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Biomedical subjects

R R Townsend

Publications and source records attributed to R R Townsend.

At least 37 records · Page 2Linked to original sources
PubMed1996

Separation of biosynthetic oligosaccharide branch isomers using high-performance liquid chromatography on a porous two-dimensional graphite stationary phase.

Oligomannosidic branch isomers (structures differing only in the branch location of a single residue) which are biosynthetic intermediates in yeast and higher eukaryotics have been separated using high-performance liquid chromatography (HPLC) on porous graphatized carbon (PGC) columns, a stationary phase of two-dimensional crystalline carbon. A mixture of two Man6GlcNAc isomers from IgM, which was determined from 1H NMR analysis, was completely separated by PGC-HPLC. Mixtures of larger yeast oligomannosidic branch isomers were also chromatographically resolved using PGC-HPLC. Man10GlcNAc and Man11GlcNAc species from invertase expressed in Pichia pastoris showed three and five peak fractions, respectively, by PGC-HPLC in agreement with the number of isomeric forms from one- and two-dimensional 1H NMR analyses of the individual sized fractions (Trimble, R. B., Atkinson, P. H., Tschopp, J. R., Townsend, R. R., and Maley, F. (1991) J. Biol. Chem. 266, 22807-22817). Selected peak fractions were further analyzed to confirm assignments using matrix- assisted laser-desorption mass spectrometry after digestion with an alpha(1 --> 2)-specific mannosidase (Aspergillus saitoi). PGC-HPLC should prove invaluable for the preparation of singular oligosaccharides to define exoglycosidase and glycosyl transferase branch specificity and for preparing standards to develop more sensitive methods for structural elucidation of oligosaccharides.

Aspergillus↗
PubMed1996

Multimode high-performance liquid chromatography of fluorescently labeled oligosaccharides from glycoproteins.

A polymeric, secondary amine stationary phase was used to develop a method for the separation of 2-aminobenzamide (AB)-labeled neutral and anionic oligosaccharides from glycoproteins. Sequential hydrophilic interaction liquid and anion exchange chromatography were performed in the same chromatographic analysis (multimode HPLC) and unambiguous separation between neutral and anionic oligosaccharides was accomplished. Improved resolution was also achieved. The oligomannosidic-type structures from bovine ribonuclease B (Man4GlcNAc-AB-Man9GlcNAc-AB) were separated not only by size, but also by the branch location of terminal Manalpha(1 --> 2)-linked residues. Sialylated lactosamine-type oligosaccharides from human alpha1 acid glycoprotein were resolved according to charge, Fuc content, and the number of Galbeta(1 --> 4)Galbeta(1 --> 3) repeats. The minor fucosylated and polylactosamine species were well separated from the major sialylated tetra-antennary oligosaccharides. Volatile mobile phases were used to minimize sample handling for matrix assisted laser desorption mass spectrometric analysis of peak fractions. Novel polylactosamine structures (3-5 repeats) from alpha1-acid glycoprotein were deduced from the molecular weight analysis. Multimode HPLC should prove useful for preparing low pmol quantities of fluorescently labeled oligosaccharides with fewer steps and minimal sample handling for mass spectrometric analysis, important requisites for structural studies of sample-limited glycoproteins.

Animals↗
PubMed1996

Calcium channel blockers and hypertension: 1. New trials.

The long-term benefits and risks of using calcium channel blockers to treat hypertension have been called into question by reports of increased incidence of myocardial infarction and death among patients taking these drugs. There is now a worldwide effort, involving more than 30 countries and 100,000 patients, to provide a comprehensive data-base for making informed decisions.

Calcium Channel Blockers↗
PubMed1996

The Beta-subunit of the rabbit H,K-ATPase:a glycoprotein with all terminal lactosamine units capped with alpha-linked galactose residues.

The beta-subunit of the gastric H,K-ATPase is the most abundant glycoprotein in the tubulovesicular compartment of the acid-secreting parietal cells. The oligosaccharides of the beta-subunit have been shown to contain fucose, N-acetylglucosamine, mannose, galactose, and N-acetylgalactosamine. Previous studies have shown that the rabbit beta-subunit is devoid of N-acetylneuraminic acid. Here we report the structural features of the N-linked oligosaccharides of the beta-subunit from rabbit H,K-ATPase. We used glycosidase digestions and analysis by high-pH anion-exchange chromatography with pulsed amperometric detection and matrix-assisted laser desorption/ionization mass spectrometry to analyze the peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase (PNGase F)- and endo-beta-N-acetylglucosaminidase H (Endo H)-released oligosaccharides. The studies showed that the oligosaccharides of the beta-subunit are a mixture of both oligomannosidic and lactosamine-type structures. The high-mannose structures were identified as Man5Man8GlcNAc2 species. A striking finding was that all the branches of the lactosamine-type structures were terminated with Galalpha-->Galbeta-->GlcNAc extensions. All of the lactosamine-type structures were found to be core fucosylated and some of them contained one to three lactosamine repeats. We propose that a part of the adaptation of the gastric beta-subunit to the acidic environment of the stomach is through providing acid-stable terminal residues on the oligosaccharides.

Amidohydrolases↗
PubMed1996

Identification of protein kinase A phosphorylation sites on NBD1 and R domains of CFTR using electrospray mass spectrometry with selective phosphate ion monitoring.

HPLC-electrospray mass spectrometry was used to identify the phosphorylated sites on a bacterially expressed cystic fibrosis transmembrane conductance regulator (CFTR) fragment containing the first nucleotide binding domain (NBD1) and the regulatory domain (R). Tryptic digests of NBD1-R (CFTR residues 404-830) were analyzed after protein kinase A (PKA) treatment for all possible peptides and phosphopeptides (a total of 118 species) containing Ser residues within "high-probability" PKA consensus sequences: R-R/K-X-S/T, R-X-X-S/T, and R-X-S/T. Three criteria were used to assign phosphorylated sites: (1) an 80-Da increase in the predicted average molecular weight of the tryptic peptides; (2) co-elution with the PO3- ion induced by stepped energy collision; and (3) the relative elution positions of the phosphorylated and unmodified peptides. Ser residues within the eight dibasic sites in the NBD1 and R domains (positions 422, 660, 700, 712, 737, 768, 795, and 813) were phosphorylated, a pattern similar to that observed for full-length CFTR. The serine at position 753, which in CFTR is phosphorylated in vivo, was not phosphorylated. The remaining potential PKA sites, Ser489, Ser519, Ser557, Ser670, and Thr788, were not phosphorylated. The "low-probability" PKA sites (those not containing an Arg residue) were not phosphorylated. The results suggest that isolated domains of CFTR developed useful models for investigating the biochemical and structural effects of phosphorylation within CFTR. The mass spectrometry approach in this study should prove useful for defining phosphorylation sites of CFTR in vitro and in vivo.

Amino Acid Sequence↗
PubMed1996

Exoglycosidase purity and linkage specificity: assessment using oligosaccharide substrates and high-pH anion-exchange chromatography with pulsed amperometric detection.

Simplified HPLC protocols to determine the activity and linkage specificity and to detect the most commonly-encountered contaminants in available exoglycosidase preparations (Jacob and Scudder, Methods Enzymol., 230, 280-300, 1994) were developed. Monosaccharides and oligosaccharides were analyzed in a single chromatographic step using high-pH anion-exchange chromatography with pulsed amperometric detection. All analyses were performed with underivatized oligosaccharide substrates and by direct injection of unprocessed, diluted enzyme digests into the chromatograph. The sialidase from Newcastle disease virus was found to release both alpha (2-->3)- and alpha (2-->6)-linked Neu5Ac from a triantennary, lactosamine-type oligosaccharide. The activity of alpha-galactosidase from green coffee beans was assayed using Gal alpha(1-->3)[Fuc-alpha(1ar2)]Gal by detection of Gal and Fuc alpha(1-->3)Gal. The linkage specificities of beta-galactosidases from Streptococcus pneumoniae and bovine testis were assessed using Gal beta(1-->3 or 4)GlcNAc beta(1-->3)beta(1-->4)Glc as substrates. Contaminating beta-N-acetylhexosaminidase activity in the beta-galactosidase preparation was assayed using an agalactobiantennary oligosaccharide. The alpha(1-->3 or 4) linkage specificity of fucosidase III from almond meal was confirmed (Scudder et al., J. Biol. Chem. 265, 16472-16477, 1990) by its inactivity against a biantennary oligosaccharide with all Fuc residues linked alpha(1-->6). An alpha-fucosidase from chicken liver was found to cleave alpha(1-->2,3 or 6)-linked Fuc residues from oligosaccharides. The activity of jack bean (Canavalia ensiformis) alpha-mannosidase was assayed with a relatively resistant substrate, Man alpha(1-->3)- Man beta(1-->4)GlcNAc. A GlcNAc beta(1-->4)-terminated triantennary oligosaccharide was used to assay for contaminating beta-N-acetylhexosaminidase activity in alpha-mannosidase preparations and to determine the linkage and branch specificity of beta-N-acetylhexosaminidase at different enzyme concentrations.

Animals↗
PubMed1996

Classic papers symposium: ambulatory blood pressure monitoring and hypertensive target organ damage.

Despite tremendous advances in detection and treatment, including both non-pharmacologic and pharmacologic methods, hypertension continues to be a major health concern. In addition to efforts at reducing mortality, recent clinical and research attention has been focused on reducing morbidity from hypertension. Therefore, such efforts are focused on the effect of an elevated blood pressure on individual target organs and in particular on the heart, brain, and kidney. Although the role of blood pressure reduction alone in preserving target organ damage from hypertension is intensely debated, there is little debate that reducing blood pressure is important. The primary manner in which the blood pressure level is determined in the clinical setting continues to be the casual office measurement of blood pressure. However, the clinical use of ambulatory blood pressure determinations, including 24-hour automated blood pressure monitoring, is increasing. Recent data suggest that the determination of ambulatory blood pressures may be a better predictor not only of mortality but also of target organ damage from hypertension. This article will review recent key clinical studies addressing the role of ambulatory blood pressure on certain cardiac, cerebral, and renal manifestations of hypertension.

Blood Pressure Monitoring, Ambulatory↗
PubMed1996

Potential role of molecular mimicry between Helicobacter pylori lipopolysaccharide and host Lewis blood group antigens in autoimmunity.

Helicobacter pylori is involved in gastritis, gastric and duodenal ulcers, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma. Earlier studies already suggested a role for autoimmune phenomena in H. pylori-linked disease. We now report that lipopolysaccharides (LPS) of H. pylori express Lewis y, Lewis x, and H type I blood group structures similar to those commonly occurring in gastric mucosa. Immunization of mice and rabbits with H. pylori cells or purified LPS induced an anti-Lewis x or y or anti-H type I response, yielding antibodies that bound human and murine gastric glandular tissue, granulocytes, adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma cells. Experimental oral infections in mice or natural infection in humans yielded anti-Lewis antibodies also. The beta chain of gastric (H+,K+)-ATPase, the parietal cell proton pump involved in acid secretion, contained Lewis y epitopes; gastric mucin contained Lewis x and y antigenic determinants. Growth in mice of a hybridoma that secretes H. pylori-induced anti-Lewis y monoclonal antibodies resulted in histopathological evidence of gastritis, which indicates a direct pathogenic role for anti-Lewis antibodies. In conclusion, our observations demonstrate that molecular mimicry between H. pylori LPS and the host, based on Lewis antigens, and provide understanding of an autoimmune mechanism for H. pylori-associated type B gastritis.

Animals↗
PubMed1996

HCO3(-)-dependent conformational change in gastric parietal cell AE2, a glycoprotein naturally lacking sialic acid.

Although the AE1 chloride/bicarbonate exchanger of the red blood cell is among the most thoroughly investigated of membrane transport proteins, less is known about the related AE2 polypeptide of parietal cells. We have studied enzymatic deglycosylation of native AE2 polypeptide in gastric mucosal membranes from pig and rabbit. Deglycosylation of AE2 was maximal at low ionic strength. Deglycosylation of AE2 in membranes was preferentially inhibited by bicarbonate compared with other anions. This inhibition was maximal at alkaline pH and was not evident after detergent solubilization of AE2. Deglycosylation of AE2 increased its susceptibility to proteolytic degradation, but the presence of bicarbonate protected against this degradation. Bicarbonate failed to inhibit deglycosylation of the membrane glycoproteins AE1 and gastric H(+)-K(+)-adenosinetriphosphatase beta-subunit or deglycosylation of the soluble glycoproteins fetuin and ribonuclease B. These data suggest that bicarbonate induces a conformational change in AE2 that can protect the polypeptide from deglycosylation and proteolysis. Pig AE2 was purified in sodium dodecyl sulfate, and its monosaccharide composition was determined after blotting onto polyvinylidene fluoride membrane. AE2 was found to be devoid of sialic acid, with a composition suggestive of the presence of lactosamine-type chains.

Animals↗
PubMed1996

Ambulatory blood pressure monitoring: coming of age in nephrology.

The number of patients undergoing ambulatory blood pressure monitoring (ABPM) and the number of publications using this technique to evaluate the risks and effects of high blood pressure on target organs has been increasing, and dramatically so, in the last 5 years. Much of this growth has centered on the role of the blood pressure load (the percentage of systolic or diastolic readings above a preset value during a specific time period) and the changes in blood pressures levels that occur, with sleep. Although many studies are focused on the interaction between blood pressure (as assessed by ABPM) and the heart, interest is growing in the application of ABPM to the practice of nephrology. This paper discusses some of the technical aspects of ABPM, followed by a review of five areas of clinical research using ABPM, and which are relevant to renal medicine: microalbuminuria, renal function, renovascular hypertension, dialysis (hemodialysis and continuous ambulatory peritoneal dialysis), and transplantation. Despite a general lack of reimbursement for performance of the ABPM procedure, the growth in its usage and the willingness of clinicians to withhold or alter therapy on the basis of ABPM readings is testimony to its clinical value in the management of hypertension.

Blood Pressure↗
PubMed1995

Purification and structure-function analysis of native, PNGase F-treated, and endo-beta-galactosidase-treated CHIP28 water channels.

CHIP28 occurs naturally in glycosylated and nonglycosylated forms. The purpose of this study was to determine the role of glycosylation in CHIP28 structure and function. A new purification procedure based on phenylboronic acid-agarose (PBA) affinity chromatography was developed to isolate CHIP28. In purified native CHIP28 from erythrocytes, approximately 50% of CHIP28 molecules were glycosylated; each mole of glycosylated CHIP28 contained 5.4 kDa of monosaccharides consisting of 2 mol of Fuc, 8 mol of Gal, 1 mol of GalN, 13 mol of GlcN, 3 mol of Man, and 1 mol of Neu5Ac. The proportions of each monosaccharide and the sensitivity to endo-beta-galactosidase indicated that CHIP28 contained polylactosaminyl oligosaccharides. Glycosylated and nonglycosylated CHIP28 remained tightly associated when solubilized in octyl beta-D-glucoside (OG) and could not be separated by conventional chromatographic procedures. To remove the sugar moiety, CHIP28 was enzymatically deglycosylated by PNGase F and purified by Q-Sepharose anion-exchange and Erythrina cristagalli lectin chromatography. High-performance size-exclusion chromatography revealed that native CHIP28 eluted as an apparent dimer, whereas deglycosylated CHIP28 eluted as an apparent monomer. In reconstituted proteoliposomes, deglycosylated CHIP28 had a single channel water permeability (pf) of 3.1 x 10(-14) cm3/s (10 degrees C), not different from that of 3.2 x 10(-14) cm3/s for native CHIP28. Circular dichroism of native and deglycosylated CHIP28 in OG revealed 45% and 48% alpha-helix, respectively; intrinsic tryptophan fluorescence showed no effects of glycosylation on tryptophan environment. Freeze-fracture electron microscopy with rotary shadowing indicated that native and deglycosylated CHIP28 assembled as tetramers in reconstituted proteoliposomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amidohydrolases↗
PubMed1995

Night call in U.S. radiology residency programs.

RATIONALE AND OBJECTIVES: We acquired information about resident call in radiology programs throughout the United States to allow programs to compare themselves with others and to learn of possible alternate approaches to similar clinical and educational needs. METHODS: A 30-question survey was mailed to the program directors of all accredited U.S. radiology residency programs. A second mailing was sent to program directors who did not respond within 3 months. The survey addressed questions of the timing of call during residency training, the frequency of call, the nature of the call experience, and the relation to fellow and faculty call. Questions regarding available technical assistance, resident clinical activities postcall, faculty review of oncall studies, and other relevant issues were included. RESULTS: One hundred sixty-six of 206 (81%) of the program directors responded. The amount and type of call taken by radiology residents was highly variable in different programs. The mean number of in-house call days per month was 2.9 (SD = 2.6), 3.3 (SD = 2.2), 2.4 (SD = 1.9), and 1.3 (SD = 1.6) for first-, second-, third-, and fourth-year residents, respectively. The nature of technical and fellow/faculty assistance available to the resident was also variable. CONCLUSION: Night call in U.S. radiology residency programs is variable but tends to be concentrated in the second year of residency. Fourth-year residents take less call than other residents, especially close to the time of the written and oral board examinations. Although program directors were satisfied with many aspects of their call systems, most indicated at least one major change they would like to make.

Data Collection↗
PubMed1995

Separation of partially desialylated branched oligosaccharide isomers containing alpha (2-->3)- and alpha (2-->6)-linked Neu5Ac.

The following two tri-sialylated triantennary oligosaccharides, which differ only in the linkage of the Neu5Ac to the uppermost branch were, individually, partially desialylated to produce all possible di- and mono-sialylated isomers. [formula: see text] A tetra-sialylated triantennary isomer, which contained an alpha (2-->6)-linked Neu5Ac to the GlcNAc on branch III, was also converted to all possible trisialylated isomers by mild acid hydrolysis as previously described (Roher et al., Anal. Biochem., 212, 7-16, 1993). The resulting branch isomers were separated using high-pH anion-exchange chromatography (HPAEC). Structures were assigned to peak fractions on the basis of the previously described effect of alpha (2-->6)- and alpha (2-->3)-linked Neu5Ac on the elution order of branched lactosamine-type oligosaccharides (Townsend et al., Anal. Biochem., 182, 1-8, 1989). No differences in the acid lability of the Neu5Ac linkage to either Gal (alpha (2-->3) or alpha (2-->6)) or GlcNAc (alpha (2-->6)) were observed. Our studies show that chemical desialylation and HPAEC is a useful approach to prepare and identify all possible sialylated branch isomers and should prove useful for defining the branch specificity of sialyltransferases and sialidases.

Acetylglucosamine↗
PubMed1995

Ejaculatory duct obstruction: diagnosis and treatment with transrectal sonography.

Ejaculatory duct obstruction is thought to be a relatively uncommon but readily treatable cause of male infertility. Obstruction is frequently caused by cysts originating from or impinging on the ejaculatory ducts. The use of transrectal sonography has facilitated identification of patients with ejaculatory duct obstruction and has led to the realization that this abnormality is more common than was previously recognized. Transrectal sonography now plays a critical role in both the diagnosis and the treatment of this condition. The purpose of this pictorial essay is to illustrate the use of transrectal sonography in the diagnosis and treatment of ejaculatory duct obstruction.

Adult↗
PubMed1994

Efficacy and safety of atenolol, enalapril, and isradipine in elderly hypertensive women.

PURPOSE: This trial was designed to evaluate the efficacy and safety of three different classes of antihypertensive agents in elderly women. PATIENTS AND METHODS: The trial had three phases: 4 to 8 weeks of placebo, 6 weeks of titration, and 16 weeks of maintenance. White women between 60 and 80 years old with sitting diastolic blood pressures (DBPs) from 95 through 114 mm Hg treated with placebo were evaluated by history, physical examination, laboratory studies, and quality-of-life interview. After double-blind randomization with low-dose atenolol, enalapril, or isradipine, the dose was increased stepwise and hydrochlorothiazide added as needed to achieve goal DBP (less than 90 mm Hg and greater than 10 mm Hg below baseline). During maintenance, patients not at goal were "stepped up," and patients with uncontrolled DBP at maximum dosage were removed from the study. The pretreatment (baseline) blood pressure of the 315 randomized participants averaged 161/100 mm Hg; 92% had been treated previously for hypertension, 15% had diabetes mellitus, 11% smoked, and 38% consumed alcohol. RESULTS: For 245 patients completing the trial, the average decrease in blood pressure during treatment was 18.2/15.6 mm Hg. Antihypertensive efficacy was similar for the monotherapy drug regimens, with 84%, 71%, and 80% of patients receiving atenolol, enalapril, and isradipine, respectively, achieving DBP goal. Of the 70 patients who did not complete the trial, 42 left because of symptoms and 19 because of uncontrolled DBP. No important, unexpected drug-induced changes in symptoms or blood chemistries were noted. Symptom frequency differed little among the three dosage levels, becoming maximal by the second visit at the same dosage level. CONCLUSION: All three drugs lowered DBP comparably, and none produced alarming effects. Thirteen percent of patients left the study because of symptoms.

Aged↗