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R R Townsend

Publications and source records attributed to R R Townsend.

At least 91 records · Page 5Linked to original sources
PubMed1988

Monosaccharide analysis of glycoconjugates by anion exchange chromatography with pulsed amperometric detection.

The method of anion exchange chromatography followed by pulsed amperometric detection (AE-PAD; Johnson, D. C., and Polta, T. Z. (1986) Chromatogr. Forum 1, 37-44) has been applied to the compositional analysis of glycoconjugates. Using 22 mM NaOH as a column effluent, underivatized fucose, galactosamine, glucosamine, galactose, glucose, and mannose were readily separated in 15 min at a flow rate of 1 ml/min. The limit of quantification of the monosaccharides was better than 100 pmol (signal to noise ratio 184:1). AE-PAD was employed to quantify the monosaccharides of several glycoproteins, glycopeptides, and oligosaccharides after hydrolysis with 2 M trifluoroacetic acid. Both neutral and amino sugars could be rapidly estimated in a single chromatographic step using AE-PAD. Complete release of N-acetylglucosamine required more vigorous hydrolysis conditions (Lee, Y. C. (1972) in Methods in Enzymology (Ginsburg, V., Ed.), Vol. 28, pp. 63-73, Academic Press, New York). In both glycopeptides and oligosaccharides, approximately one less residue of Man than predicted was determined. Both AE-PAD and liquid chromatographic analysis of borate-monosaccharide complexes with fluorometric detection (Mikami, H., and Ishida, Y. (1983) Bunseki Kagaku 32, E207-E210) gave similar quantification of mannose and other sugars. The capability of rapid, sensitive quantification of underivitized monosaccharides should facilitate structural analysis of glycoconjugates.

Amino Sugars↗
PubMed1988

Separation of positional isomers of oligosaccharides and glycopeptides by high-performance anion-exchange chromatography with pulsed amperometric detection.

High-performance anion-exchange (HPAE) chromatography under alkaline conditions (pH congruent to 13) has been found to efficiently separate neutral oligosaccharides (triose to undecaose) according to molecular size, sugar composition, and linkage of monosaccharide units. The method was able to resolve 1----3, 1----4, and 1----6 positional isomers of neutral oligosaccharides, which are defined as having the same number, type, sequence, and anomeric configurations of monosaccharides but differing in the linkage position of a single sugar. From correlating structural features of different oligosaccharides and retention times, we deduced that at least two factors are operative to determine the superior resolution of oligosaccharides by this type of chromatography: (i) the relative acidities of the hydroxyl groups and (ii) the accessibility of oxyanions of the oligosaccharides to the functional groups of the stationary phase. Splitting of peaks attributable to mutarotation was not observed. Reducing oligosaccharides were much more retained than their reduced counterparts. Linkage of Fuc(alpha 1-3) to GlcNAc of oligosaccharides markedly decreased retention times. Positional isomers of two branched monosaccharides, which differed by 1----6 and 1----4 linkages, were widely separated. The separation of 1----3 and 1----4 positional isomers of both tetrasaccharides and glycopeptides containing undecasaccharides demonstrated the significant improvement in resolution of HPAE compared to previous chromatographic methods by either reverse-phase or amine-bonded stationary phases. Picomole quantities of underivatized oligosaccharides have been detected by triple-pulse amperometric detection, which produced similar responses for a wide range of structures. Quantification of two triantennary glycopeptides from bovine fetuin by using either detector response or 1H NMR was comparable. The N-glycanase-catalyzed release of two 1----4 and 1----3 positional isomers of an undecasaccharide from a tryptic glycopeptide of bovine fetuin could be observed and quantified by direct injection of the enzyme mixture into the chromatograph.

Carbohydrate Conformation↗
PubMed1988

Acute appendicitis: sonographic criteria based on 250 cases.

Two hundred and fifty consecutive patients with suspected appendicitis were examined with graded compression sonography. The initial diagnostic criterion for appendicitis was visualization of a noncompressible appendix; this was later modified to include the dimensions of the visualized appendix. The appendix was visualized in 91 of 250 patients (36%). Five adult patients with sonographically visible appendixes that were 6 mm or less in maximal diameter had either benign clinical follow-up (three patients) or a histologically normal appendix removed at surgery (two patients). However, two patients with appendixes measuring 6 mm in diameter and multiple appendicoliths had surgically confirmed acute appendicitis. Of 84 patients with visible appendixes measuring greater than 6 mm in maximal diameter, 78 had surgically confirmed acute appendicitis. In the remaining six, symptoms resolved spontaneously, and no surgery was required. In the absence of compelling clinical findings or an appendicolith, adult patients with maximal appendiceal diameters of 6 mm or less should undergo a period of close observation rather than immediate surgery. A diagnosis of appendicitis can be made in adult patients with persistent right lower quadrant pain and a visualized appendix greater than 6 mm in diameter.

Acute Disease↗
PubMed1988

Sonography of nonconjoined monoamniotic twin pregnancies.

Five nonconjoined monoamniotic twin pregnancies were identified prenatally by sonography. In all cases the diagnosis was made when umbilical cords of the two fetuses were seen to be entangled. One monoamniotic twin pregnancy was not recognized prenatally because the cords were not seen to be entangled. Although monoamniotic twins frequently die related to cord knotting, sonographic visualization of cord entanglement does not imply impending demise. Visualization of cord entanglement appears to be specific for the diagnosis of monoamnionicity, but the sensitivity is not known. Prenatal diagnosis allows informed planning of obstetrical monitoring and mode of delivery.

Female↗
PubMed1988

Factors affecting prenatal sonographic estimation of weight in extremely low birthweight infants.

Because critical management decisions are based on sonographic estimation of fetal weight in fetuses less than 1000 g, we sought to evaluate the accuracy of birthweight prediction in this range and to identify factors affecting this accuracy. Fetal weight was estimated using several published methods in 53 fetuses with birthweights less than 1000 g. Standard deviations greater than 12.3% indicate more random error in the sonographic weight prediction than has been reported in higher weight groups. No statistically significant differences were found between patient groups with decreased, normal, or increased amniotic fluid volume or portable examination. There was a trend toward lower mean deviation (2.9 vs 6.0%) and standard deviation (8.9 vs. 15.0%) in studies with scan quality judged "good" compared with "poor" based on ability to visualize anatomic landmarks.

Amniotic Fluid↗
PubMed1988

Membrane thickness in ultrasound prediction of chorionicity of twin gestations.

A retrospective review of sonograms performed on 75 twin gestations was performed to evaluate the ability of sonography to distinguish monochorionic from dichorionic gestations based on the thickness of the membrane separating the fetuses. Clinical or pathologic evidence of chorionicity and amnionicity was available in all cases. A thick membrane had a predictive value of 83% for dichorionicity and was seen in 89% of the first sonograms obtained on dichorionic gestations. Of third trimester dichorionic pregnancies, a thick membrane was seen in only 52%. A thin membrane on the initial study had a predictive value for monochorionic diamniotic pregnancy of 83%, but was seen in only 54% of cases. There was 100% intraobserver and 91% interobserver concordance in interpretation of membrane thickness. Technical factors important in interpretation of membrane thickness are discussed. The appearance of the membrane can be useful in sonographic evaluation of chorionicity and amnionicity in twin gestations, but should be used in conjunction with all other information available.

Amnion↗
PubMed1988

Sonographic identification of autosomal recessive polycystic kidney disease associated with increased maternal serum/amniotic fluid alpha-fetoprotein.

A patient with no family history of renal disease was referred for obstetric sonography because of elevated maternal serum and amniotic fluid alpha-fetoprotein (AFP), and was proved to be carrying a fetus with autosomal recessive polycystic kidney disease. Review of records of previous cases with a sonographic diagnosis of this disease revealed one other case with elevated and one case with normal amniotic fluid AFP. Sonographers should be aware of the potential diagnosis of this condition in patients with elevated AFP, even in gestations not known to be at risk. Because the sonographic diagnosis of this disease can be difficult in gestations known to be at risk, AFP determination may prove to be a useful diagnostic adjunct in these pregnancies as well.

Adult↗
PubMed1987

Synthesis of D-galactosamine derivatives and binding studies using isolated rat hepatocytes.

Derivatives of glycosides of D-galactosamine were prepared in order to study further the binding requirement of the Gal/GalNAc receptor in mammalian hepatocytes. These structures included N-propanoyl, N-benzoyl, and N,N-phthaloyl derivatives of 2-hydroxyethyl 2-amino-2-deoxy-beta-D-galactopyranoside, 6-amino-hex-1-yl 2-deoxy-2-(trifluoroacetamido)-beta-D-galactopyranoside, the mono- and di-O-methyl derivatives of allyl 2-acetamido-2-deoxy-beta-D-galactopyranoside, and allyl 2-acetamido-2,4-dideoxy-4-fluoro-alpha-D-galactopyranoside. The inhibition results confirmed some of our previous findings on the involvement of the hydroxyl groups, and provided new information on the involvement of the N-substituent, as well as on the requirement of hydrogen bonding of the 4-hydroxyl group in binding.

Animals↗
PubMed1986

Binding of N-linked bovine fetuin glycopeptides to isolated rabbit hepatocytes: Gal/GalNAc hepatic lectin discrimination between Gal beta(1,4)GlcNAc and Gal beta(1,3)GlcNAc in a triantennary structure.

Glycopeptides were isolated from bovine fetuin after digestion with Pronase, aminopeptidase M, and carboxypeptidase Y. The glycopeptides were derivatized with tert-butyloxycarbonyltyrosine and separated on the basis of peptide by using reverse-phase high-performance liquid chromatography. Using 400-MHz 1H NMR, the asialotriantennary oligosaccharides at each of the three N-linked glycosylation sites were found to be combinations of the following two structures in which the third branch is either Gal beta(1,4)GlcNAc or Gal beta(1,3)GlcNAc: (formula; see text) The asialotriantennary glycopeptides containing all beta(1,4)-lactosamine as the branches were designated Gal beta(1,4)GlcNAc-TRI while triantennary glycopeptides containing beta(1,3)-lactosamine as branch III were termed Gal beta(1,3)GlcNAc-TRI. The Gal beta(1,3)GlcNAc unit was localized predominantly to the branch III arm on the basis of a downfield shift (-0.027 ppm) in the H-1 and upfield shift (0.01 ppm) in the NAc methyl signals from the branch III GlcNAc resulting from Gal beta(1,3) instead of Gal beta(1,4) substitution. Revised assignments are proposed for the H-1's of Gal residues 6 (delta 4.464) and 8 (delta 4.471) [Vliegenthart, J. F. G., Dorland, L., & van Halbeek, H. (1983) Adv. Carbohydr. Chem. Biochem. 41, 209-373] in a Gal beta(1,4)GlcNAc-TRI. The proportion of Gal beta(1,3)GlcNAc-TRI glycopeptides from the Asn-Asp, Asn-Gly, and Asn-Cys sites was found to be 40%, 60%, and 20%, respectively. Analysis of the binding of these glycopeptides, containing from 20% to 60% Gal beta(1,3)GlcNAc as branch III, to rabbit hepatocytes revealed that the greater the proportion of Gal beta(1,3)GlcNAc, the lower the affinity of the mixture. The Kd for Gal beta(1,4)GlcNAc-TRI was found to be between 3.6 and 5.4 nM (P = 0.10) with a mean of 4.4 nM from binding data analyzed by using the LIGAND program [Munson, P. J., & Rodbard, D. (1980) Anal. Biochem. 107, 220-239] and computer simulations of the binding of two ligands as a mixture to one receptor site. The Kd of Gal beta(1,3)GlcNAc-TRI oligosaccharide, prepared by hydrazinolysis, was found to be 305 nM from inhibition studies.

Acetylgalactosamine↗
PubMed1986

Technical factors responsible for "placental migration": sonographic assessment.

Whether placental migration occurs is debatable. To determine the incidence of placenta previa in early pregnancy and the frequency of placental migration, a retrospective analysis was undertaken of 2,087 consecutive obstetrical sonograms obtained during a 23-month period. Among 849 patients scanned between 14 and 26 weeks after conception, placenta previa was diagnosed using ultrasonography (US) in 53 patients (6.2%). This diagnosis was confirmed at surgery in 15 patients (1.8%); there were 12 cases of central previa and three cases of partial previa. Retrospective analysis revealed that in 25 cases, technical factors, such as an overly distended urinary bladder (19 cases) or focal uterine contractions (six cases), were responsible for the false-positive diagnoses. In the 13 remaining patients, however, there were no obvious technical difficulties to account for the diagnosis of previa. Migration did not occur in any of the patients with central previa. It appears that placental migration may occur, although less frequently than has previously been reported. Furthermore, this study suggests that migration does not occur in patients with central previa.

False Positive Reactions↗
PubMed1985

Different modes of ligand binding to the hepatic galactose/N-acetylgalactosamine lectin on the surface of rabbit hepatocytes.

A study of the binding of three different 125I-labeled, galactose-terminated ligands to the hepatic galactose/N-acetylgalactosamine-specific lectin found on the surface of rabbit hepatocytes revealed that the different ligands manifest different physical parameters of binding. Asialoorosomucoid (125I-ASOR) binding was best described as involving two independent classes of binding sites on rabbit hepatocytes, with 161 000 sites/cell with a dissociation constant of 0.44 nM and 292 000 sites/cell with a Kd of 9.7 nM. Asialotriantennary glycopeptide purified from human alpha-1 protease inhibitor and modified with tyrosine at the N-terminus to permit radioiodination (TRI) [Lee, Y. C., Townsend, R. R., Hardy, M. R., Lönngren, J., Arnarp, J., Haraldsson, M., & Lönn, H. (1983) J. Biol. Chem. 258, 199-202] was also found to bind to two apparent classes of binding sites but with different binding parameters: 292 000 sites/cell of Kd = 1.47 nM and 982 000 sites/cell of Kd = 25.3 nM. A synthetic ligand, alpha,beta-diaspartamide of tris[(beta-lactosyloxy)methyl](6-aminohexanamido)methane (di-tris-lac) containing six nonreducing galactose residues [Lee, R. T., Lin, P., & Lee, Y. C. (1984) Biochemistry 23, 4255-4261], was found to bind to 817 000 sites/cell of Kd = 0.63 nM and 1.23 X 10(6) sites/cell of Kd = 25.3 nM. Thus, there were many more total binding sites for TRI or di-tris-lac on the surface of rabbit hepatocytes than there were for asialoorosomucoid, although the dissociation constants were similar for all three ligands.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylgalactosamine↗
PubMed1984

Determination of the sialylation pattern of human fibrinogen glycopeptides with fast atom bombardment.

Sialylated biantennary glycopeptides from human fibrinogen were analyzed with fast atom bombardment mass spectrometry. The mass spectrometric conditions used in the positive mode showed predominantly molecular ions with no fragment ions due to the loss of sialic acid. Standard mixtures of glycopeptides with zero, one, and two sialic acid residues revealed a linear relationship between ion abundance and molar fraction. The desorption efficiency varied according to the number of sialic acid residues in these biantennary glycopeptides. The relative abundance of different molecular ion species differing only in amino acid content was in agreement with chemical analysis. Sensitivity, precision, and requirements for sample preparation were assessed. Both assignment of molecular weights and quantification of individual glycopeptide species from human fibrinogen were obtained. The glycopeptides from human fibrinogen were found to consist of a mixture of equal amounts of monosialylated and disialylated species with no asialoglycopeptides.

Fibrinogen↗
PubMed1984

Rapid release of galactose-terminated ligands after endocytosis by hepatic parenchymal cells: evidence for a role of carbohydrate structure in the release of internalized ligand from receptor.

We have compared the behavior of 125I-labeled asialoorosomucoid (ASOR) and 125I-labeled asialotriantennary glycopeptide (TRI) from alpha-1-protease inhibitor in the perfused rat liver. These two ligands are recognized and internalized by the Gal/GalNAc receptor system of hepatocytes. We found that both ligands could be efficiently degraded when continuously perfused through a liver at 37 degrees C. However, as much as 51% of the internalized 125I-labeled TRI was rapidly (t1/2 = 3 min) released intact into the perfusion medium under conditions that permitted ligand-receptor association. In contrast, ASOR was totally degraded under these conditions. When dissociation was promoted (i.e., in the presence of GalNAc or EGTA), as much as 57% of the internalized ASOR was rapidly released (also intact). In each case, the shorter the initial internalization phase, the greater the proportion of ligand that was subsequently released. Neither the degree of receptor occupancy nor the temperature (16-37 degrees C) during the initial internalization phase influenced the rate or extent of the release process. Electron microscopic localization of internal ASOR-horseradish peroxidase conjugate demonstrated that peripheral endosomes were the major compartment labeled during exocytosis. We conclude that substantial fractions of both ligands rapidly return to the cell surface after internalization, ASOR bound to receptor and most of TRI free from receptor. The remaining ligand molecules enter the lysosomal pathway.

Animals↗
PubMed1983

Binding and endocytosis of glycoproteins and neoglycoproteins by isolated rabbit hepatocytes.

Rabbit hepatocytes were isolated by a collagenase perfusion technique, and used to study the binding and endocytosis of the glycoprotein, asialo-orosomucoid, and the neoglycoprotein, Gal39-bovine serum albumin. Both of these proteins contain exposed galactosyl residues, and were avidly bound by the lectin on the hepatic parenchymal cell surface. Steady state and kinetic experiments performed at 2 degrees C and at 37 degrees C revealed the presence of two apparent classes of binding sites totalling 4.7 X 10(5) sites/cell at 2 degrees C, and 6.3 X 10(5) sites/cell at 37 degrees C. At 37 degrees C, both classes of sites participated in internalization of bound ligand. The cells were capable of internalizing about 60 000 molecules/min per cell. The process appeared to be first-order, with a rate constant k = 0.098 min-1 and t1/2 = 7.1 +/- 0.6 min. Binding could be inhibited by galactose-containing compounds, EGTA, and by anti-(hepatic lectin) immunoglobulin G. The inhibition by antibody appeared to be reversible upon removal of antibody-containing medium.

Animals↗
PubMed1983

Binding of synthetic oligosaccharides to the hepatic Gal/GalNAc lectin. Dependence on fine structural features.

A series of synthetic oligosaccharides, resembling natural N-acetyllactosamine type glycans, were tested for their ability to inhibit the binding of labeled ligand to the mammalian hepatic lectin on rabbit hepatocytes at 2 degrees C. A dramatic hierarchy of inhibitory potency (tetraantennary greater than triantennary much greater than biantennary much greater than monoantennary) could be demonstrated. The range of concentration required for 50% inhibition of labeled ligand binding extended from approximately 1 mM, for the monoantennary oligosaccharides, to approximately 1 nM for triantennary oligosaccharides, even though the absolute Gal concentration increased only 3-fold. It was found that the number of Gal residues/cluster and their branching mode are major determinants of binding affinity of ligands to the hepatic lectin on the surface of hepatocytes.

Animals↗
PubMed1983

Stimulation of fibrinogen biosynthesis by fibrinogen fragments D and E.

Infusions of either fibrinogen fragment D or fibrinogen fragment E into rabbits were followed by increases in fibrinogen synthesis determined by the rate of incorporation of 75Se-selenomethionine into circulating fibrinogen. The degree of stimulation was proportional to the amount of protein infused. When 4.5 mg of each fibrinogen fragment was administered separately to different groups of animals, fibrinogen fragment D was associated with a fourfold increase in fibrinogen synthesis above that in the control animals compared with 1.5-fold increase induced by fragment E. Fragments D and E were assayed for bound sialic acid, the absence of which facilitates binding, transport and catabolism of many circulating glycoproteins by the liver. Fibrinogen fragment D contained 1.3% sialic acid compared to 1.4% in fragment E. These data indicate conservation of sialic acid during plasmic digestion of fibrinogen. The capacity of these glycopolypeptide fragments to stimulate fibrinogen synthesis appears unrelated to the nearly identical quantities of N-acetyl neuraminic acid found in each fragment.

Animals↗
PubMed1982

Carbohydrate structure of human fibrinogen. Use of 300-MHz 1H-NMR to characterize glycosidase-treated glycopeptides.

The carbohydrate composition of fibrinogen and constituent S-carboxymethylated chains was determined. All the carbohydrate on the parent molecule could be accounted for on the B beta and gamma chains. The A alpha chain was found to be devoid of carbohydrate. Glycopeptides were prepared from fibrinogen, B beta, and gamma chains by pronase digestion and subsequent chromatography on Sephadex G-50. The 300-MHz 1H-NMR spectra of glycopeptides from all three sources were consistent with biantennary type oligosaccharide chains. The glycopeptides resulting from exoglycosidase digestions were examined with 300-MHz 1H-NMR spectroscopy, and the changes in the signals caused by selective removal of sugar residues were studied. This technique enabled us to assign each anomeric proton to the corresponding monosaccharide unit independent of previous work. Our results indicate that fibrinogen glycopeptides are of the biantennary type, and are in complete agreement with the previously reported peak assignments (Vliegenthart, J. F. G., van Halbeek, H., and Dorland, L. (1981) Pure Appl. Chemn. 53, 45-77). Affinity chromatography of the glycopeptides on concanavalin A-Sepharose also showed that the glycopeptides from fibrinogen are greater than 95% biantennary oligosaccharide chains. Based on carbohydrate composition, 1H-NMR spectroscopy, sequential exoglycosidase digestion, and gas chromatography-mass spectrometry of derived partially methylated alditol acetates, we propose that fibrinogen contains four oligosaccharide chains of the structure shown below. (formula, see text)

Carbohydrate Sequence↗