High-pH anion-exchange chromatography of glycoprotein-derived carbohydrates.
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Anion Exchange Resins↗
Biomedical subjects
R R Townsend
Publications and source records attributed to R R Townsend.
Plasma renin activity and insulin sensitivity in normotensive subjects.
Insulin sensitivity in essential hypertension and atherosclerosis has elicited substantial research interest. Hyperinsulinemia in prospective studies identifies patients at risk for the development of coronary atherosclerosis. Plasma renin activity has also been shown to predict myocardial infarction, and correlates inversely with insulin sensitivity in hypertensive patients. The current study investigated the relationship between plasma renin activity and insulin sensitivity in 25 normotensive subjects. Using the euglycemic clamp technique, the data show a significant inverse correlation between supine plasma renin activity and insulin-mediated glucose uptake (r = -0.44; P = .027). Significant relationships were also found between plasma renin activity and fasting insulin concentration and fasting serum triglycerides. Reductions in relative skeletal muscle blood flow during insulin infusion have been reported in insulin-resistant compared to insulin-sensitive subjects. Skeletal muscle is the main site of insulin action during euglycemic clamp study and angiotensin II increases skeletal muscle blood flow. Increasing plasma renin activity may reflect a metabolic compensation which offsets the relative reduction in insulin-mediated enhancement in skeletal muscle blood flow in insulin resistance by generation of angiotensin II, which in turn promotes skeletal muscle blood flow.
Adult↗
The incidence of varicoceles in the general population when evaluated by physical examination, gray scale sonography and color Doppler sonography.
The association between male subfertility and varicocele has long been recognized. Physical examination is the most widely used method for detecting varicoceles but other techniques may be more sensitive. Color Doppler sonography has the theoretical advantage of allowing direct demonstration of reversed flow in the testicular veins. The frequency of detection of reversed flow by color Doppler ultrasound in a normal population is, however, unknown. We evaluated 34 asymptomatic young men with physical examination, standard scrotal sonography and color Doppler evaluation of the scrotal veins. Findings were correlated with semen analysis. Of the subjects 15% had varicoceles by physical examination and 18% by scrotal sonography, while 35% had retrograde flow by color Doppler evaluation. The majority of men in this population did not exhibit retrograde flow within the gonadal veins when evaluated by color Doppler sonography. The physiological significance of retrograde flow, however, remains to be defined.
Adolescent↗
Case report: bilateral adrenal pheochromocytoma.
Pheochromocytoma remains a clinical challenge to diagnose and manage. In addition, the association of multiple endocrine neoplasia syndromes with pheochromocytoma require the clinician's awareness to evaluate patients with pheochromocytoma (especially when bilateral) for abnormalities in thyroidal C-cell function with serum calcitonin determinations. The authors present a case of a 42-year-old woman initially diagnosed with, and treated for, cranial artery vasculitis because she had a stroke and a history of rheumatoid arthritis and asthma. Subsequent evaluation of episodic blood pressure increases, headache, and tachycardia revealed biochemical evidence of catecholamine overproduction. Bilateral adrenal masses were found on computed tomography scanning, and the functional nature of the adrenal masses was confirmed by a meta-Iodobenzylguanidine scan. Upon further evaluation, an elevated serum calcitonin concentration was demonstrated, which increased greatly with pentagastrin stimulation. C-cell hyperplasia was demonstrated by subsequent thyroidectomy, confirming the diagnosis of multiple endocrine neoplasia 2A. The difficulty in arriving at a correct diagnosis, the subsequent management, including bilateral adrenalectomy and thyroidectomy, and newer insight into the genetic abnormalities of multiple endocrine neoplasia 2A are discussed.
Adrenal Gland Neoplasms↗
Changes in lipolytic sensitivity following repeated epinephrine infusion in humans.
The lipolytic response to epinephrine is increased in lean but not obese subjects during conditions of increased circulating catecholamines such as starvation and critical illness. We evaluated the effect of repeated epinephrine treatment (0.03 micrograms.kg-1.min-1 for 30 min thrice daily) on basal lipolysis and the lipolytic response to epinephrine in lean and obese subjects. Lipolytic rates were determined using [2H5]glycerol during basal conditions and 60 min of epinephrine infusion before and after 6 days of repeated epinephrine treatment. Basal lipolysis and the lipolytic response to epinephrine were higher in obese (2.47 +/- 0.51 mumol.kg-1.min-1 and 106 +/- 21 mumol/kg, respectively) than lean (1.45 +/- 0.15 mumol.kg-1.min-1 and 70 +/- 12 mumol/kg, respectively; P = 0.08 obese vs. lean) subjects. Repeated epinephrine treatment decreased basal glycerol rate of appearance and the lipolytic response to epinephrine in lean and obese subjects. Plasma insulin concentration increased after repeated epinephrine treatment in both obese (14.3 +/- 0.6 to 18.2 +/- 1.6 microU/ml; P < 0.05) and lean (8.4 +/- 1.3 to 11.1 +/- 1.5 microU/ml; P < 0.01) subjects. Repeated epinephrine treatment suppresses basal and epinephrine-stimulated lipolysis. Increased plasma insulin concentration may be the mechanism.
Adult↗
Color Doppler evaluation of urethral diverticulum.
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Adult↗
Monosaccharide and oligosaccharide analysis of proteins transferred to polyvinylidene fluoride membranes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
We have developed an intermediate method toward the complete carbohydrate analysis of proteins, which should be universally applicable to all proteins and independent of sample matrix. Using only Coomassie Blue-stained proteins which have been electroblotted onto polyvinylidene fluoride membranes, we report a strategy for: (i) determining unequivocally whether a protein is glycosylated; (ii) obtaining a complete monosaccharide composition; (iii) oligosaccharide mapping which separates most forms according to size, charge and isomerity; and (iv) sequentially releasing and analyzing specific classes of oligosaccharides with endoglycosidases. The method was shown to be applicable to a variety of well characterized soluble glycoproteins and to the membrane-bound protein, the gastric H+, K(+)-ATPase. The monosaccharide composition of the H+,K(+)-ATPase revealed the absence of N-acetylneuraminic or N-glycolylneuraminic acids and a monosaccharide composition which indicated O-linked sugar chains. Oligomannosidic/hybrid and biantennary oligosaccharides were sequentially released and analyzed from one electroblotted band of recombinant tissue plasminogen activator using endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F2, respectively. Sialylated polylactosamine structures were identified and quantified by analyzing high performance liquid chromatography profiles of oligosaccharides first released by peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase and then treated with endo-beta-galactosidase, using a single, stained band of recombinant erythropoietin. This recombinant erythropoietin was found to contain eight times more tetrasialylated oligosaccharides than previously reported (Sasaki, H., Bothner, B., Dell, A., and Fukuda, M. (1987) J. Biol. Chem. 262, 12059-12076); 47% of released oligosaccharides were identified as polylactosamine structures.
Animals↗
Identification, quantification, and characterization of glycopeptides in reversed-phase HPLC separations of glycoprotein proteolytic digests.
Monosaccharide analysis by high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC/PAD) was used to identify the glycopeptides in the reversed-phase (RP-HPLC) separation of a bovine fetuin tryptic digest (1.6 nmol). This method, requiring no sample derivatization, identified four asparagine-linked (N-linked) glycopeptides and at least seven serine/threonine-linked (O-linked) glycopeptides. Glycopeptide identification was confirmed by Edman sequencing. Monosaccharide quantification of each glycopeptide suggested that all of the N-linked glycopeptides were the complex type and all the O-linked glycopeptides were sialylated. We determined that glycopeptides could be prepared by acidic reversed-phase chromatography with less than 3% loss of N-acetylneuraminic acid (Neu5Ac). The N-linked glycopeptides of bovine fetuin were prepared, digested with N-glycosidase F (PNGase F), and their oligosaccharides analyzed by HPAEC/PAD. These oligosaccharide profiles revealed that the Asn-138 oligosaccharide attachment site contained the majority of the disialylated and monosialylated oligosaccharides. The Asn-158 oligosaccharide attachment site contained the majority of the tetrasialylated oligosaccharides.
Amino Acid Sequence↗
The role of the renin-angiotensin system in insulin sensitivity in normotensive subjects.
Nonobese patients with essential hypertension have been reported to be insulin resistant. Because blockade of the renin-angiotensin system has been demonstrated to improve insulin sensitivity in hypertensive patients, the authors evaluated the effects of angiotensin-II administration and of angiotensin-converting enzyme inhibition with enalapril on insulin-mediated glucose uptake in normotensive men. The results showed no change in insulin sensitivity with either 0.3 ng/kg/min or 1 ng/kg/min angiotensin-II administration or with enalapril therapy. The authors conclude that neither physiologic amounts of angiotensin-II nor converting enzyme inhibition alter insulin sensitivity in normotensive men.
Adult↗
Pressor doses of angiotensin II increase insulin-mediated glucose uptake in normotensive men.
The effect of pressor doses of angiotensin II infused intravenously on insulin-mediated glucose uptake was determined in normotensive men. A 3-h hyperinsulinemic euglycemic clamp was employed in 14 normotensive subjects to determine insulin-mediated glucose uptake with or without an infusion of angiotensin II (approximately 15 ng.kg-1.min-1), which increased blood pressure by 20/15 mmHg (systolic/diastolic). Addition of angiotensin II increased whole body glucose uptake by 15% (9.2 +/- 0.5 vs. 10.8 +/- 0.8 mg.kg-1 x min-1; P = 0.011), and glucose oxidation (determined by indirect calorimetry) by 25% (4.0 +/- 0.3 vs. 4.9 +/- 0.4 mg.kg-1 x min-1; P < 0.05) over insulin alone. There was no significant increase in hepatic glucose output during angiotensin II infusion (2.2 +/- 0.1 vs. 2.4 +/- 0.1 mg.kg-1 x min-1; P = NS). We conclude that angiotensin II in pressor doses increases insulin-mediated glucose disposal and oxidation. The mechanism for this may involve a redirection of blood flow into skeletal muscle during angiotensin II infusion or a direct biochemical action of angiotensin II. Although performed in lean normotensive subjects, these results cast doubt on a significant role for angiotensin II in the insulin resistance associated with essential hypertension.
Adult↗
Evaluation of elevated blood pressure.
The clinical laboratory is essential to the successful and complete evaluation of the patient with hypertension. A reasonably thorough knowledge of the important physiologic components of blood pressure regulation, in conjunction with a good history and physical examination and a simple panel of screening tests serve to detect or rule out the presence of secondary forms of hypertension in the majority of patients.
Adrenal Gland Diseases↗
Insulin resistance and coronary artery disease.
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Antihypertensive Agents↗
Case report: stimulation of severe hypertension as a means of malingering.
Severe hypertension frequently requires hospitalization. Although hospital admission is undesirable for most patients, others are motivated to achieve hospitalization status for secondary gain. The authors present a case of a prison inmate who simulated severe hypertension with a combination of Valsalva's maneuver and arm flexion, on the background of preexisting essential hypertension and mitral valve prolapse. His secondary intent--to avoid his prison duties--classifies him as a malingerer. The differences between this malingering and the psychiatric diagnosis of Munchausen syndrome are discussed. The contributions of essential hypertension and mitral valve prolapse to the self-induced blood pressure elevations also are considered.
Adult↗
Insulin enhances pressor responses to norepinephrine in rat mesenteric vasculature.
Hyperinsulinemia and insulin resistance have been proposed to play a role in human and experimental hypertension. To characterize this relation further, we examined the pressor responses to periarterial nerve stimulation (PNS) and norepinephrine infusion in the isolated mesenteric vasculature of the normal rat before and after insulin (10, 100, and 1,000 microunits/ml) infusion. The pressor responses to PNS were similar before and after insulin, except at the highest dose of insulin (1,000 microunits/ml) and the highest frequency of PNS (10 Hz). However, insulin significantly increased the pressor responses to norepinephrine. This increase in responsiveness was evident at all doses of insulin studied. In contrast, insulin did not affect the pressor responses to either angiotension II or serotonin administration. The mechanism or mechanisms for the augmented pressor response to norepinephrine after insulin infusion remain to be determined. However, the selectivity of the response for norepinephrine and the absence of a marked pressor increase after PNS indicate that the mechanism probably involves either the alpha-receptor itself or an amplification of the postreceptor signal transduction. The role of chronic hyperinsulinemia and insulin resistance in the pathogenesis of essential hypertension requires further study.
Angiotensin II↗
Reassessment of noradrenergic function in salt-induced hypertension in Dahl rats.
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Animals↗
Structure of oligosaccharides on Saccharomyces SUC2 invertase secreted by the methylotrophic yeast Pichia pastoris.
Saccharomyces SUC2 invertase, secreted by the methylotrophic yeast Pichia pastoris and purified to homogeneity from the growth medium by DE-52 chromatography, appeared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a diffuse ladder of species at 85-90 kDa, while the secreted Saccharomyces form migrated as a broad band from 100 to 150 kDa. Endo-beta-N-acetylglucosaminidase H released the Pichia invertase carbohydrate generating a 60-kDa protein with residual Asn-linked GlcNAcs and oligosaccharides separated on Bio-Gel P-4 into Man8-11GlcNAc. Nearly 75% of the oligosaccharides were equally distributed between Man8,9GlcNAc, while 17% were Man10GlcNAc and 8% were Man11GlcNAc. Oligosaccharide pools were analyzed for homogeneity by high-pH anion-exchange chromatography, and structures were assigned using 500 MHz one- and two-dimensional 1H NMR spectroscopy. Pichia Man8GlcNAc was the same isomer as found in Saccharomyces, which arises by removing the alpha 1,2-linked terminal mannose from the middle arm of the lipid-oligosaccharide Man9GlcNAc (Byrd, J. C., Tarentino, A. L., Maley, F., Atkinson, P. H., and Trimble, R. B. (1982) J. Biol. Chem. 257, 14657-14666). The Man9GlcNAc pool was 5% lipid-oligosaccharide precursor and 95% Man8GlcNAc isomer with a terminal alpha 1,6-linked mannose on the lower-arm alpha 1,3-core-linked residue (Hernández, L. M., Ballou, L., Alvarado, E., Gillece-Castro, B. L., Burlingame, A. L., and Ballou, C. E. (1989) J. Biol. Chem. 264, 11849-11856). An alpha 1,2-linked mannose on the new alpha 1,6-linked branch in Man9GlcNAc provided 80% of the Man10GlcNAc, which is the structure on Saccharomyces invertase (Trimble, R. B., and Atkinson, P. H. (1986) J. Biol. Chem. 261, 9815-9824). A minor Man10GlcNAc (12%) and the principal Man11GlcNAc (82%) were the major Man9,10GlcNAc with novel alpha 1,2-linked mannoses on the preexisting alpha 1,2-linked termini. Although Pichia glycans did not have terminal alpha 1,3-linked mannoses as found on Saccharomyces core oligosaccharides, over 60% of the structures were isometric configurations unique to lower eukaryotes.
Carbohydrate Sequence↗
Separation of high-mannose isomers from yeast and mammalian sources using high-pH anion-exchange chromatography.
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Animals↗
Analysis of glycoprotein oligosaccharides using high-pH anion exchange chromatography.
The natural heterogeneity of glycoprotein glycans requires that chromatography be an essential part of structural elucidation. The isomeric nature of oligosaccharide structure requires chromatography which is selective for not only composition, size and anomerity, but also ring substitutions and branching configurations. HPAEC is sensitive to these structural features and thus, has become an important new method for understanding the elusive function of glycoprotein glycans.
Amino Acid Sequence↗