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Biomedical subjects

R A Stein

Publications and source records attributed to R A Stein.

At least 19 recordsLinked to original sources
PubMed2001

Real-time kinetics of ligand/cell surface receptor interactions in living cells: binding of epidermal growth factor to the epidermal growth factor receptor.

We describe a system for extending stopped-flow analysis to the kinetics of ligand capture and release by cell surface receptors in living cells. While most mammalian cell lines cannot survive the shear forces associated with turbulent stopped-flow mixing, we determined that a murine hematopoietic precursor cell line, 32D, is capable of surviving rapid mixing using flow rates as great as 4.0 mL/s, allowing rapid processes to be quantitated with dead times as short as 10 ms. 32D cells do not express any endogenous epidermal growth factor (EGF) receptor or other ErbB family members and were used to establish monoclonal cell lines stably expressing the EGF receptor. Association of fluorescein-labeled H22Y-murine EGF (F-EGF) to receptor-expressing 32D cells was observed by measuring time-dependent changes in fluorescence anisotropy following rapid mixing. Dissociation of F-EGF from EGF-receptor-expressing 32D cells was measured both by chase experiments using unlabeled mEGF and by experiments in which equilibrium was perturbed by dilution. Comparison of these dissociation experiments showed that little, if any, ligand-induced dissociation occurs in the chase dissociation experiments. Data from a series of association and dissociation experiments, performed at various concentrations of F-EGF in the nanomolar range and at multiple cell densities, were simultaneously analyzed using global analysis techniques and fit to a two independent receptor-class model. Our analysis is consistent with the presence of two distinct receptor populations having association rate constants of k(on1) = 8.6 x 10(6) M(-1) s(-1) and k(on2) = 2.4 x 10(6) M(-1) s(-1) and dissociation rate constants of k(off1) = 0.17 x 10(-2) s(-1) and k(off2) = 0.21 x 10(-2) s(-1). The magnitudes of these parameters suggest that under physiological conditions, in which cells are transiently exposed to nanomolar concentrations of ligand, ligand capture and release may function as the first line of regulation of the EGF receptor-induced signal transduction cascade.

Amino Acid Substitution↗
PubMed2001

An analytical approach to the measurement of equilibrium binding constants: application to EGF binding to EGF receptors in intact cells measured by flow cytometry.

In ligand binding studies, ligand depletion often limits the accuracy of the results obtained. This problem is approached by employing the simple observation that as the concentration of receptor in the assay is reduced, ligand depletion is also reduced. Measuring apparent K(D)'s of a ligand at multiple concentrations of receptor with extrapolation to infinitely low receptor concentration takes ligand depletion into account and, depending on the binding model employed, yields a K(D) within the defined limits of accuracy. We apply this analysis to the binding of epidermal growth factor (EGF) to the EGF receptor expressed in intact 32D cells, using a homogeneous fluorescein-labeled preparation of EGF and measuring binding by flow cytometry. Binding isotherms were carried out at varying cell densities with each isotherm fit to the generally applied model with two independent binding sites. Examination of the variation in the K(D)'s versus cell density yields a high-affinity site that accounts for 18% of the sites and a lower affinity site that accounts for the remainder. However, further examination of these data suggests that while consistent with each individual isotherm, the simple model of two independent binding sites that is generally applied to EGF binding to the EGF receptor is inconsistent with the changes in the apparent K(D)'s seen across varying cell densities.

Animals↗
PubMed2001

Cardiovascular risks of sexual activity.

The risks associated with sexual activity relate in part to the pathophysiology of the cardiovascular response to sexual activity. This review addresses this subject and reviews studies that directly address the cardiovascular risk associated with sexual activity.

Blood Pressure↗
PubMed2001

Dipyridamole-induced ST-segment elevation indicative of transmural myocardial ischemia--a case report.

Dipyridamole nuclear myocardial perfusion test is a safe and effective alternative to exercise nuclear perfusion testing for detecting myocardial ischemia. It is the procedure of choice in selected patients who are unable to exercise adequately. Intravenous dipyridamole causes coronary vasodilation with resultant maldistribution and heterogeneity of coronary flow in the presence of significant coronary artery disease. True ischemia, causing symptoms or ST-segment depression, is uncommon, in part because there is no increase in myocardial oxygen demand. A patient in whom myocardial ischemia developed, manifested by ST-segment elevation, during dipyridamole stress testing is described. Scintigraphic images illustrated a myocardial perfusion defect, which was consistent with coronary angiographic findings. This case report addresses the importance of dipyridamole-induced ST-segment elevation, its correlation with angiographic findings, and the need for continued hemodynamic and electrocardiographic monitoring in patients following dipyridamole infusion.

Coronary Angiography↗
PubMed2001

Cryptogenic ischemic stroke and paradoxical embolism: should a patent foramen ovale be closed? Case report and literature review.

Paradoxical embolism is a well-recognized cause of stroke. While the diagnosis in the majority of the cases with a patent foramen ovale is presumptive, numerous treatment strategies have been described. However, there is no single approach that has been overwhelmingly recommended for these patients. A patient is described who presented with ischemic stroke. Transesophageal echocardiography revealed a thrombus that straddled a patent foramen ovale. Anticoagulation with intravenous heparin resulted in resolution of thrombus and neurologic deficit. The literature regarding diagnosis and treatment of paradoxical embolism in the presence of patent foramen ovale is reviewed.

Aged↗
PubMed2001

Multi-morphology wide QRS tachycardias in a patient without structural heart disease: an unusual presentation of ventricular tachycardia.

Ventricular tachycardia occurs infrequently in patients without structural heart disease. These tachycardias commonly originate from the right ventricular outflow tract. However, the presence of more than one arrhythmogenic substrate in a patient without structural heart disease is rare. We report such a 48-year old patient with no structural heart disease who presented with three morphology wide QRS tachycardia. A brief review of the pathophysiology and management is discussed.

Anti-Arrhythmia Agents↗
PubMed2000

Cardiovascular response to sexual activity.

The cardiovascular response to coitus and the risk of an acute cardiac event related to sexual activity is of clinical importance, especially now that effective pharmacologic treatment of male erectile dysfunction permits older men to resume active sex lives. Early studies by Masters and Johnson of young subjects engaging in coitus in laboratory settings reported that heart rates and systolic blood pressures were at near maximum exercise levels. Subsequent data from studies by Hellerstein and Friedman and by Stein in men with coronary artery disease, using ambulatory electrocardiographic recordings during coitus at home, demonstrated significantly lower heart rate and blood pressure responses to coitus. The associated myocardial oxygen demand of coitus in these men was found to in the range of moderate activities, often achieved or exceeded during their workday. The cardiovascular risk of coitus was addressed in the recent ONSET study in which myocardial infarction (MI) patients were interviewed shortly after their MI about potential triggering activities or events. Coitus was noted to represent a very low absolute risk of being a trigger for MI, but had an increased relative risk of 2.5 for the subjects in their study. Sexual intercourse will, in most men, represent only a moderate "stress" on the heart in terms of the responses that impact on myocardial oxygen requirement (heart rates, and systolic blood pressure). In patients with coronary artery atherosclerosis, coitus, compared with vigorous physical activity and intense emotional responses, represents a small risk of triggering an acute MI.

Coitus↗
PubMed2000

Management of sexual dysfunction in patients with cardiovascular disease: recommendations of the Princeton Consensus Panel.

Sexual dysfunction is highly prevalent in both sexes and adversely affects patients' quality of life and well being. Given the frequent association between sexual dysfunction and cardiovascular disease, in addition to the potential cardiac risk of sexual activity itself, a consensus panel was convened to develop recommendations for clinical management of sexual dysfunction in patients with cardiovascular disease. Based upon a review of the research and presentations by invited experts, a classification system was developed for stratification of patients into high, low, and intermediate categories of cardiac risk. The large majority of patients are in the low-risk category, which includes patients with (1) controlled hypertension; (2) mild, stable angina; (3) successful coronary revascularization; (4) a history of uncomplicated myocardial infarction (MI); (5) mild valvular disease; and (6) no symptoms and <3 cardiovascular risk factors. These patients can be safely encouraged to initiate or resume sexual activity or to receive treatment for sexual dysfunction. An important exception is the use of sildenafil in patients taking nitrates in any form. Patients in the intermediate-risk category include those with (1) moderate angina; (2) a recent MI (<6 weeks); (3) left ventricular dysfunction and/or class II congestive heart failure; (4) nonsustained low-risk arrhythmias; and (5) >/=3 risk factors for coronary artery disease. These patients should receive further cardiologic evaluation before restratification into the low- or high-risk category. Finally, patients in the high-risk category include those with (1) unstable or refractory angina; (2) uncontrolled hypertension; (3) congestive heart failure (class III or IV); (4) very recent MI (<2 weeks); (5) high-risk arrhythmias; (6) obstructive cardiomyopathies; and (7) moderate-to-severe valvular disease. These patients should be stabilized by specific treatment for their cardiac condition before resuming sexual activity or being treated for sexual dysfunction. A simple algorithm is provided for guiding physicians in the management of sexual dysfunction in patients with varying degrees of cardiac risk.

Adult↗
PubMed2000

Management of sexual dysfunction in patients with cardiovascular disease: recommendations of The Princeton Consensus Panel.

Sexual dysfunction is highly prevalent in both sexes and adversely affects patients' quality of life and well being. Given the frequent association between sexual dysfunction and cardiovascular disease, in addition to the potential cardiac risk of sexual activity itself, a consensus panel was convened to develop recommendations for clinical management of sexual dysfunction in patients with cardiovascular disease. Based upon a review of the research and presentations by invited experts, a classification system was developed for stratification of patients into high, low, and intermediate categories of cardiac risk. The large majority of patients are in the low-risk category, which includes patients with (1) controlled hypertension; (2) mild, stable angina; (3) successful coronary revascularization; (4) a history of uncomplicated myocardial infarction (MI); (5) mild valvular disease; and (6) no symptoms and <3 cardiovascular risk factors. These patients can be safely encouraged to initiate or resume sexual activity or to receive treatment for sexual dysfunction. An important exception is the use of sildenafil in patients taking nitrates in any form. Patients in the intermediate-risk category include those with (1) moderate angina; (2) a recent MI (<6 weeks); (3) left ventricular dysfunction and/or class II congestive heart failure; (4) nonsustained low-risk arrhythmias; and (5) >/=3 risk factors for coronary artery disease. These patients should receive further cardiologic evaluation before restratification into the low- or high-risk category. Finally, patients in the high-risk category include those with (1) unstable or refractory angina; (2) uncontrolled hypertension; (3) congestive heart failure (class III or IV); (4) very recent MI (<2 weeks); (5) high-risk arrhythmias; (6) obstructive cardiomyopathies; and (7) moderate-to-severe valvular disease. These patients should be stabilized by specific treatment for their cardiac condition before resuming sexual activity or being treated for sexual dysfunction. A simple algorithm is provided for guiding physicians in the management of sexual dysfunction in patients with varying degrees of cardiac risk.

Humans↗
PubMed2000

Structural characterization of penicillin-binding protein-related factor A (PrfA) from Bacillus species.

The prfA genes of Bacillus stearothermophilus and Bacillus subtilis are in an operon downstream of the ponA gene encoding penicillin-binding protein 1 (PBP1), a major enzyme involved in peptidoglycan synthesis. The specific function of the 23- to 24-kDa PrfA protein is unknown but this protein plays some role in nucleoid segregation and the functions of PrfA and PBP1 are interrelated. We overexpressed B. stearothermophilus and B. subtilis PrfA in Escherichia coli and purified the proteins to homogeneity by cation exchange and gel filtration chromatography. The protein is a monomer in solution, and circular dichroism spectroscopy revealed an abundance of beta-sheet secondary structure. Crystals of B. stearothermophilus PrfA were also obtained and diffracted X-rays to 1.8 A resolution.

Bacillus subtilis↗
PubMed2000

Evolutionary analysis of the ErbB receptor and ligand families.

We have compared all available deduced protein sequences of the ErbB family of receptors and their ligands. Analysis of the aligned sequences of the receptors indicates that there are some differences in the receptors that are specific to invertebrates. In addition, comparison of the vertebrate ErbB receptors suggest that a gene duplication event generated two ancestral receptors, the ErbB3/ErbB4 precursor and the ErbB1/ErbB2 precursor. Subsequent gene duplications of these precursors generated the four receptors present in mammals. Analysis of the sequences for the known ligands of the ErbB receptors suggests that the vertebrate ligands segregate into the ErbB1 ligands and the ErbB3/ErbB4 ligands, paralleling the evolution of the receptors; however, it is difficult to ascertain any correlation between the invertebrate and the vertebrate ligands. Even though ErbB3 is kinase-impaired, there is significant conservation of the kinase domain within the vertebrate lineage (human, rat, and F. rubripes), suggesting some function for this domain other than kinase activity, such as mediating protein-protein interactions that are involved in receptor dimerization and/or activation of the kinase domain of the heterodimerization partner. To date, no ligand for ErbB2 has been identified, and comparison of the extracellular domains of ErbB2 reveals two regions that are not conserved across the mammalian species. These two regions of divergence align with sequences in ErbB1 that have been shown to be proximal to the amino-terminus and to the carboxyl-terminal region, respectively, of bound EGF. Further, one of these regions contains an insertion, relative to the other members of the mammalian ErbB family, which might affect the ligand binding site and provide a structural basis for this receptor's apparent inability to bind ligand independently.

Amino Acid Sequence↗
PubMed2000

Cardiovascular exercise and wellness. Exercise training for cardiac rehabilitation patients: meeting the challenges of the millennium.

Exercise training for cardiac rehabilitation has evolved over the past decades in response to a growing knowledge base in exercise physiology, an expanding understanding to the knowledge base of coronary disease, and a change in the patients presenting for cardiac rehabilitation. The patient population has changed from a post myocardial infarction patient group, to patients who have had coronary artery revascularization (coronary artery bypass surgery or percutaneous transluminal coronary angioplasty) with the implantation of intraarterial stents. Program goals have evolved from enhancing endurance fitness in deconditioned patients to initiating the long-term adoption of an active exercising lifestyle with the use of strength training to complement endurance training. An increased understanding of behavioral issues in the adoption of an active lifestyle will influence the evolution of cardiac rehabilitation exercise training. During the next several years, it is anticipated that the patient population will change to include patients with significant left ventricular systolic dysfunction and congestive heart failure. The exercise training programs will then further evolve to reflect the successful exercise training formats utilized in the multicenter trials of exercise training for patients with congestive heart failure. (c) 2000 by CHF, Inc.

Journal Article↗
PubMed1997

Cardiovascular response to sudden strenuous exercise: an exercise echocardiographic study.

We studied the response in left ventricular (LV) internal dimensions and posterior wall thickness during the performance of sudden strenuous exercise without warm-up (SSE) and sudden strenuous exercise with warm-up (SSEw) in 15 healthy, untrained college-aged males (26 +/- 5.0 yr). Measurements of left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD), stroke dimension (SD = LVEDD-LVESD), fractional shortening (FS = SD/EDD) and posterior wall thickness (PWT) were obtained from continuous 2-D targeted on M-mode echocardiography. Continuous EKG and blood pressure were obtained at rest and during the final 10 s of SSE (30 s of upright leg cycle ergometry of 400 W at 80 rpm). SSEw was preceded by warm-up exercise (6 min of graded leg cycle exercise of 2-min stages initial load 30 W, increasing in 30-W increments at 60 rpm, followed immediately by SSE). Our findings revealed that there were no significant differences in the LV internal dimensions (LVEDD, LVESD, FS, PWT), HR max, RPP max, ECG, and MAP max between SSE and SSEw. Sudden strenuous exercise without warm-up is not associated with a reduced LV function. The results of this study are contradictory to previous findings that have suggested that SSE is associated with transient global left ventricular (LV) dysfunction.

Adult↗
PubMed1996

5'-(p-fluorosulfonylbenzoyl)-2'(or 3')-(methylanthraniloyl)adenosine, fluorescent affinity labels for adenine nucleotide binding sites: interaction with the kinase active site of the receptor for epidermal growth factor.

We have found that the epidermal growth factor (EGF) receptor kinase can utilize the fluorescent ATP derivative, methylanthraniloyl ATP, as a substrate. On the basis of this observation, together with our previous studies that showed that 5'-(p-fluorosulfonylbenzoyl)adenosine (5'-FSBAdo) is a highly specific affinity label for the ATP site of the kinase domain of the EGF receptor, we prepared new derivatives of 5'-FSBAdo, 5'-(p-fluorosulfonyl)-2'(or 3')-(methylanthraniloyl)adenosine (FSBMantAdo), as fluorescent affinity labels for adenine nucleotide binding sites, and in particular for the ATP site of the EGF receptor. The two products were purified by HPLC and were characterized by UV-Vis absorbance spectroscopy, mass spectrometry, nuclear magnetic resonance spectroscopy, and fluorescence spectroscopy. Incubation of membrane vesicles containing the EGF receptor with either the 2' or 3' derivative resulted in irreversible inhibition of the receptor kinase activity, as assessed by autophosphorylation assays. Preincubation of vesicles with AMP imidodiphosphate (AMPPNP), a hydrolysis-resistant ATP analog, prior to treatment with FSBMantAdo resulted in the protection of the receptor kinase activity' from FSBMantAdo inactivation. Steady state fluorescence spectra (with excitation at 360 nm) revealed a blue shift in the emission maximum of partially purified FSBMantAdo-labeled receptor (426 nm), as compared with the emission maximum of free FSBMantAdo (441 nm) in aqueous solution, suggesting that the receptor-bound label is in a relatively low polarity environment. These studies show that FSBMantAdo is a specific affinity label for the ATP site of the EGF receptor. FSBMantAdo may also prove useful as a fluorescent affinity label for other ATP binding sites.

Adenine Nucleotides↗