PubMed Health⌕ Search

Biomedical subjects

R A Field

Publications and source records attributed to R A Field.

At least 19 recordsLinked to original sources
PubMed2001

Overexpression, purification, crystallization and data collection on the Bordetella pertussis wlbD gene product, a putative UDP-GlcNAc 2'-epimerase.

The Boredetella pertussis wlbD gene product is a putative uridine-5-diphosphate N-acetylglucosamine (UDP-GlcNAc) 2'-epimerase involved in Band A lipopolysaccharide biosynthesis. The wlbD gene is homologous to Escherichia coli rffE (32% identical), an established UDP-GlcNAc 2'-epimerase that is involved in enterobacterial common antigen (ECA) formation. The structure of the rffE protein reveals an unexpected role for a bound sodium ion in orientating a substrate-binding alpha-helix in the enzyme active site. Whilst key active-site residues in rffE are present in the wlbD sequence, the sodium-binding residues outside the active site are absent. This raises questions about the modulation of enzyme activity in these two enzymes. The wlbD gene from B. pertussis has been cloned and overexpressed in E. coli and the resulting protein has been purified to homogeneity. In the current study, crystals of the mutant Gln339Arg wlbD enzyme have been obtained by sitting-drop vapour diffusion. Uncomplexed Gln339Arg and UDP-GlcNAc complex data sets have been collected in-house on a rotating-anode generator to 2.1 A. Combined, the data sets identify the space group as P2(1)2(1)2(1), with unit-cell parameters a = 78, b = 91, c = 125 A, alpha = beta = gamma = 90 degrees. The asymmetric unit contains two monomers and 53% solvent.

Antigens, Bacterial↗
PubMed2001

Hydrolase and sialyltransferase activities of trypanosoma cruzi trans-sialidase towards NeuAc-alpha-2,3-gal-Gal-beta-O-PNP.

NeuAc-alpha-2,3-Gal-beta-O-PNP has been synthesised and its ability to act as a substrate for the hydrolase and transferase activities of Trypanosoma cruzi trans-sialidase have been investigated. The turn-over of this compound shows marked differences from the behaviour of NeuAc-MU. In addition, distinct differences in the action of T. cruzi trans-sialidase and Clostridium perfringens neuraminidase on NeuAc-alpha-2,3-Gal-beta-O-PNP were apparent.

Clostridium perfringens↗
PubMed2001

Synthetic mannosides act as acceptors for mycobacterial alpha1-6 mannosyltransferase.

A series of synthetic mannosides was screened in a cell-free system for their ability to act as acceptor substrates for mycobacterial mannosyltransferases. Evaluation of these compounds demonstrated the incorporation of [14C]Man from GDP-[14C]Man into a radiolabeled organic-soluble fraction and analysis by thin layer chromatography and autoradiography revealed the formation of two radiolabeled products. Each synthetic acceptor was capable of accepting one or two mannose residues, resulting in a major and a minor mannosylated product. Both products from each acceptor were isolated and their mass was confirmed by fast-atom bombardment-mass spectrometry (FABMS). Characterization of each mannosylated product by exo-glycosidase digestion. acetolysis and linkage analysis by gas chromatography mass spectrometry of partially per-O-methylated alditols, revealed only alpha1-6-linked products. In addition. the antibiotic amphomycin selectively inhibited the formation of mannosylated products suggesting polyprenolmonophosphate-mannose (C15 50-P-Man) was the immediate mannose donor in all mannosylation reactions observed. The ability of synthetic disaccharides to act as acceptor substrates in this system, is most likely due to the action of a mycobacterial polyprenol-P-Man:mannan alpha1-6 mannosyltransferase involved in the biosynthesis of linear alpha1-6-linked lipomannan.

Carbohydrate Sequence↗
PubMed2000

RmlC, the third enzyme of dTDP-L-rhamnose pathway, is a new class of epimerase.

Deoxythymidine diphosphate (dTDP)-L-rhamnose is the precursor of L-rhamnose, a saccharide required for the virulence of some pathogenic bacteria. dTDP-L-rhamnose is synthesized from glucose-1-phosphate and deoxythymidine triphosphate (dTTP) via a pathway involving four distinct enzymes. This pathway does not exist in humans and the enzymes involved in dTDP-L-rhamnose synthesis are potential targets for the design of new therapeutic agents. Here, the crystal structure of dTDP-6-deoxy-D-xylo-4-hexulose 3,5 epimerase (RmlC, EC5.1.3.13) from Salmonella enterica serovar Typhimurium was determined. The third enzyme of the rhamnose biosynthetic pathway, RmlC epimerizes at two carbon centers, the 3 and 5 positions of the sugar ring. The structure was determined by multiwavelength anomalous diffraction to a resolution of 2.17 A. RmlC is a dimer and each monomer is formed mainly from two beta-sheets arranged in a beta-sandwich. The structure of a dTDP-phenol-RmlC complex shows the substrate-binding site to be located between the two beta-sheets; this site is formed from residues of both monomers. Sequence alignments of other RmlC enzymes confirm that this region is very highly conserved. The enzyme is distinct structurally from other epimerases known and thus, is the first example of a new class of carbohydrate epimerase.

Amino Acid Sequence↗
PubMed2000

Palatability of wethers fed an 80% barley diet processed at different ages and of yearling wethers grazed on native range.

Seasonal availability of lamb in the Western United States contributes to a large fluctuation in lamb supply and value. However, alternatives to fall marketing may not be practical unless palatability traits are acceptable. A 3-yr study was conducted to investigate 1) the effects of slaughter age (7 to 8; 10 to 11; or 14 to 15 mo) on carcass and palatability characteristics of wethers fed an 80% barley diet (Exp. 1); and 2) the effects of finishing on range or on an 80% barley diet on carcass and palatability traits of 14- to 15-mo-old wethers (Exp. 2). In Exp. 1, no differences (P = .27) were detected in flavor intensity or longissimus muscle area among slaughter age groups, but fat depth was greater (P < .05) for 7- to 8-mo-old wethers than for 10- to 11- or 14- to 15-mo-old wethers. Year x slaughter age interactions were detected (P < .10) for hot carcass weight, Warner-Bratzler shear value, body wall thickness, and percentage kidney fat. Hot carcass weight was greater (P < .05) for 14- to 15-mo-old wethers than for both groups of younger wethers in yr 1, did not differ (P = .53) among slaughter ages in yr 2, and was greater (P < .05) for 10- to 11- than for 14- to 15-mo-old wethers in yr 3. Warner-Bratzler shear values did not differ (P > .10) among slaughter ages in yr 1 and 3, but shear values for 14- to 15-mo-old wethers were greater (P < .05) than for both younger slaughter age groups in yr 2. Percentage kidney fat was lower (P < .05) for 14- to 15- than for 7- to 8-mo-old wethers in all years. In Exp. 2, flavor intensity of the meat did not differ (P = .35) between finishing systems, but longissimus muscle area was greater (P = .02) for range-finished wethers than for wethers fed an 80% barley diet. Year x finishing treatment interactions were detected (P < .10) for shear values, body wall thickness, percentage kidney fat, and fat depth. Shear values were greater (P = .10) for range-finished wethers than for wethers fed an 80% barley diet in yr 1, but did not differ (P > .55) in yr 2 and 3. Body wall and fat measurements were greater (P < .10) for wethers fed an 80% barley diet than for range-finished wethers in all years except yr 3, when fat depth did not differ (P = .47). Overall, slaughtering wethers fed an 80% barley diet or range-finished wethers at older ages produced acceptable carcasses with desirable meat palatability traits.

Aging↗
PubMed1999

The GPI biosynthetic pathway as a therapeutic target for African sleeping sickness.

African sleeping sickness is a debilitating and often fatal disease caused by tsetse fly transmitted African trypanosomes. These extracellular protozoan parasites survive in the human bloodstream by virtue of a dense cell surface coat made of variant surface glycoprotein. The parasites have a repertoire of several hundred immunologically distinct variant surface glycoproteins and they evade the host immune response by antigenic variation. All variant surface glycoproteins are anchored to the plasma membrane via glycosylphosphatidylinositol membrane anchors and compounds that inhibit the assembly or transfer of these anchors could have trypanocidal potential. This article compares glycosylphosphatidylinositol biosynthesis in African trypanosomes and mammalian cells and identifies several steps that could be targets for the development of parasite-specific therapeutic agents.

Aminoacyltransferases↗
PubMed1999

N-Substituted analogues of S-nitroso-N-acetyl-D,L-penicillamine: chemical stability and prolonged nitric oxide mediated vasodilatation in isolated rat femoral arteries.

Previous studies show that linking acetylated glucosamine to S-nitroso-N-acetyl-D,L-penicillamine (SNAP) stabilizes the molecule and causes it to elicit unusually prolonged vasodilator effects in endothelium-denuded, isolated rat femoral arteries. Here we studied the propanoyl (SNPP; 3 carbon side-chain), valeryl (SNVP; 5C) and heptanoyl (SNHP; 7C) N-substituted analogues of SNAP (2C), to further investigate other molecular characteristics that might influence chemical stability and duration of vascular action of S-nitrosothiols. Spectrophotometric analysis revealed that SNVP was the most stable analogue in solution. Decomposition of all four compounds was accelerated by Cu(II) and cysteine, and neocuproine, a specific Cu(I) chelator, slowed decomposition of SNHP. Generation of NO from the compounds was confirmed by electrochemical detection at 37 degrees C. Bolus injections of SNAP (10 microl; 10(-8)-10(-3) M) into the perfusate of precontracted, isolated rat femoral arteries taken from adult male Wistar rats (400-500 g), caused concentration-dependent, transient vasodilatations irrespective of endothelial integrity. Equivalent vasodilatations induced by SNVP and SNHP were transient in endothelium-intact vessels but failed to recover to pre-injection pressures at moderate and high concentrations (10(-6)-10(-3) M) in those denuded of endothelium. This sustained effect (> 1 h) was most prevalent with SNHP and was largely reversed by the NO scavenger, haemoglobin. We suggest that increased lipophilicity of SNAP analogues with longer sidechains facilitates their retention by endothelium-denuded vessels; subsequent slow decomposition within the tissue generates sufficient NO to cause prolonged vasodilatation. This is a potentially useful characteristic for targeting NO delivery to areas of endothelial damage.

1-Octanol↗
PubMed1999

Man alpha1-2 Man alpha-OMe-concanavalin A complex reveals a balance of forces involved in carbohydrate recognition.

We have determined the crystal structure of the methyl glycoside of Man alpha1-2 Man in complex with the carbohydrate binding legume lectin concanavalin A (Con A). Man alpha1-2 Man alpha-OMe binds more tightly to concanavalin A than do its alpha1-3 and alpha1-6 linked counterparts. There has been much speculation as to why this is so, including a suggestion of the presence of multiple binding sites for the alpha1-2 linked disaccharide. Crystals of the Man alpha1-2 Man alpha-OMe-Con A complex form in the space group P2(1)2(1)2(1) with cell dimensions a = 119.7 A, b = 119.7 A, c = 68.9 A and diffract to 2. 75A. The final model has good geometry and an R factor of 19.6% (Rfree= 22.8%). One tetramer is present in the asymmetric unit. In three of the four subunits, electron density for the disaccharide is visible. In the fourth only a monosaccharide is seen. In one subunit the reducing terminal sugar is recognized by the monosaccharide site; the nonreducing terminal sugar occupies a new site and the major solution conformation of the inter-sugar glycosidic linkage conformation is adopted. In contrast, in another subunit the non reducing terminal sugar sits in the so called monosaccharide binding site; the reducing terminal sugar adopts a different conformation about its inter-sugar glycosidic linkage in order for the methyl group to access a hydrophobic pocket. In the third subunit, electron density for both binding modes is observed. We demonstrate that an extended carbohydrate binding site is capable of binding the disaccharide in two distinct ways. These results provide an insight in to the balance of forces controlling protein carbohydrate interactions.

Carbohydrate Conformation↗
PubMed1998

A synthetic acceptor substrate for Trypanosoma brucei UDP-Gal: GPI anchor side-chain alpha-galactosyltransferases.

A synthetic analogue of a trisaccharide fragment of the Trypanosoma brucei Variant Surface Glycoprotein (VSG) GlycosylPhosphatidyInositiol (GPI) anchor, Gal-alpha-1,3(Man-alpha-1,6)-Man-alpha-O-octyl (1), serves as a substrate for two T. brucei alpha-galactosyltransferases. The principle tetrasaccharide product derived from (1) contains a Gal-alpha-1,2-Gal linkage.

Animals↗
PubMed1998

Solution structure of the complex between the B-subunit homopentamer of verotoxin VT-1 from Escherichia coli and the trisaccharide moiety of globotriaosylceramide.

We report the solution structure of the carbohydrate-binding B subunit of verotoxin VT-1 (VTB) from enterohemorrhagic Escherichia coli in association with the trisaccharide Galalpha1-4Galbeta1-4Glcbeta1-O-trimethylsilylethyl , determined by use of stable isotope-assisted NMR techniques. In contrast to the crystal structure of the complex which predicts three binding sites per monomer, only one of these sites is observed with substantial occupancy by the trisaccharide in solution.

Bacterial Toxins↗
PubMed1998

New conformational constraints in isotopically (13C) enriched oligosaccharides.

Multidimensional heteronuclear NMR studies have been applied to the resonance assignment and conformational analysis of 13C-enriched Neu5Acalpha2-3Galbeta1-4Glc. It is demonstrated that three-dimensional ROESY-HSQC experiments provide through-space distance restraints which cannot be observed with conventional homonuclear 1H techniques due to resonance overlap. In particular, connectivities demonstrating the existence of the "anti" conformation about the Galbeta1-4Glc glycosidic linkage are unambiguously observed. It is shown that 13C isotopic enrichment of the trisaccharide at a level >95% enables straightforward measurement of trans-glycosidic 1H-13C and 13C-13C coupling constants and a Karplus-type relation is derived for the latter. In total 15 conformational restraints were obtained for the trisaccharide in aqueous solution, all of which were in excellent agreement with theoretical parameters computed from a 5 ns molecular dynamics simulation of the glycan.

CA-19-9 Antigen↗
PubMed1998

Derivation of an equation to estimate marrow content of bovine cervical vertebrae.

Marrow content of bovine cervical vertebrae from Choice- and Select-grade carcasses weighing 294 to 343 kg was determined so that a method to monitor the amount of marrow in meat from advanced meat/bone separation machinery and recovery (AMR) systems could be developed. The marrow determination requires cleaning and then ashing bones. Because a large difference in ash content of bone and bone marrow exists and because cartilage content of cervical vertebrae in Choice and Select beef is relatively constant, it was possible to derive the following equation: Weight of marrow = [weight of cartilage (% ash in cartilage - % ash in bone) + % ash in bone (total weight) - (total ash)]/[(% ash in bone - % ash in marrow)]. Constants for ash in fresh bone, marrow, and cartilage were 58.51, .57, and 2.14% with SD of 2.23, .15, and .30%, respectively. A cartilage content of 9.5% along with cervical vertebrae weight and total ash weight were also used to calculate 33.9% marrow in cervical vertebrae. Means for marrow pressed or centrifuged from bovine cervical vertebrae were lower than those obtained from the equation. Therefore, pressing and centrifuging left some marrow in spongy bone. Our ashing method for determining the amount of marrow in whole cervical vertebrae should be useful for determining marrow remaining in cervical vertebrae of bone cakes from AMR systems. Percentage ash in pressed bones is higher and the calculated marrow content is lower when pressed bones are compared to cervical vertebrae that are not pressed. The amount of marrow in whole cervical vertebrae minus the amount left in cervical vertebrae from bone cakes equals the amount in meat from AMR systems.

Analysis of Variance↗
PubMed1997

An examination of some derivatives of S-nitroso-1-thiosugars as vasodilators.

A number of S-nitrosated compounds derived from 1-thiosugars (glucose, galactose, xylose, maltose, and lactose) have been prepared and characterized. Most of the compounds obtained were unstable either as solids or in solution. However, S-nitroso-1-thio-2,3,4,6-tetra-O-acetylglucopyranose was stable enough to examine as a vasodilator using an isolated rat tail artery model. It also proved effective in human cutaneous vascular smooth muscle relaxation when delivered transdermally.

Animals↗
PubMed1997

Structure of a glycoconjugate in solution and in complex with an antibody Fv fragment.

By use of heteronuclear (13C, 1H) NMR methods, the three-dimensional structure and dynamics of the glycoconjugate estrone-3-glucuronide (E3G) uniformly 13C enriched in the glucuronic acid moiety has been probed both in free solution and in association with an anti-E3G antibody single-chain Fv fragment. The glycan is found to exist in multiple conformations in free solution, with particularly large torsional fluctuations about the glycosidic linkage psi. Resonance assignments and distance restraints for the glycoconjugate in the bound state were obtained from heteronuclear proton-carbon-carbon-proton-COSY and isotope-edited NOESY techniques, respectively. Quantitation of the NOE data with a full-relaxation matrix approach showed that the antibody selects a conformation from the solution repertoire which does not correspond with either of the two lowest energy conformations of the free glycan, and the internal energy of the glycan in the bound state is estimated to be at most approximately 15 kcal/mol higher than the global minimum energy conformation. The glucuronide moiety undergoes a stacking interaction with an aromatic ring in the binding site, and both ring-current shifts and nuclear Overhauser effects computed from the predicted bound-state conformation are in good agreement with experiment. The bound-state conformation is also in good agreement with preliminary x-ray data on a related complex.

Carbohydrate Conformation↗
PubMed1997

Hydrophobic mannosides act as acceptors for trypanosome alpha-mannosyltransferases.

A series of hydrophobic mannosides were synthesized and tested for their ability to act as acceptor substrates for mannosyltransferases in a Trypanosoma brucei cell-free system. The thiooctyl alpha-mannosides and octyl alpha-mannosides all accepted single mannose residues in alpha-linkage, as judged by thin layer chromatography of the products before and after jack bean alpha-mannosidase digestion. The mannosylation reactions were inhibited by amphomycin, suggesting that the immediate donor was dolichol-phosphate-mannose (Dol-P-Man) in all cases. The transferred alpha-mannose residues were shown to be both alpha 1-2 and alpha 1-6 linked by Aspergillus phoenicis alpha-mannosidase and acetolysis treatments, respectively. These data suggest that the compounds can act as acceptor substrates for the Dol-P-Man dependent alpha 1-2 and alpha 1-6 mannosyltransferases of the GPI biosynthetic pathway and/or the dolichol-cycle of protein N-glycosylation. One of the compounds, Man alpha 1-6 Man alpha 1-O-(CH2)7CH3, inhibited endogenous GPI biosynthesis in the cell-free system, suggesting that it could be a substrate for the trypanosome Dol-P-Man:Man2GlcN-Pl alpha 1-2 mannosyltransferase.

Animals↗