Search PubMed⌕ Search

Biomedical subjects

R A Stein

Publications and source records attributed to R A Stein.

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

Thermal inactivation of the protein tyrosine kinase of the epidermal growth factor receptor.

It has been shown previously that the EGF-stimulable protein tyrosine kinase in a membrane preparation from A431 cells was inactivated by heat shock (45 degrees C), under conditions where EGF binding was unaffected [Carpenter et al. (1979) J. Biol. Chem. 254, 4884-4891]. A later study found that in intact cells, the protein tyrosine kinase of the EGF receptor was insensitive to heat shock [Liu & Carpenter (1992) Biochem. J. 286, 541-547]. We have extended these previous studies to better understand the thermal stability of the protein tyrosine kinase of the EGF receptor. We have measured the rate of inactivation of the kinase in membrane vesicles in the physiological to heat shock temperature range (37-45 degrees C). At 45 degrees C, the protein tyrosine kinase is rapidly inactivated with a rate of approximately 0.14 min-1. There is, however, protection against inactivation by incubation of the EGF receptor with AMPPNP, a hydrolysis-resistant ATP analog. At 45 degrees C, the rate of inactivation of nucleotide-bound receptor is an order of magnitude lower than the rate of inactivation of unoccupied receptor. Analysis of the temperature dependence of inactivation between 37 and 45 degrees C yields an activation energy, E(a), of 42 kcal/mol, an activation Gibbs free energy, delta G(++), between 23 and 22 kcal/mol, an activation enthalpy, delta H(++), of 42 kcal/mol, and an activation entropy, delta S(++), of 60 cal/(K.mol). The signs and magnitudes of the thermodynamic parameters suggest that inactivation is more likely due to some local reorganization within the kinase domain than to a simple chemical process. Further, the kinetic data show that the receptor is stabilized against inactivation by binding adenine nucleotide.

Adenylyl Imidodiphosphate↗
PubMed1996

Activation of creatine kinase-B and phospholamban gene expression in transformed latissimus dorsi muscle: evaluation of mRNA by polymerase chain reaction.

Latissimus dorsi muscle (LDM) transformation following chronic stimulation is the critical requirement for its use in cardiac assist procedures. In order to identify one or two molecular markers that can be used to effectively monitor the LDM transformation, the modulation in the expression of creatine kinase (CK) and phospholamban (PLB) genes by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was examined. Continuous in situ stimulation of left LDM was performed in four dogs for a period of 10 weeks after a vascular delay period of 2 weeks following surgery. For RT-PCR, gene-specific radiolabeled primers and equal amounts of cDNA synthesized from total RNA extracted from the LDM biopsies obtained at 4, 7, and 10 weeks of stimulation were used. A 2.6-fold increase in creatine kinase (brain type) (CK-B) mRNA was observed at transformed LDM compared to the control (P = 0.004) following 10 weeks of stimulation. On the contrary, a 30% decline was observed in creatine kinase (muscle type) (CK-M) mRNA level. An increase up to eight-fold was also observed in PLB mRNA in stimulated LDM compared to the contralateral muscle (P = 0.002). The PLB mRNA level in transformed LDM reached plateau and became comparable to that of normal heart after 7 weeks of stimulation. However, a sustained increase in CK-B mRNA level was observed until 10 weeks of stimulation. The level of beta-actin mRNA used as control remained the same in both stimulated and control samples. Thus the increase in CK-B and PLB mRNA and downregulation of CK-M mRNA in transformed LDM, demonstrated here by RT-PCR, indicate a switch from anaerobic to aerobic potential of transformed LDM along with a change towards slow-twitch phenotype and provide valuable markers to monitor the effectiveness of muscle transformation in cardiomyoplasty.

Actins↗
PubMed1995

Intracoronary ST-T alternans during coronary balloon angioplasty.

To evaluate the presence and clinical significance of electrical alternans during PTCA, intracoronary electrocardiography (ic-ECG) was performed in 65 consecutive lesions. ST-T alternans, defined as a beat-to-beat difference in the ST elevation > or = 1 mm, was present in five lesions (7.7%), all in the proximal left anterior descending (LAD) coronary artery. The phenomenon was seen only after 130 sec (mean 174 +/- 57) of balloon inflation. Only two of the five showed ST-T alternans simultaneously on both surface and ic-ECG. One of five patients had premature ventricular contractions following ST-T alternans. Three of these five lesions required a second balloon inflation with duration of > or = 300 sec; there was no ST-T alternans on the second inflation in any of these lesions. We conclude: (1) the detection of ST-T alternans during PTCA is enhanced by use of ic-ECG, (2) electrical alternans during PTCA was seen only in proximal LAD lesions, implicating a large amount of ischemic myocardium as a requisite for the phenomenon, (3) electrical alternans is not immediate, requiring a certain duration of balloon occlusion and hence ischemia to occur, and (4) the absence of ST-T alternans during second balloon inflations suggests ischemic preconditioning may abolish this phenomenon.

Adult↗
PubMed1994

Light distribution of ocular endophotocoagulator probes and its surgical implications.

Ophthalmic endophotocoagulator probe cone angle affects the spot size, working distance, laser output power requirement, tissue exposure time, and uniformity of tissue irradiance, which all affect ease and safety of clinical use. The cone angle and irradiance distribution of several ophthalmic endophotocoagulator delivery systems were studied by directing the laser energy emitted by them on a CCD video sensor at several angles of incidence. The irradiances followed a Gaussian distribution. The measured irradiances were compared to a mathematical model of tissue irradiance that adjusted for the cone angle, probe-tissue distance, and angle of incidence. Using this model, laser irradiances produced by endophotocoagulator probes can be estimated under a wide variety of conditions. At highly oblique angles of incidence, wide-angle probes produce unexpectedly higher and uneven tissue irradiance. When numerous characteristics are considered, probes with a cone angle in the range of 10 degrees have many advantages over narrower or wider angle probes.

Fiber Optic Technology↗
PubMed1993

The effect of corneal thickness on applanation tonometry.

Simultaneous manometry and Perkins tonometry were performed at 10.0, 20.0, and 30.0 mm Hg on 15 eyes on which intraocular procedures were performed. There was a statistically significant relationship between corneal thickness and the error of Perkins tonometry. Thin corneas produced underestimations of the intraocular pressure by as much as 4.9 mm Hg, whereas thick corneas produced overestimations by as much as 6.8 mm Hg. Measuring the corneal thickness is necessary to interpret properly the results of Goldmann applanation tonometry, particularly in eyes with thin corneas.

Anthropometry↗
PubMed1993

Foreign bodies in the foot.

A symptomatic foreign object lodged in the foot can be a difficult problem. The object's location and shape are the most important factors to identify before removal of the object is attempted. Many different imaging modalities have been used to locate the foreign objects in the foot. The authors briefly reviewed several common imaging modalities. Computed tomography appears to be superior to the other imaging modalities discussed in visualizing and localizing foreign objects in the foot. Magnetic resonance scanning is probably equal to computed tomography but is less readily available and more expensive and is contraindicated with ferrous metal foreign bodies.

Adult↗
PubMed1992

Changes in left ventricular size, wall thickness, and function in anemic patients treated with recombinant human erythropoietin.

Left ventricular size and function were evaluated in 15 anemic chronic hemodialysis patients before and after the administration of recombinant human erythropoietin (rHuEPO). All patients were studied with two-dimensional and M-mode echocardiographic examinations before the initiation of rHuEPO (T1) and at 28 +/- 7 weeks of rHuEPO therapy (T2). The two-dimensional targeted M-mode echocardiographic measurements obtained were: end-diastolic dimension (EDD); end-systolic dimension (ESD); stroke dimension (SD); dimensional shortening (SD/EDD); systolic posterior wall thickness (PWs); diastolic posterior and interventricular septal thickness; end-systolic wall stress (ESWS); and left ventricular mass. Mean hematocrit in these patients increased almost 50%. The EDD decreased from a mean value (+/- SEM) of 6.41 +/- 0.33 to 4.93 +/- 0.21 cm (p less than 0.05). ESD decreased from a mean value of 4.16 +/- 1.2 to 2.77 +/- 0.06 cm (p less than 0.05). The calculated mean SD decreased slightly but not significantly from 2.21 +/- 0.69 to 2.19 +/- 0.60 cm. The calculated SD/EDD increased from a mean 0.35 +/- 0.09 to 0.44 +/- 0.07 (p less than 0.05). ESWS fell from 59.2 +/- 12.2 to 37.6 +/- 9.3 gm/cm2 (p less than 0.01), and left ventricular mass fell (p less than 0.05) from 347 +/- 15.2 to 227 +/- 59 gm. There was no significant difference in resting heart rate or systolic blood pressure between T1 and T2. The increase in dimension shortening reflects afterload reduction, as indicated by the fall in end-systolic wall stress.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Segmentation of high-frequency ultrasound images of atherosclerotic coronary arteries.

High-frequency epicardial echocardiography (HFE) is an ultrasound imaging technique capable of visualizing coronary arteries in cross-section. Use of this imaging technique in the intra-operative setting can provide information about the structure of coronary arteries. As a result of time constraints imposed in this setting, there is a need for rapid, on-line analysis of HFE images. An algorithm for segmenting HFE images of coronary arteries into wall and luminal regions is described in this paper. The algorithm is more objective and time efficient than manual methods. A validation study with post-mortem human hearts demonstrated an excellent correlation (r = 0.99 for luminal areas, r = 0.99 for wall areas) between HFE images segmented with the algorithm and by manual methods, and a good correlation (r = 0.90 for luminal areas, r = 0.86 for wall areas) between HFE images segmented with the algorithm and manually segmented images of histological samples of the corresponding coronary arterial segments. Use of the algorithm for intra-operative HFE image segmentation may reduce the amount of time required for HFE image analysis and allow for an increased amount of data collection and analysis in the operating room.

Algorithms↗
PubMed1991

Exercise in hemodialysis patients after treatment with recombinant human erythropoietin.

To assess the effect of substantial increases in blood hemoglobin (Hb) caused by treatment with recombinant human erythropoietin (rhEPO) on exercise capacity in maintenance hemodialysis patients, we evaluated 10 patients (7 men and 3 women) at a mean age of 44.3 +/- 8.4 years on maintenance hemodialysis for a mean of 29.7 +/- 30.2 months by treadmill exercise to exhaustion. The patients were tested before administration of rhEPO and after a minimum 1 g/dl rise in Hb. With a change in Hb from 7.1 +/- 1.4 to 9.8 +/- 2.1 g/dl, peak oxygen consumption (VO2 peak) with exercise increased 50.3 +/- 9% (T1 = 15.1 +/- 5.3, T2 = 22.7 +/- 4.6 ml O2/kg/min, p less than 0.05). Respiratory exchange ratio (RER) at a given submaximal exercise level (3 mph, 6% of elevation) decreased significantly (T1 = 1.13 +/- 0.24, T2 = 0.92 +/- 0.08, p less than 0.05). The rhEPO-mediated increase in Hb was associated with an increased VO2 peak--an improvement of the peak exercise capacity and a reduced submaximal RER--reflecting a reduction in anaerobic metabolism at activities of daily living.

Adult↗
PubMed1991

Lipid and lipoprotein profiles relate to peak aerobic power in spinal cord injured men.

The relationship between peak VO2 and serum lipids, lipoproteins, and apolipoproteins was assessed in nine traumatic spinal cord injured (SCI), active, male volunteers. Mean (SD) age, height, and weight were 30.6 (11.6) yr, 171.1 (11.3) cm, and 74.2 (12.5) kg, respectively. Peak VO2 (X = 2.13 l.min-1) was assessed by a graded arm-crank test to maximum, percent body fat (X = 28.7%) by densitometry, and lipid profile by measures of serum triglycerides, total cholesterol (TC), high and low density lipoprotein cholesterol (HDL-C and LDL-C, respectively), apolipoproteins (apoA-1, apoB), and various ratios of lipids, lipoproteins, and apolipoproteins. Significant inverse relationships emerged between peak VO2 and TC/HDL-C (r = -0.86; P less than 0.01), apoB/apoA-1 (r = -0.75; P less than 0.05), triglycerides (r = -0.73; P less than 0.05), and LDL-C/HDL-C (r = -0.72; P less than 0.05). Direct correlations (P less than 0.05) were demonstrated between peak VO2 and apoA-1/apoB (r = 0.71) and HDL-C/apoA-1 (r = 0.64). The present results indicate that, for active, mid-to-lower thoracic SCI men, the putative atherogenic and antiatherogenic lipid, lipoprotein, and apolipoprotein indices are significantly related to peak VO2 in a manner similar to that described for the able-bodied. These findings indicate the relevance of aerobic fitness assessment in planning CHD prevention strategies for the SCI.

Adult↗
PubMed1990

Time-resolved rotational dynamics of phosphorescent-labeled myosin heads in contracting muscle fibers.

We have measured the microsecond rotational motions of myosin heads in contracting rabbit psoas muscle fibers by detecting the transient phosphorescence anisotropy of eosin-5-maleimide attached specifically to the myosin head. Experiments were performed on small bundles (10-20 fibers) of glycerinated rabbit psoas muscle fibers at 4 degrees C. The isometric tension and physiological ATPase activity of activated fibers were unaffected by labeling 60-80% of the heads. Following excitation of the probes by a 10-ns laser pulse polarized parallel to the fiber axis, the time-resolved emission anisotropy of muscle fibers in rigor (no ATP) showed no decay from 1 microsecond to 1 ms (r infinity = 0.095), indicating that all heads are rigidly attached to actin on this time scale. In relaxation (5 mM MgATP but no Ca2+), the anisotropy decayed substantially over the microsecond time range, from an initial anisotropy (r0) of 0.066 to a final anisotropy (r infinity) of 0.034, indicating large-amplitude rotational motions with correlation times of about 10 and 150 microseconds and an overall angular range of 40-50 degrees. In isometric contraction (MgATP plus saturating Ca2+), the amplitude of the anisotropy decay (and thus the amplitude of the microsecond motion) is slightly less than in relaxation, and the rotational correlation times are about twice as long, indicating slower motions than those observed in relaxation. While the residual anisotropy (at 1 ms) in contraction is much closer to that in relaxation than in rigor, the initial anisotropy (at 1 microsecond) is approximately equidistant between those of rigor and relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Effects of exercise on the signal-averaged electrocardiogram in coronary artery disease.

The effects of exercise on the signal-averaged electrocardiogram (SAECG) were investigated in 52 patients with stable coronary artery disease. The SAECG was recorded before and immediately after the exercise test and analyzed at 25 to 250 Hz and 40 to 250 Hz. All patients had SAECG with noise level less than or equal 0.8 microV at 25 Hz and less than or equal to 0.6 microV at 40 Hz and with the difference in noise level between control SAECGs and SAECGs after exercise less than or equal to 0.2 to 0.3 microV. Twenty-eight patients developed ST changes consistent with transient subendocardial ischemia that persisted during the SAECG recording after exercise. There was no significant difference between control SAECGs and SAECGs after exercise in patients with or without a positive exercise test. The absence of significant change on the SAECG was not related to the presence or absence of prior myocardial infarction, site of infarction, development of exercise-induced ventricular arrhythmias or presence of an abnormal recording at baseline. These data suggest that exercise-induced electrophysiologic changes and ventricular arrhythmias may not be related to the anatomic-electrophysiologic substrate that underlies late potentials on the SAECG.

Arrhythmias, Cardiac↗