaccharide precursors and the Helix pomatia column was used to determine and compare the rate of incorporation of labeled precursors in the A-active glycoconjugates from these cells.
- Detection of colorectal carcinoma-associated antigens using specific antibodies against human milk oligosaccharidesLaw, Kevin L. (Virginia Polytechnic Institute and State University, 1986)Rabbit antibodies against human milk sialyltetrasaccharide b Galßl—3[NeuAca2—6]GlcNAcßl—3Galßl—4Glc) and sialyltetrasaccharide a (NeuAca2-3Ga|ßl·3GlcNAcß1-3Galßl-4Glc) were used to detect their homologous haptens as gangliosides in the human colorectal carcinoma cell line SW'lll6. Sialyltetrasaccharide b-ceramide was detected in the monosialylganglioside fraction from human meconium and a total ganglioside fraction from SWlll6 cells on thin layer chromatograms by radioimmune staining using anti-sialyltetrasaccharide . b. Sialyltetrasaccharide b-ceramide was not detected in a total Iipid extract from normal intestinal mucosa, thus suggesting that it may represent another tumor·associated antigen. Two novel disialylgangliosides recently reported in human colonic adenocarcinoma — disialylIactotetraosylceramide (NeuAc¤2·3Ga|ß1-3[NeuAca2·6]GlcNAcßl-3 Galßl-4Glcl-lCer) and disialyl Lea (NeuAc¤2-3Ga|B1—3[NeuAc¤2-6 (Fucal·4)]GIcNAcßl-3Ga|ßl-4Glcl·lCer) -· both contain the sialyltetrasaccharide a and b structures, either of which may represent the biosynthetic precursor of these disialylgangliosides. The anti·sialy|tetrasaccharide a antibody specifically recognizes its reduced homologous hapten and was used in a radioimmmune binding assay to detect sialyltetrasaccharide a as a reduced and tritiated, ganglioside·derived sialyloligosaccharide from SWlll6 cells. Sialyltetrasaccharide a-ceramide was recently detected in human embryonal carcinoma cells, and it is the biosynthetic precursor of the sialyl Lea antigen, a tumor-associated ganglioside in SWH16 cells. This report confirms the existence of sialyltetrasaccharide a-ceramide in SW11l6 cells.
- Economic Comparisons Between an Even-Aged and an Uneven-Aged Loblolly Pine Silvicultural SystemCafferata, Michael J.S. (Virginia Tech, 1997-05-28)This study compares financially optimal uneven-aged and even-aged silvicultural regimes of loblolly pine (Pinus Taeda). Uneven-aged regimes which maximize net present value (NPV) are found by quantifying the effects of diameter distribution (Q factor), maximum diameter, cutting cycle, and residual basal area on NPV. For the benchmark inputs, the regime yielding the highest NPV had a maximum diameter of 12 inches, residual basal area of 45 ft²/acre, and a cutting cycle of 11 years. Financially optimal even-aged regimes are taken from published literature of even-aged silviculture. Even-aged and uneven-aged silvicultural regimes are simulated starting from, 1) bare land, 2) a balanced uneven-aged loblolly pine stand, and 3) a mature even-aged loblolly pine stand. For the three starting conditions and selected benchmark variable values, simulation of even-aged silviculture yields NPVs of $877, $2,152 and $3,400 per acre and simulation of uneven-aged silviculture yields NPVs of $644, $2,084, and $2,569 per acre. Sensitivity analysis shows, for the levels of the variables tested, that even-aged silviculture yields higher NPVs than uneven-aged silviculture when starting from bare land or from a mature even-aged stand. When starting from an uneven-aged stand, for the variable values tested, uneven and even-aged silviculture are financially very competitive. Aside from the aesthetic benefits of avoiding clearcutting under uneven-aged silviculture, non-timber considerations between loblolly pine silvicultural systems are not well documented. Resource professionals hold opinions often in direct conflict with each other regarding the non-timber costs and benefits of even-aged and uneven-aged silviculture when considering wildlife, soil and water, and catastrophic damage events.
- Glycolipids in mouse F9 teratocarcinoma cells: some changes associated with retinoic acid-induced differentiationGorbea, Carlos M. (Virginia Tech, 1991-01-15)To investigate the changes in glycolipid biosynthesis during early embryogenesis mouse F9 teratocarcinoma cells were induced to differentiate in vitro in the presence of retinoic acid. Control embryonal carcinoma cells and their differentiated derivatives, RNF9 cells, were metabolically-radiolabeled with [6-3H]galactose or [6-3H]glucosamine, and their glycolipids were compared. The neutral and acidic glycolipid fractions from both cell lines were subjected to ozonolysis and alkali fragmentation or endoglycoceramidase digestion to release the glycolipid-derived oligosaccharides. The neutral oligosaccharides were separated according to size by gel filtration and high performance liquid chromatography. These analyses indicated that differentiated F9 cells synthesized less high molecular weight oligosaccharides (containing more than 5 sugar residues) relative to controls. Serial lectin affinity chromatography on columns of immobilized Helix pomatia. Wisteria f1oribunda. Griffonia simplicifolia-I and Ricinus communis-' agglutinins followed by reduction and permethylation revealed that globoside (GaINAcβ1, 3Galα1, 4Galβ1, 4Glc) and lactose (Galβ1,4Glc) are the principal glycolipid-derived oligosaccharides synthesized by F9 and RNF9 cells. An increased biosynthesis of these components was observed in RNF9 cells relative to controls. These changes paralleled the reduced biosynthesis of Forssman pentasaccharide (GaiNAcα 1 ,3GaINAcβ1 ,3Galα1 ,4Galβ1 ,4Glc) reported previously. Normalization of the incorporation of 3H-monosaccharides in glycolipid-derived oJigosaccharides to the number of cells indicated a 2-6 fold increase in the incorporation of radioactive precursors in RAlF9 cells relative to F9 controls, suggesting that an enhancement in glycosphingolipid biosynthesis accompanies the differentiation of F9 cells. The monosialylganglioside-derived oligosaccharides obtained from F9 and RAlF9 cells were separated by anion exchange chromatography. A reduced biosynthesis of high molecular weight components was observed in RAlF9 cells when compared with undifferentiated F9. Lectin affinity chromatography on immobilized Maackia amurensis agglutinin followed by reduction and permethylation indicated a dramatic increase in the synthesis of GM1 (Galβ1,3Ga1NAcβ1,4[NeuAcα2,3] Galβ1,4Glc) and GM3 (NeuAcα2,3Galβ1,4Glc) in RAlF9 cells relative to controls. These changes were accompanied by a decrease in the synthesis of sialyltetrasaccharide a (NeuAcα2,3Galβ1 ,3GlcNAcβ1 ,3Galβ1,4Glc) and sialylparagloboside (NeuAcα2,3Galβ1 ,4GlcNAβ1 ,3Galβ1 ,4Glc) in the differentiated cells. These observations are in agreement with previous reports in leukemic and human embryonal carcinoma cell lines and may be related to the growth arrest and antigenic changes associated with F9 differentiation. In the work reported herein, serial lectin affinity chromatography in concert with permethylation analysis prove to be powerful methods for the isolation and characterization of glycolipid-derived oligosaccharides. The application of these methods has allowed the unequivocal identification of main glycosphingolipid components as well as of some representing less than 1 % of the total glycolipids synthesized by two cell lines. This information should provide the basis for further studies involving glycosyltransferas.
- Micro lipid droplet precursors of milk lipid globulesDeeney, Jude T. (Virginia Tech, 1985-01-05)The lipid in milk (milk fat) is found in the form of droplets known as milk lipid globules (MLG). These milk lipid globules are encompassed by a unit membrane known as the milk lipid globule membrane (MLGM) which is derived from the apical plasma membrane of the mammary epithelial cell during secretion. In lactating mammary epithelial cells, immediate precursors of milk lipid globules appear to be cytoplasmic lipid droplets (CLD). These cytoplasmic lipid droplets have diameters >1 μm and are characterized by an electron dense, granular surface coat. A previously unrecognized group of structures with diameters <.5 μm, which resemble cytoplasmic lipid droplets in matrix and surface coat appearance, has been observed. The surface coat of these triacylglycerol containing structures, termed micro lipid droplets (μLD), was similar to that of cytoplasmic lipid droplets in enzyme and polypeptide composition. Morphological evidence suggested that these small structures may originate from rough endoplasmic reticulum (RER) and fuse with cytoplasmic lipid droplets. Immunochemical studies showed homology of certain proteins among the rough endoplasmic reticulum, micro lipid droplets and cytoplasmic lipid droplets, which supported the possibility of an endoplasmic reticulum origin of these droplets. The rate of incorporation of [1-¹⁴C]-palmitate and [1,2,3-³H]-glycerol into lipid of RER, μLD, CLD and MIG fractions suggested a possible translocation pathway of triacylglycerols from the rough endoplasmic reticulum to cytoplasmic lipid droplets. The micro lipid droplets seem to provide triacylglycerols to support growth of cytoplasmic lipid droplets. In addition, morphological evidence suggested that these micro lipid droplets can be secreted directly in a manner similar to cytoplasmic lipid droplets, providing for the small lipid globules in milk. Little is known concerning the biochemical processes of milk lipid secretion but it is thought that butyrophilin, a glycoprotein found in milk lipid globule membrane, may play a role. After treatment of mammary epithelial cells with tunicamycin, butyrophilin content of this membrane is reduced. Thus a method for the study of the physiological role of this glycoprotein is proposed.
- Monosialylgangliosides from human meconium: characterization using specific anti-oligosaccharide antibodiesPrieto-Trejo, Pedro Antonio (Virginia Polytechnic Institute and State University, 1986)Rabbit antisera directed against human milk sialyloligosaccharides were used to detect specific monosialylgangliosides from the lipid fraction of human meconium. Gangliosides of this fraction were detected after thin layer chromatography by immuno-staining with specific anti-oligosaccharide sera. The monosialylganglioside fraction of human meconium was subjected to ozonolysis and alkali-fragmentation and the resulting ganglioside-derived oligosaccharides were reduced with NaB [³H]₄ and partially separated using paper chromatography. The [³H]-oligosaccharide alditols were assayed for binding to specific anti-oligosaccharide sera in a direct-binding radioimmunoassay using nitrocellulose filters to collect immune-complexes. Radiolabeled oligosaccharide alditols which were recognized by specific antisera were affinity purified by eluting nitrocelIulose filters containing antibody-oligosaccharide complexes or using columns of immobilized anti-oligosaccharide antibodies. Structural analyses of two sialyl[³H]tetrasaccharide alditols obtained in this way were carried out with sequential enzymatic degradation using specific exoglycosidases. The products of enzymatic digestions were identified by cochromatography in paper with known standards. Data obtained from these experiments are consistent with the presence of the following, previously unidentified gangliosides in human meconium: NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc-cer Galβ1-3[NeuAcα2-6]GlcNAcα1-3Galβ1-4Glc-cer
- Structural analysis of glycolipid-derived oligosaccharides from metabolically radiolabelled colorectal carcinoma SW1116 cellsTarrago-Trani, Maria T. (Virginia Tech, 1991)This dissertation describes the analysis of the carbohydrate portion of glycosphingolipids from colorectal carcinoma cells, SW1116, by metabolically labelling the cells with radioactive monosaccharide precursors. SW1116 cells (1 x 10⁶) metabolically labelled with 222 μCi/ml of either 6-[³H]-D-galactose (25 Ci/mMol) or 6-[³H]-D-glucosamine (38 Ci/mMol) for 30 hours, incorporated 1%-3% of the radioactivity into their glycoconjugates. Approximately 63% of the radioactivity recovered in the glycoconjugates corresponded to glycolipids when cells were labelled with 6-[³H]-D-galactose, and about 12% when cells were radiolabelled with 6- [³H]-D-glucosamine. Metabolically radiolabelled glycolipids were separated into neutral (88-91% of the radioactivity recovered in glycolipids) and acidic (9-12% of the radioactivity in glycolipids) fractions by ion exchange chromatography. Glycolipids in these fractions were subjected to ozonolysis and alkali fragmentation to release the oligosaccharide chains from the ceramide portion. Oligosaccharides obtained from the neutral glycolipids were separated into single components by a combination of high performance liquid chromatography (HPLC) and Ricinus communis agglutinin I (RCA-I)-agarose affinity chromatography. Oligosaccharides were identified based on the monosaccharide composition, methylation analysis, and exoglycosidase digestions. Major glycolipid components present in the neutral fraction were, glucosylceramide (Glc-Cer), galactosylceramide(Gal-Cer), galabiosylceramide (Galαl-4Gal-Cer), lacto-N-tetraosylceramide (Galβ1-3GIcNAcβ1-3Galβ1-4Glc-Cer), Lea- pentaglycosylceramide (Galβ1-3[Fucal-4]GlcNAcβ1-3Galβ1-4Glc-Cer), HIpentaglycosylceramide (Fucal-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc-Cer), a difucosylated lacto-N-tetraosylceramide, and a fucosylated lacto-Nnorhexaglycosylceramide. Minor components detected in this fraction corresponded to lactosylceramide (Galfp1-4Glc-Cer), lacto-Nneotetraosylceramide (Galβ1-4GlcNAcβ1-3Galβ1-4Glc-Cer), and fucosylated and difucosylated lacto-N-neotetraosylceramides. The acidic fraction was separated into monosialylgangliosides and _ disialylgangliosides by ion exchange chromatography. Monosialyloligosaccharides were further purified on HPLC, and biochemically characterized by methylation analysis, exoglycosidase digestions, and monosaccharide composition. The major component of this fraction corresponded to the sialyl-Lea glycolipid (NeuAcα2-3Galβ1-3[Fucαl-4]GlcNAcβ1-3Galβ1-4Glc-Cer) as previously reported by Magnani et al. [183]. GM3 (NeuAcα2-3Galβ1-4Glc-Cer) (0.42% of radioactivity recovered in glycolipids), sialyltetraosylceramide a (NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc-Cer) (0.46% of radioactivity in glycolipids), sialyltetraosylceramide b (Galβ1-3[NeuAcα2-6]GIcNAcβ1-3Galβ1- 4Glc-Cer) (0.21% of radioactivity in glycolipids), and sialyllated fucosylhexaglycosylceramide, were present in minor quantities. Results from this study demonstrate that metabolic radiolabelling provides a method for the structural analysis of glycolipids, as sensitive as the immunostaining procedures, as unmistakable as physical techniques (Mass Spectrometry, and Nuclear Magnetic Resonance), and that permits the identification of the majority of glycolipids expressed by a cell line, using relatively small number of cells in culture (6 x 10⁶). Application of this method could be extended to the study of changes in glycolipid accompanying oncogenic transformation and differentiation, glycolipid biosynthesis, intracellular sorting of glycolipids, recycling and turnover.
- Studies on pyridine nucleotide-dependent processes in Haemophilus influenzaeDenicola-Seoane, Ana (Virginia Polytechnic Institute and State University, 1989)Haemophilus influenzae and related species have a unique requirement for externally-provided NAD; therefore, several pyridine nucleotide-requiring enzymes become important for the survival of these pathogens. Haemophilus influenzae ATP:NMN adenylyltransferase was partially purified 15-fold with a 27% yield using dye affinity chromatography. Affinity chromatography was also used to purify NAD kinase from Haemophilus influenzae, 18-fold with a 32% yield. Substrate specificity studies of these enzymes demonstrated the enzymes to function with 3-acetylpyridine analogs of their respective substrates. A membrane-bound NMN glycohydrolase was demonstrated in Haemophilus influenzae. The enzyme functions with 3-acetylpyridine mononucleotide as a substrate, and is inhibited effectively by 3-aminopyridine mononucleotide. The possible involvement of this enzyme in the transport of NMN into the cytoplasm is discussed Growth inhibition studies demonstrated that 3-aminopyridine mononucleotide is a potent inhibitor of growth of the organism and could inhibit growth by inhibiting the transport of NMN. The previously reported inhibition of growth by the 3-aminopyridine adenine dinucleotide was attributed to the formation of the mononucleotide through the reaction catalyzed by the Haemophilus influenzae periplasmic nucleotide pyrophosphatase. A cytosolic lactate dehydrogenase, specific for D(-)-lactate was purified to electrophoretic homogeneity 2100-fold with a 14% yield. The purified enzyme was demonstrated to be a tetramer of M, = 135,000. It catalyzes essentially the reduction of pyruvate with very low activity observed for the oxidation of D(-)-lactate. An optimum pH of 7.2 was determined for the reduction of pyruvate with NADH as the coenzyme. Several NADH analogs, altered either in the pyridine or purine moiety, functioned as coenzymes. Coenzyme-competitive inhibition by adenosine derivatives demonstrated important interactions of the pyrophosphate region of the coenzyme in binding with the enzyme. Several structural analogs of NADH and pyruvate were evaluated as selective inhibitors of the enzyme. Chemical modification of the purified D-lactate dehydrogenase was effectively achieved by micromolar concentrations of several N-alkylmaleimides. Positive chain length effects in the inactivation by maleimides indicated the presence of a hydrophobic region close to the sulfhydryl groups being modified. The product of the reaction catalyzed by D-lactate dehydrogenase, D(-)-lactate, provides the substrate for a membrane-bound D-lactate oxidase. The D-lactate oxidase converts D(-)-lactate back to pyruvate and transfers electrons to the respiratory chain. No cytosolic L(+)-lactate dehydrogenase was found in Haemophilus influenzae; however, the organism possesses an L-lactate oxidase associated with the cell membrane. The L-lactate oxidase is also part of the respiratory chain, and utilizes exogenous L(+)-lactate to give pyruvate for the organism to use as a carbon source. Haemophilus influenzae and related species have a unique requirement for externally-provided NAD; therefore, several pyridine nucleotide-requiring enzymes become important for the survival of these pathogens. Haemophilus influenzae ATP:NMN adenylyltransferase was partially purified 15-fold with a 27% yield using dye affinity chromatography. Affinity chromatography was also used to purify NAD kinase from Haemophilus influenzae, 18-fold with a 32% yield. Substrate specificity studies of these enzymes demonstrated the enzymes to function with 3-acetylpyridine analogs of their respective substrates. A membrane-bound NMN glycohydrolase was demonstrated in Haemophilus influenzae. The enzyme functions with 3-acetylpyridine mononucleotide as a substrate, and is inhibited effectively by 3-aminopyridine mononucleotide. The possible involvement of this enzyme in the transport of NMN into the cytoplasm is discussed. Growth inhibition studies demonstrated that 3-aminopyridine mononucleotide is a potent inhibitor of growth of the organism and could inhibit growth by inhibiting the transport of NMN. The previously reported inhibition of growth by the 3-aminopyridine adenine dinucleotide was attributed to the formation of the mononucleotide through the reaction catalyzed by the Haemophilus influenzae periplasmic nucleotide pyrophosphatase. A cytosolic lactate dehydrogenase, specific for D(-)-lactate was purified to electrophoretic homogeneity 2100-fold with a 14% yield. The purified enzyme was demonstrated to be a tetramer of M, = 135,000. It catalyzes essentially the reduction of pyruvate with very low activity observed for the oxidation of D(-)-lactate. An optimum pH of 7.2 was determined for the reduction of pyruvate with NADH as the coenzyme. Several NADH analogs, altered either in the pyridine or purine moiety, functioned as coenzymes. Coenzyme-competitive inhibition by adenosine derivatives demonstrated important interactions of the pyrophosphate region of the coenzyme in binding with the enzyme. Several structural analogs of NADH and pyruvate were evaluated as selective inhibitors of the enzyme. Chemical modification of the purified D-lactate dehydrogenase was effectively achieved by micromolar concentrations of several N-alkylmaleimides. Positive chain length effects in the inactivation by maleimides indicated the presence of a hydrophobic region close to the sulfhydryl groups being modified. The product of the reaction catalyzed by D-lactate dehydrogenase, D(-)-lactate, provides the substrate for a membrane-bound D-lactate oxidase. The D-lactate oxidase converts D(-)-lactate back to pyruvate and transfers electrons to the respiratory chain. No cytosolic L(+)-lactate dehydrogenase was found in Haemophilus influenzae; however, the organism possesses an L-lactate oxidase associated with the cell membrane. The L-lactate oxidase is also part of the respiratory chain, and utilizes exogenous L(+)-lactate to give pyruvate for the organism to use as a carbon source.