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Biomedical subjects

R J Simpson

Publications and source records attributed to R J Simpson.

At least 361 records · Page 20Linked to original sources
PubMed1985

The murine plasma cell antigen PC-1: purification and partial amino acid sequence.

The murine plasma cell alloantigen PC-1 is selectively expressed on B lymphocytes in their terminal phase of differentiation into antibody-secreting cells. Previous work on an analytical scale has shown that PC-1 consists of two apparently identical disulfide-bonded polypeptides, each of Mr 115,000. In this paper, we describe the generation of a monoclonal antibody to PC-1 and its use in the preparative isolation of PC-1 by affinity chromatography. Final purification to apparent homogeneity was achieved by preparative polyacrylamide gel electrophoresis. It was estimated that NS-1 myeloma cells possess 1 to 4 X 10(5) PC-1 monomers per cell on their surface. The yield of PC-1 after purification was approximately 10(5) monomers per cell. Purified PC-1 was digested with trypsin, and the resulting peptides were separated by reversed-phase high-performance liquid chromatography. Purified peptides were sequenced with a gas-phase sequencer.

Amino Acid Sequence↗
PubMed1984

High-performance liquid chromatography of amino acids, peptides and proteins. LVIII. Application of reversed-phase high-performance liquid chromatography to the separation of tyrosine-specific phosphorylated polypeptides related to human growth hormone.

Procedures for the purification of native and phosphorylated human growth hormone (hGH), S-carboxymethylated hGH, and hGH tryptic peptides, based exclusively on reversed-phase chromatography have been developed. Combinations of several volatile ion-pairing systems with small- and large-pore n-alkylsilicas were exploited in a general strategy, which allowed high recoveries of various hGH-related polypeptides from enzymatic incubations, as well as rapid desalting of samples following chemical modifications of the native protein, such as reductive alkylation in 6 M guanidine hydrochloride. The influence of the elution conditions on retention behaviour of phosphorylated hGH and its tryptic peptides in reversed-phase high-performance liquid chromatography is discussed.

Chromatography, High Pressure Liquid↗
PubMed1984

The inhibition of erythrocyte glyceraldehyde-3-phosphate dehydrogenase. In situ PMR studies.

The kinetics of inhibition of human erythrocyte glyceraldehyde-3-phosphate dehydrogenase by iodoacetate were studied in the intact cell and in vitro. The kinetics were determined using 1H-NMR to follow solvent exchange of 1H and 2H at the C-2 position of lactate. The exchange occurs via a series of enzyme-catalysed reactions, including that catalysed by glyceraldehyde-3-phosphate dehydrogenase. A direct assay with quenching of the inhibition was also used to check the results. Iodoacetate was shown to act as an active site-directed inhibitor of the dehydrogenase. The enzyme inhibition patterns, which are characterised by a binding step and a kinetic step, are similar in situ and in vitro. Membrane binding, however, was found to alter the inhibition pattern for the enzyme in vitro.

Deuterium↗
PubMed1984

Studies of Fe3+ transport across isolated intestinal brush-border membrane of the mouse.

Mouse intestinal brush-border membrane vesicles take up iron from media containing 59Fe3 +-nitrilotriacetic acid. The iron uptake by the vesicles represents accumulation of iron which relates to an osmotically active space. Uptake is linearly related to vesicle protein concentration and is inhibited by low incubation temperature and low medium free Fe3+ concentrations. Experiments with the lipid soluble iron ligand 8-hydroxyquinoline and with Triton X-100 imply that the uptake is rate limited by membrane transport.

Animals↗
PubMed1984

Reproducibility and treatment of exercise-induced ventricular tachycardia.

Fourteen patients with exercise-induced ventricular tachycardia (VT) underwent serial treadmill testing, and those with reproducible arrhythmia were treated with a beta-adrenergic blocking agent. In 11 patients (79%), VT of similar rate, morphologic characteristics and duration was reproduced on 2 consecutive treadmill tests performed 1 to 14 days apart. Beta blockade prevented recurrent VT during acute testing in 10 of 11 patients and during chronic therapy in 9. Eight patients had a consistent relation between a critical sinus rate and the onset of VT. In these patients, successful therapy correlated with preventing achievement of the critical sinus rate during maximal exercise. Thus, serial exercise testing is an appropriate means of assessing efficacy of therapy in patients with exercise-induced VT, provided that reproducibility is established on 2 control tests before beginning treatment. Therapy with beta-blocking agents is effective, especially when guided by the presence of a critical sinus rate-VT relation.

Adult↗
PubMed1984

In situ cyanogen bromide cleavage of N-terminally blocked proteins in a gas-phase sequencer.

Herein we describe a procedure for the in situ cyanogen bromide cleavage of N-terminally blocked proteins which have been immobilised onto the glass fiber sample disk of the gas-phase sequencer. In this manner, new amino terminii suitable for automated Edman degradation can be generated. Cytochrome C was cleaved using this method on the carboxyl side of methionine residues 65 and 80. This allowed sequence analysis to begin simultaneously at residues 66 and 81 (Table 1). This procedure offers an alternative sequencing tactic for methionine-containing proteins which are N-terminally blocked.

Amino Acid Sequence↗
PubMed1984

The receptor for transferrin on murine myeloma cells: one-step purification based on its physiology, and partial amino acid sequence.

The receptor for transferrin is one of the major surface proteins of proliferating lymphocytes and other cells. It binds ferrotransferrin from serum and endocytoses it into an acidic nonlysosomal intracellular compartment where iron is released, but in which apotransferrin remains tightly bound to its receptor. Recycling of the apotransferrin-receptor complex to the cell surface is associated with a return to neutral pH and concomitant loss of affinity of apotransferrin for its receptor. Apotransferrin is then free to leave the cell and initiate a new cycle. We have exploited this cycle in a novel method for the purification of the receptor for transferrin. Murine myeloma cells were lysed in nonionic detergent, and the lysate passed over a column of ferrotransferrin-agarose at pH 7.4. After washing with sodium acetate at pH 5.0, iron was removed with sodium citrate pH 5.0 and desferrioxamine. Upon returning the pH to neutrality, the receptor was eluted and found to be homogeneous by SDS-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. The degree of purification was estimated to be at least 3,000-fold, and the calculated yield was 10 to 20%. The purified receptor was capable of binding to transferrin. The receptor was digested with trypsin, and the resulting peptides were separated by reversed-phase high performance liquid chromatography in NH4HCO3. Selected peptides were rechromatographed in 0.1% trifluoroacetic acid, and their amino acid sequences were determined.

Amino Acid Sequence↗
PubMed1983

High-performance liquid chromatography of amino acids, peptides and proteins. XLVII. Analytical and semi-preparative separation of several pituitary proteins by high-performance ion-exchange chromatography.

The chromatographic separation of several pituitary proteins on a Mono-Q anion-exchange column is described. The effect of eluent pH and buffer composition on the resolution is demonstrated with several standard proteins. The experimental data indicate that good protein recoveries and resolution can be obtained on this essentially monodisperse microparticulate ion-exchange resin when the pH of the eluent is chosen ca. 0.5 pH units below the pI of the most basic component in the mixture. With this new column separations are ca. 30 times faster than with conventional cellulosic anion exchangers at similar sample loads.

Animals↗
PubMed1983

Centrifugal analysis of undiluted packed human erythrocyte lysates. Studies of the association of glyceraldehyde-phosphate dehydrogenase with the membrane fraction.

Concentrated human erythrocyte lysates (greater than 99% initial haematocrit) were subjected to high centrifugal fields. This caused the membrane fraction to separate from the cytoplasmic portion, due to the lower density of the former. Enzyme distribution data indicated that glyceraldehyde-phosphate dehydrogenase was predominantly in the cytoplasmic fraction.

Cell Fractionation↗
PubMed1983

Vagal syncope during recurrent pulmonary embolism.

The mechanism for syncope during pulmonary embolism is not well understood. We describe two patients with transient sinus bradycardia and atrioventricular (AV) block during syncope from recurrent pulmonary embolism. Consciousness was regained each time the rhythm returned to normal. We believe that the syncope and bradyarrhythmia was caused by a parasympathetic reflex, since simultaneous slowing of the sinus rate with concomitant AV block is a common manifestation of increased vagal tone. Such a reflex is consistent with known cardiac reflexes, may occur frequently, and may be one of the mechanisms for syncope in patients with pulmonary embolism.

Bradycardia↗
PubMed1983

Safety of multiple bolus loading of intravenous disopyramide.

Although a single intravenous bolus of disopyramide is an effective antiarrhythmic, side effects occur in some patients. We tested the safety of multiple bolus loading for intravenous disopyramide in 10 patients with frequent premature ventricular beats. Concurrent with a 1.0 mg/kg/hr infusion, a bolus of 0.5 mg/kg of disopyramide was given over 5 minutes. Up to three additional boluses were given 5 minutes after the first bolus unless a 50% reduction in premature ventricular beats or side effects occurred. The infusion was continued for 3 hours and was then decreased to 0.4 mg/kg/hr for 15 hours. All patients had a 50% reduction in premature ventricular beats and attained therapeutic blood levels. Three patients with a history of controlled congestive heart failure developed either hypotension or pulmonary edema. Hypotension and pulmonary edema following intravenous disopyramide is more related to the pharmacology of the drug than to the loading scheme employed.

Adult↗
PubMed1983

Exercise-induced distal atrioventricular block.

Three patients with 1:1 atrioventricular (AV) conduction at rest developed fixed 2:1 or 3:1 AV block during treadmill exercise testing. Electrophysiologic study documented block distal to the AV node in all three patients, and suggested that the exercise-induced block occurred because of increased atrial rate and abnormal refractoriness of the His-Purkinje conduction system. The findings in these three patients suggest that high grade AV block appearing during exercise reflects conduction disease of the His-Purkinje system rather than of the AV node, even in the absence of bundle branch block. Patients with this diagnosis should be considered for permanent cardiac pacing.

Aged↗
PubMed1983

Phosphorylation of human growth hormone by the epidermal growth factor-stimulated tyrosine kinase.

In the present study, we have demonstrated that human growth hormone (hGH) can be phosphorylated by the epidermal growth factor (EGF)-stimulated tyrosine kinase of A431 cell membranes. Phosphotyrosine was the predominant phosphoamino acid released from phosphorylated hGH on partial acid hydrolysis. All five tyrosine-containing tryptic peptides of hGH are also phosphorylated by the EGF-stimulated tyrosine kinase. The highest phosphate incorporation was found for peptide T4 (residues 20-38), which is distinguished by a high frequency of acidic amino acids. The phosphorylated peptides have been characterized by HPLC and two-dimensional mapping on paper. Comparison with the labeled peptides obtained on tryptic digestion of phosphorylated hGH suggests that tyrosine phosphorylation is restricted to two tryptic peptides, T4 (tyrosine-28 or -35) and T6 (tyrosine-42). It is suggested that the absence of early insulin-like activity in the naturally occurring Mr 20,000 variant of hGH, which has an internal deletion spanning residues 32-46, may be a consequence of the loss of the tyrosine phosphorylation sites at residues 35 and 42.

Amino Acid Sequence↗
PubMed1983

Nitrogen Redistribution during Grain Growth in Wheat (Triticum aestivum L.) : IV. Development of a Quantitative Model of the Translocation of Nitrogen to the Grain.

Translocation of nitrogen was measured in wheat (Triticum aestivium L. cv SUN 9E) plants grown without an exogenous supply of nitrogen from the time that the flagleaf began to emerge, and a model of nitrogen translocation was constructed to describe translocation on one day during the linear period of grain growth. Nitrogen for grain development was derived entirely by the redistribution of nitrogen from vegetative organs. Leaves contributed 40%, glumes 23%, stem 23%, and roots 16% of the nitrogen incorporated by the grains on the fifteenth day after anthesis. Less than 50% of the nitrogen exported from leaves was translocated directly to the grain via the phloem, the rest was translocated to the roots and was cycled in the roots and exported to the shoot in the transpiration stream. Nitrogen imported by leaves and glumes via the xylem was not accumulated in these organs but was transferred to the phloem for reexport from the organs. A large proportion (60%) of the nitrogen in the transpiration stream was cycled in the glumes. The glumes were also a major source of nitrogen for grain development. It was considered likely that this organ always plays an important role in nitrogen metabolism in wheat.

Journal Article↗
PubMed1983

The electrophysiological substrate of atrial fibrillation.

The electrophysiological substrate that predisposes the human atrium to sustain atrial fibrillation is incompletely understood. However, abnormalities of atrial size, refractory period and conduction are important precursors to this arrhythmia. The propensity of various disease states, cardioactive drugs and the autonomic nervous system to potentiate atrial fibrillation may be explained by changes in these electrophysiological properties.

Animals↗
PubMed1983

Expression of the cDNA for alpha 1-acid glycoprotein, a rat plasma protein, in Escherichia coli.

Polyadenylated RNA was isolated from acute-phase liver and transcribed into cDNA. After homopolymer tailing this was cloned into the PstI site of pBR322 which was used to transform E. coli RR1 cells. Clones containing cDNA corresponding to acute-phase proteins were identified by differential hybridization with 32P-labelled normal and acute-phase cDNA. A clone synthesizing about 10 ng of alpha 1-acid glycoprotein per E. coli colony was detected using a solid-phase based immunochemical procedure. The identification of the clone was verified by competition assay with authentic alpha 1-acid glycoprotein isolated from serum and by restriction analysis of the cDNA inserted into pBR322.

Animals↗