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Richard A Stein

Publications and source records attributed to Richard A Stein.

17 recordsLinked to original sources
PubMed2006

Insights into the evolution of the ErbB receptor family and their ligands from sequence analysis.

BACKGROUND: In the time since we presented the first molecular evolutionary study of the ErbB family of receptors and the EGF family of ligands, there has been a dramatic increase in genomic sequences available. We have utilized this greatly expanded data set in this study of the ErbB family of receptors and their ligands. RESULTS: In our previous analysis we postulated that EGF family ligands could be characterized by the presence of a splice site in the coding region between the fourth and fifth cysteines of the EGF module and the placement of that module near the transmembrane domain. The recent identification of several new ligands for the ErbB receptors supports this characterization of an ErbB ligand; further, applying this characterization to available sequences suggests additional potential ligands for these receptors, the EGF modules from previously identified proteins: interphotoreceptor matrix proteoglycan-2, the alpha and beta subunit of meprin A, and mucins 3, 4, 12, and 17. The newly available sequences have caused some reorganizations of relationships among the ErbB ligand family, but they add support to the previous conclusion that three gene duplication events gave rise to the present family of four ErbB receptors among the tetrapods. CONCLUSION: This study provides strong support for the hypothesis that the presence of an easily identifiable sequence motif can distinguish EGF family ligands from other EGF-like modules and reveals several potential new EGF family ligands. It also raises interesting questions about the evolution of ErbB2 and ErbB3: Does ErbB2 in teleosts function differently from ErbB2 in tetrapods in terms of ligand binding and intramolecular tethering? When did ErbB3 lose kinase activity, and what is the functional significance of the divergence of its kinase domain among teleosts?

Amino Acid Motifs↗
PubMed2005

Solution structure of the cytoplasmic domain of erythrocyte membrane band 3 determined by site-directed spin labeling.

The cytoplasmic domain of the anion exchange protein (cdb3) serves as a critical organizing center for protein-protein interactions that stabilize the erythrocyte membrane. The structure of the central core of cdb3, determined by X-ray crystallography from crystals grown at pH 4.8, revealed a compact dimer for residues 55-356 and unresolved N- and C-termini on each monomer [Zhang et al. (2000) Blood 96, 2925-2933]. Given that previous studies had suggested a highly asymmetric structure for cdb3 and that pH dependent structural transitions of cdb3 have been reported, the structure of cdb3 in solution at neutral pH was investigated via site-directed spin labeling in combination with conventional electron paramagnetic resonance (EPR) and double electron electron resonance (DEER) spectroscopies. These studies show that the structure of the central compact dimer (residues 55-356) is indistinguishable from the crystal structure determined at pH 4.8. N-Terminal residues 1-54 and C-terminal residues 357-379 are dynamically disordered and show no indications of stable secondary structure. These results establish a structural model for cdb3 in solution at neutral pH which represents an important next step in characterizing structural details of the protein-protein interactions that stabilize the erythrocyte membrane.

Anion Exchange Protein 1, Erythrocyte↗
PubMed2005

Calcium binding by the essential virulence factor BAD-1 of Blastomyces dermatitidis.

BAD-1 (Blastomyces adhesin 1), a 120-kDa protein of Blastomyces dermatitidis, functions as an adhesin, immune modulator, and essential virulence factor. Structurally, BAD-1 is composed of a short N-terminal region, a core of 30 tandem repeats critical for virulence, and a C-terminal epidermal growth factor domain that binds the protein to yeast cell surface chitin. Each of the 30 acidic residue-rich tandem repeats contains a sequence that resembles the calcium-binding loop of the EF-hand domain found in many calcium-binding proteins. Here, we investigated the binding of calcium by BAD-1 and its biological significance. Yeast washed with double distilled H2O released surface-bound BAD-1, but EGTA washes were an order of magnitude more efficient, suggesting an interaction between BAD-1 and calcium. Immobilized BAD-1 was stained with ruthenium red dye, an indicator of calcium-binding proteins. In equilibrium dialysis, BAD-1 bound 45Ca2+ with an affinity of 0.41 x 10(-5) m and a capacity of 27 calcium/mol. Mass spectrometry confirmed this capacity. Elevated [Ca2+] diminished BAD-1 solubility. Upon deletion of its C-terminal epidermal growth factor-like domain, BAD-1 resisted aggregation by elevated [Ca2+] but retained its affinity and capacity for calcium. Removing 20 copies of the tandem repeat, however, sharply reduced the capacity of BAD-1 for calcium. Growth of the bad-1 null yeast was inhibited by 5 mm EGTA, and re-expression of BAD-1 in trans or the addition of exogenous purified BAD-1 restored growth. Thus, BAD-1 is a high capacity calcium-binding protein. This property contributes to the structure and function of BAD-1, as well as to B. dermatitidis acquisition of calcium from the environment.

Amino Acid Sequence↗
PubMed2005

Dipolar coupling between nitroxide spin labels: the development and application of a tether-in-a-cone model.

A tether-in-a-cone model is developed for the simulation of electron paramagnetic resonance spectra of dipolar coupled nitroxide spin labels attached to tethers statically disordered within cones of variable halfwidth. In this model, the nitroxides adopt a range of interprobe distances and orientations. The aim is to develop tools for determining both the distance distribution and the relative orientation of the labels from experimental spectra. Simulations demonstrate the sensitivity of electron paramagnetic resonance spectra to the orientation of the cones as a function of cone halfwidth and other parameters. For small cone halfwidths (< approximately 40 degrees ), simulated spectra are strongly dependent on the relative orientation of the cones. For larger cone halfwidths, spectra become independent of cone orientation. Tether-in-a-cone model simulations are analyzed using a convolution approach based on Fourier transforms. Spectra obtained by the Fourier convolution method more closely fit the tether-in-a-cone simulations as the halfwidth of the cone increases. The Fourier convolution method gives a reasonable estimate of the correct average distance, though the distance distribution obtained can be significantly distorted. Finally, the tether-in-a-cone model is successfully used to analyze experimental spectra from T4 lysozyme. These results demonstrate the utility of the model and highlight directions for further development.

Anisotropy↗
PubMed2005

Measuring chromosome dynamics on different time scales using resolvases with varying half-lives.

The bacterial chromosome is organized into multiple independent domains, each capable of constraining the plectonemic negative supercoil energy introduced by DNA gyrase. Different experimental approaches have estimated the number of domains to be between 40 and 150. The site-specific resolution systems of closely related transposons Tn3 and gammadelta are valuable tools for measuring supercoil diffusion and analysing bacterial chromosome dynamics in vivo. Once made, the wild-type resolvase persists in cells for time periods greater than the cell doubling time. To examine chromosome dynamics over shorter time frames that are more closely tuned to processes like inducible transcription, we constructed a set of resolvases with cellular half-lives ranging from less than 5 min to 30 min. Analysing chromosomes on different time scales shows domain structure to be dynamic. Rather than the 150 domains detected with the Tn3 resolvase, wild-type cells measured over a 10 min time span have more than 400 domains per genome equivalent, and some gyrase mutants exceed 1000.

Chromosomes, Bacterial↗
PubMed2005

Organization of supercoil domains and their reorganization by transcription.

During a normal cell cycle, chromosomes are exposed to many biochemical reactions that require specific types of DNA movement. Separation forces move replicated chromosomes into separate sister cell compartments during cell division, and the contemporaneous acts of DNA replication, RNA transcription and cotranscriptional translation of membrane proteins cause specific regions of DNA to twist, writhe and expand or contract. Recent experiments indicate that a dynamic and stochastic mechanism creates supercoil DNA domains soon after DNA replication. Domain structure is subsequently reorganized by RNA transcription. Examples of transcription-dependent chromosome remodelling are also emerging from eukaryotic cell systems.

Bacterial Proteins↗
PubMed2004

Transcription-induced barriers to supercoil diffusion in the Salmonella typhimurium chromosome.

Transcription and replication both influence and are influenced by superhelical changes in DNA. Explaining how supercoil movement is channeled in living chromosomes has been a major problem for 30 years. Transcription of membrane-associated proteins leads to localized hypersupercoiling of plasmid DNA, and this behavior indicates the presence of aberrant supercoil diffusion. Using the lambda Red recombination system, we constructed model domains in the Salmonella typhimurium chromosome to analyze supercoiling dynamics of regions encoding membrane proteins. Regulation of Tn10-derived tetracycline resistance involves a repressor, TetR, and a membrane-bound export pump, TetA. Strains deficient in TetR activity had 60-fold higher transcription levels (from P(A)) than TetR-positive strains. High tetA transcription caused a 10- to 80-fold decrease in the gammadelta resolution efficiency for the domain that includes the Tet module. Replacing tetA with genes encoding cytosolic proteins LacZ and Kan also caused the appearance of supercoil diffusion barriers in a defined region of the chromosome. In strains containing a functional TetR located next to a regulated lacZ reporter (P(R)tetR-P(A)lacZ), induction of transcription with chlortetracycline caused a 5-fold drop in resolution efficiency in the test domain interval. A short half-life resolvase showed that barriers appeared and disappeared over a 10- to 20-min span. These studies demonstrate the importance of transcription in chromosome structure and the plasticity of supercoil domains in bacterial chromosomes.

Antiporters↗
PubMed2004

The clinical use of supine left lateral leg cycle ergometry for exercise electrocardiographic testing in women, as used in exercise echocardiography.

BACKGROUND: Two-dimensional exercise echocardiography is commonly performed in the supine (SUP) left lateral posture (SLL) to optimize imaging quality. OBJECTIVES: To assess the clinical use of SLL cycle ergometry, we determined the metabolic, cardiovascular, and hemodynamic responses during graded leg cycle ergometry in women, performed in SLL, commonly used during exercise echocardiography, and compared our findings with those obtained during upright (UP) and SUP leg cycle ergometry. METHODS: A total of 21 apparently healthy women performed leg cycle ergometry in 3 distinct postures (UP, SUP, and SLL). RESULTS: Peak oxygen consumption and peak achieved workload during SLL cycle ergometry were significantly lower compared with UP cycle ergometry, indicating that in SLL physical work capacity would be underestimated compared with that obtained for the UP and SUP postures. The peak heart rate was significantly lower in SLL and the peak systolic blood pressure higher, compared with UP. Maximal rate pressure product was not significantly different among the postures, indicating the equivalent value of SLL with regard to augmenting myocardial oxygen demand during exercise. Stroke volume (calculated from aortic Doppler flow integral) was higher at rest, and increased to a smaller extent during exercise, in the SUP posture and SLL compared with UP. Similar peak values were attained for the 3 postures. CONCLUSIONS: SLL is not a suitable modality for the assessment of functional status or for the derivation of target heart rates for exercise training in UP posture. However, SLL leg cycle ergometry exercise provokes a comparable stimulus for the detection of coronary artery disease, as it will increase myocardial oxygen demand to the same extent as UP leg cycle exercise.

Adult↗
PubMed2003

Mechanism of chaperone function in small heat-shock proteins. Fluorescence studies of the conformations of T4 lysozyme bound to alphaB-crystallin.

To further develop the mechanistic understanding of small heat-shock protein (sHSP) chaperone activity, we investigate the nature of the intermediate states recognized by alpha-crystallin and the conformations that are stably bound. The model substrates consist of a set of well characterized, destabilized T4 Lysozyme (T4L) mutants that have been shown to differentially bind alpha-crystallin in a manner that reflects their free-energy of unfolding. A new approach for the detection of complex formation is introduced based on the conformational sensitivity of the fluorescent probe bimane, site-specifically introduced in T4L. Emission spectra of bimane-labeled T4L reveal two distinct patterns of intensity changes upon binding that depend on the molar ratio of alpha-crystallin to T4L. This directly demonstrates the two-mode nature of the binding process by the alpha-crystallins. Biphasic binding isotherms, obtained and analyzed over a wide range of T4L concentrations, demonstrate a substantially quenched bimane fluorescence in the low affinity-bound T4L that is similar to the quenching level observed due to denaturant unfolding. Furthermore, the pattern of intensity changes that occur upon binding of a T4L variant, bimane-labeled at an alternative solvent-exposed site, establishes a direct correlation between the quenching level observed in binding and unfolding. The results can be interpreted in terms of a model where alpha-crystallin binds at least two conformationally distinct non-native states of T4L, one of which is substantially unfolded and is bound with low affinity. A high affinity binding mode to compact states may be relevant to chaperone function in the lens, where protein damage is unlikely to cause global unfolding.

Bacteriophage T4↗
PubMed2003

Endothelial dysfunction, erectile dysfunction, and coronary heart disease: the pathophysiologic and clinical linkage.

Our rapidly expanding knowledge regarding the biology of the endothelial cell and the pathophysiology of coronary heart disease (CHD) and endothelial dysfunction indicates important common factors and overlapping clinical presentations. We are, in effect, presented with a new paradigm-that of varying vascular manifestations of disease linked to the functioning of endothelial cell lining. This article reviews these areas of advancement and addresses their clinical implications regarding erectile dysfunction and CHD, as well as the role of the phosphodiesterase-5 inhibitor sildenafil.

Journal Article↗
PubMed2002

The effect of vardenafil, a potent and highly selective phosphodiesterase-5 inhibitor for the treatment of erectile dysfunction, on the cardiovascular response to exercise in patients with coronary artery disease.

OBJECTIVES: The effect of vardenafil, a potent and highly selective phosphodiesterase-5 (PDE5) inhibitor, on symptom-limited exercise time, time to first awareness of angina, and time to ischemic threshold (ST-segment depression > or =1 mm from baseline) during exercise tolerance testing (ETT) was examined in patients with stable coronary artery disease (CAD). BACKGROUND: Erectile dysfunction (ED) is common among men with CAD. PDE5 inhibition is increasingly the preferred treatment option for ED. However, the effect of PDE5 inhibition on exercise-induced ischemia in CAD patients has received limited prospective evaluation. METHODS: In this double-blind, crossover, single-dose multicenter study, 41 men with reproducible stable exertional angina due to ischemic CAD received vardenafil 10 mg or placebo, followed by ETT (5 to 10 metabolic equivalents [METS], Bruce protocol) 1 h postdose. Sublingual nitrate use was prohibited for > or =24 h pre- and postexercise study days. End points included symptom-limited treadmill exercise time, time to first awareness of angina, time to ischemic threshold, and safety. RESULTS: Relative to placebo, vardenafil 10 mg did not alter exercise treadmill time (427 +/- 105 s vs. 433 +/- 109 s, p = 0.39), or time to first awareness of angina (292 +/- 110 s vs. 291 +/- 123 s, p = 0.59), but significantly prolonged time to ischemic threshold (334 +/- 108 s vs. 381 +/- 108, p = 0.0004). At peak exercise, vardenafil 10 mg did not alter blood pressure, heart rate, or rate-pressure product relative to placebo. The most common adverse events (facial flushing and headache) were of mild or moderate intensity, and short-lived. CONCLUSIONS: Vardenafil 10 mg did not impair the ability of patients with stable CAD to exercise at levels equivalent or greater than that attained during sexual intercourse (average of 2.5 to 3.3 METS).

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2002

Rotational dynamics of the epidermal growth factor receptor.

We have examined the rotational mobility of SL-EGF, a bifunctional adduct of bis(sulfo-N-succinimidyl)-[(15)N,(2)H(16)]-doxyl-2-spiro-4'-pimelate and [Lys3,Tyr22]-murine epidermal growth factor, bound to the EGF receptor in A431 membrane vesicles. The linear EPR spectrum indicated that there was essentially no free SL-EGF in the bound complex preparation. To better define the rotational mobility of the SL-EGF bound to the EGF receptor, ST-EPR spectra were obtained at multiple Zeeman field modulation frequencies. Global analysis with a uniaxial rotational diffusion model of the ST-EPR data yielded two minima that have differences in rotational mobility and in orientation of the SL-EGF relative to the membrane normal axis. The rotational mobilities of the two rotational species are consistent with monomers and dimers or somewhat larger oligomers, such as trimers or tetramers, arguing against a role for higher order receptor clustering in receptor activation. Considering the two minima and previous observations that A431 membrane vesicles contain two distinguishable ligand-binding populations, the ST-EPR spectra were fit with a model having two uniaxial rotating species. This yielded two components that were similar to those obtained from the two original one-component fits, either fast or slow rotational mobility, with different orientations. The model-dependent results obtained suggest that there are potential conformational and rotational differences in the two populations and provide a plausible description for the origin of high- and low-affinity EGF-binding sites that can be tested in future experiments.

Animals↗
PubMed2002

PrfA protein of Bacillus species: prediction and demonstration of endonuclease activity on DNA.

The prfA gene product of Gram-positive bacteria is unusual in being implicated in several cellular processes; cell wall synthesis, chromosome segregation, and DNA recombination and repair. However, no homology of PrfA with other proteins has been evident. Here we report a structural relationship between PrfA and the restriction enzyme PvuII, and thereby produce models that predict that PrfA binds DNA. Indeed, wild-type Bacillus stearothermophilus PrfA, but not a catalytic site mutant, nicked one strand of supercoiled plasmid templates leaving 5'-phosphate and 3'-hydroxyl termini. This activity, much lower on linear or relaxed circular double-stranded DNA or on single-stranded DNA, is consistent with a role for this protein in chromosome segregation, DNA recombination, or DNA repair.

Amino Acid Sequence↗
PubMed2002

Managing concomitant cardiac disease and erectile dysfunction.

Early studies of peak heart rates and blood pressure during coitus led physicians to believe that sexual activity represents a significant risk to patients with cardiovascular disease. Subsequent studies indicated, however, that the heart rate during coitus was no higher than the rate during unaccustomed physical exercise or associated with anger. The absolute risk of myocardial infarction (MI) in a patient with a history of MI has been found to be 10 per million per hour, and the doubling of this risk in the 2 hours following coitus has a negligible impact on annual risk. Coronary artery disease (CAD) is a powerful indicator of the presence of erectile dysfunction (ED), and the risk factors for ED are similar to those for CAD. Studies of sildenafil citrate use in patients with a history of cardiovascular disease have found sildenafil to be safe and effective, except for an absolute contraindication in the concomitant use of nitrates. Physicians should become familiar with the clinical guidelines for classifying ED patients with a history of cardiovascular disease as high risk, intermediate or indeterminate risk, and low risk. The guidelines permit physicians MIlow risk while deferring the resumption of sexual activity among higher risk patients pending further evaluation.

Journal Article↗
PubMedDate not supplied

Dynamic left ventricular outflow obstruction: a reversible mechanical complication of acute myocardial infarction.

Dynamic left ventricular outflow obstruction is a less well-known mechanical complication of acute myocardial infarction. Early diagnosis is important because initiation of proper management and avoidance of precipitating factors can lead to complete recovery. A patient is described who presented with acute myocardial infarction and in whom significant left ventricular outflow tract obstruction developed. Adequate therapy with beta blockers and calcium channel blockers led to complete resolution of the obstruction.

Adrenergic beta-Antagonists↗
PubMedDate not supplied

Left ventricular intracavitary mass and pericarditis secondary to metastatic renal cell carcinoma--a case report.

Cardiac metastases from renal cell carcinoma are a well-recognized entity. However, this phenomenon is extremely rare in the absence of vena caval extension. The authors report a patient who after successful resection of renal cell carcinoma presented with left ventricular mass causing left ventricular outflow tract obstruction. There was also metastatic pericardial and intramyocardial involvement. Such a unique combination of cardiac metastasis, in the same patient, has not been reported previously.

Aged↗