10 results on '"Rachofsky E"'
Search Results
2. Dual source computed tomography coronary angiography in new onset cardiomyopathy.
- Author
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Srichai, M.B., Fisch, M., Hecht, E., Slater, J., Rachofsky, E., Hays, A.G., Babb, J., Jacobs, J.E., Srichai, M.B., Fisch, M., Hecht, E., Slater, J., Rachofsky, E., Hays, A.G., Babb, J., and Jacobs, J.E.
- Abstract
Contains fulltext : 110977.pdf (publisher's version ) (Open Access), AIM: To evaluate safety and utility of coronary computed tomography angiography (CCTA) compared to invasive coronary angiography (ICA) in new cardiomyopathy. METHODS: Eighteen patients (mean age 56.5 years, 10 males) who presented for evaluation of new onset heart failure with evidence of systolic dysfunction (ejection fraction < 40%) on echocardiography and recent ICA were prospectively enrolled. Patients with known coronary artery disease, atrial fibrillation, creatinine > 1.5 g/dL, and contraindication to intravenous contrast administration were excluded. CCTA was performed using a dual source 64-slice scanner. Mean heart rate was 75 beats per minute. Stenosis was graded for each coronary segment as: none, mild (< 50%), moderate (50%-70%), severe (> 70%), or non-evaluable. Ischemic cardiomyopathy (ICM) was diagnosed if severe stenosis was present in the left main, proximal left anterior descending artery, or two or more major arteries. RESULTS: Two patients were diagnosed with ICM by ICA. CCTA correctly identified 2 patients with ICM and 16 patients as non-ICM. CCTA successfully evaluated 240/246 coronary segments with an accuracy of 97.5%, sensitivity 70%, specificity 98.7%, positive predictive value of 70%, and negative predictive value of 98.7% for identifying severe stenosis on a per-segment level. CONCLUSION: Dual source 64-slice multi-detector CCTA is a safe, accurate, and non-invasive technique for diagnosing ICM in patients presenting during the acute phase of newly diagnosed cardiomyopathy.
- Published
- 2012
3. CASSCF Investigation of Electronic Excited States of 2-Aminopurine
- Author
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Rachofsky, E. L., Ross, J. B. A., Krauss, M., and Osman, R.
- Abstract
2-Aminopurine is a highly fluorescent analogue of adenine that can be incorporated synthetically into DNA with little perturbation of the native double-helical structure. The sensitive dependence of the quantum yield of this fluorophore on nucleic acid conformation has made it an invaluable probe of DNA structure, dynamics, and interactions. To assist in the development of models for the molecular interpretation of fluorescence measurements, the electronic structure of 2-amino-9-methylpurine has been calculated in the ground state and the lowest singlet ππ* and nπ* excited states. These computations employed the complete active space multiconfigurational self-consistent field method (CASSCF), supplemented by multiconfigurational quasi-degenerate perturbation theory (MCQDPT). The predicted energies for ππ* excitation and emission and nπ* excitation are in good agreement with previous experimental values. The permanent molecular dipoles of the ground and ππ* excited states are similar in magnitude and direction, consistent with experimental observations of weak solvatochromic shifts in ππ* absorption and emission spectra. However, the permanent dipole of the nπ* state is rotated approximately 60° relative to that of the ground state, implying that the nπ* excitation energy will increase in more polar solvents due to the relative destabilization of this state by unfavorably oriented solvent dipoles. This result demonstrates that the blue-shift of the nπ* state in polar solvents, which is commonly attributed to the effect of hydrogen bonding, can arise entirely from a general solvent effect. The energy of a radiationless vibronic transition from the ππ* state to the nπ* state will increase in more polar solvents, provided that the solvent does not rearrange during the transition. Consequently, the efficiency of fluorescence quenching by vibronic coupling between the ππ* and nπ* states is predicted to decrease significantly in such solvents. The geometry of the fluorescent emitting state, obtained by CASSCF optimization of the ππ* state, is moderately buckled due to the occupation of an antibonding orbital localized to C6. This buckling implies an out-of-plane vibration during the relaxation of the ππ* state, which is required for vibronic coupling between this state and the nπ* state. Such a solvent-sensitive intramolecular quenching mechanism may account for the observed dependence of the fluorescence lifetime of 2-aminopurine on the local environment both in pure solvents and in DNA.
- Published
- 2001
4. Primary Cardiac Sarcoma Involving the Mitral Valve, an Insidious Form of Heart Failure.
- Author
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Prol T, Petro J, Jain H, Raja S, Rachofsky E, Koulogiannis KP, and Horgan S
- Published
- 2020
- Full Text
- View/download PDF
5. Dual source computed tomography coronary angiography in new onset cardiomyopathy.
- Author
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Srichai MB, Fisch M, Hecht E, Slater J, Rachofsky E, Hays AG, Babb J, and Jacobs JE
- Abstract
Aim: To evaluate safety and utility of coronary computed tomography angiography (CCTA) compared to invasive coronary angiography (ICA) in new cardiomyopathy., Methods: Eighteen patients (mean age 56.5 years, 10 males) who presented for evaluation of new onset heart failure with evidence of systolic dysfunction (ejection fraction < 40%) on echocardiography and recent ICA were prospectively enrolled. Patients with known coronary artery disease, atrial fibrillation, creatinine > 1.5 g/dL, and contraindication to intravenous contrast administration were excluded. CCTA was performed using a dual source 64-slice scanner. Mean heart rate was 75 beats per minute. Stenosis was graded for each coronary segment as: none, mild (< 50%), moderate (50%-70%), severe (> 70%), or non-evaluable. Ischemic cardiomyopathy (ICM) was diagnosed if severe stenosis was present in the left main, proximal left anterior descending artery, or two or more major arteries., Results: Two patients were diagnosed with ICM by ICA. CCTA correctly identified 2 patients with ICM and 16 patients as non-ICM. CCTA successfully evaluated 240/246 coronary segments with an accuracy of 97.5%, sensitivity 70%, specificity 98.7%, positive predictive value of 70%, and negative predictive value of 98.7% for identifying severe stenosis on a per-segment level., Conclusion: Dual source 64-slice multi-detector CCTA is a safe, accurate, and non-invasive technique for diagnosing ICM in patients presenting during the acute phase of newly diagnosed cardiomyopathy.
- Published
- 2012
- Full Text
- View/download PDF
6. Conformation and dynamics of normal and damaged DNA.
- Author
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Rachofsky EL, Ross JB, and Osman R
- Subjects
- Base Pairing, Calcium chemistry, Calcium metabolism, DNA metabolism, DNA Damage, Fluorescence, Hydrogen Bonding, Kinetics, Nucleic Acid Conformation, Potassium Iodide chemistry, Proteins chemistry, Proteins metabolism, Solvents chemistry, Spectrometry, Fluorescence, Thermodynamics, Titrimetry methods, 2-Aminopurine chemistry, DNA chemistry, Models, Molecular
- Abstract
The genetic information that determines the structure and function of living organisms is encoded in the nucleotide sequence of double-stranded DNA molecules. Despite an apparent structural homogeneity displayed by DNA, subtle local variations in structure and dynamics are functionally significant. Short sequences exhibit specificity for regulatory and catalytic proteins, which mediate fundamental processes necessary to the survival of the cell. However, the molecular basis for specific recognition is still incompletely understood. The "indirect readout" mechanism suggests that the relative propensity of DNA to undergo structural deformations induced by the protein contributes to specific protein-DNA recognition. Although the hypothesis was originally formulated to explain recognition of specific nucleic acid sequences by DNA-binding proteins, it may have particular application to the recognition of DNA damage, because damaged sites in DNA have different equilibrium structure and dynamics from undamaged DNA. In this work, we review the approaches that we took to investigate the questions of sequence- and damage-dependent structure and dynamics of DNA. We describe a statistical thermodynamic model that relates DNA configurational flexibility to sequence-specific protein-DNA binding. The model provides a theoretical basis for interpreting experimental measurements of DNA dynamics. We describe results from MCSCF calculations of the excited states of 2-aminopurine (2AP), which provide the theoretical basis for the intramolecular mechanism of quenching as well as the effect of environment on this process. We then describe our investigations of the effect of stacking, base pairing, and base dynamics on the fluorescence of 2-AP in model systems, which allow us to develop the relationships between steady-state and time-resolved fluorescence parameters on the one hand and local structural and dynamic properties of DNA on the other hand. Finally, we describe the application of the experimental approach to study the conformational heterogeneity of DNA abasic sites, a commonly occurring type of DNA damage. We demonstrate the power of the experimental algorithm to characterize the physical differences between undamaged and damaged DNA, as well as the effects of nucleic acid sequence in both of these contexts. Thus, the work described herein comprises a combination of theoretical and experimental approaches to the problem of sequence- and damage-dependent DNA deformation.
- Published
- 2001
- Full Text
- View/download PDF
7. Conformation and dynamics of abasic sites in DNA investigated by time-resolved fluorescence of 2-aminopurine.
- Author
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Rachofsky EL, Seibert E, Stivers JT, Osman R, and Ross JB
- Subjects
- Calcium chemistry, Carbon-Oxygen Lyases chemistry, Cations, Divalent chemistry, DNA Damage, DNA-(Apurinic or Apyrimidinic Site) Lyase, Deoxyribonuclease IV (Phage T4-Induced), Escherichia coli enzymology, Kinetics, N-Glycosyl Hydrolases chemistry, Oligonucleotides chemistry, Solutions, Spectrometry, Fluorescence methods, Thermodynamics, Titrimetry, Uracil-DNA Glycosidase, 2-Aminopurine chemistry, DNA chemistry, DNA Glycosylases, DNA Repair, Escherichia coli Proteins, Nucleic Acid Conformation
- Abstract
Abasic sites are highly mutagenic lesions in DNA that arise as intermediates in the excision repair of modified bases. These sites are generated by the action of damage-specific DNA glycosylases and are converted into downstream intermediates by the specific activity of apurinic/apyrimidinic endonucleases. Enzymes in both families have been observed in crystal structures to impose deformations on the abasic-site DNA, including DNA kinking and base flipping. On the basis of these apparent protein-induced deformations, we propose that altered conformation and dynamics of abasic sites may contribute to the specificity of these repair enzymes. Previously, measurements of the steady-state fluorescence of the adenine analogue 2-aminopurine (2AP) opposite an abasic site demonstrated that binding of divalent cations could induce a conformational change that increased the accessibility of 2AP to solute quenching [Stivers, J. T. (1998) Nucleic Acids Res. 26, 3837-44]. We have performed time-resolved fluorescence experiments to characterize the states involved in this conformational change. Interpretation of these studies is based on a recently developed model attributing the static and dynamic fluorescence quenching of 2AP in DNA to aromatic stacking and collisional interactions with neighboring bases, respectively (see the preceding paper in this issue). The time-resolved fluorescence results indicate that divalent cation binding shifts the equilibrium of the abasic site between two conformations: a "closed" state, characterized by short average fluorescence lifetime and complex decay kinetics, and an "open" state, characterized by monoexponential decay with lifetime approximately that of the free nucleoside. Because the lifetime and intensity decay kinetics of 2AP incorporated into DNA are sensitive primarily to collisional interactions with the neighboring bases, the absence of dynamic quenching in the open state strongly suggests that the fluorescent base is extrahelical in this conformation. Consistent with this interpretation, time-resolved quenching studies reveal that the open state is accessible to solute quenching by potassium iodide, but the closed state is not. Greater static quenching is observed in the abasic site when the fluorescent base is flanked by 5'- and 3'-thymines than in the context of 5'- and 3'-adenines, indicating that 2AP is more stacked with the neighboring bases in the former sequence. These results imply that the conformation of the abasic site varies in a sequence-dependent manner. Undamaged sequences in which the abasic site is replaced by thymine do not exhibit an open state and have different levels of both static and dynamic quenching than their damaged homologues. These differences in structure and dynamics may be significant determinants of the high specific affinity of repair enzymes for the abasic site.
- Published
- 2001
- Full Text
- View/download PDF
8. Probing structure and dynamics of DNA with 2-aminopurine: effects of local environment on fluorescence.
- Author
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Rachofsky EL, Osman R, and Ross JB
- Subjects
- Adenosine chemistry, Adenosine Triphosphate chemistry, Antimetabolites chemistry, Cytidine chemistry, Guanosine chemistry, Guanosine Triphosphate chemistry, Hydrogen Bonding, Kinetics, Models, Chemical, Quantum Theory, Solutions, Solvents, Structure-Activity Relationship, Thermodynamics, Thymidine chemistry, Titrimetry, 2-Aminopurine chemistry, DNA chemistry, Spectrometry, Fluorescence methods
- Abstract
2-Aminopurine (2AP) is an analogue of adenine that has been utilized widely as a fluorescence probe of protein-induced local conformational changes in DNA. Within a DNA strand, this fluorophore demonstrates characteristic decreases in quantum yield and emission decay lifetime that vary sensitively with base sequence, temperature, and helix conformation but that are accompanied by only small changes in emission wavelength. However, the molecular interactions that give rise to these spectroscopic changes have not been established. To develop a molecular model for interpreting the fluorescence measurements, we have investigated the effects of environmental polarity, hydrogen bonding, and the purine and pyrimidine bases of DNA on the emission energy, quantum yield, and intensity decay kinetics of 2AP in simple model systems. The effects of environmental polarity were examined in a series of solvents of varying dielectric constant, and hydrogen bonding was investigated in binary mixtures of water with 1,4-dioxane or N,N-dimethylformamide (DMF). The effects of the purine and pyrimidine bases were studied by titrating 2AP deoxyriboside (d2AP) with the nucleosides adenosine (rA), cytidine (rC), guanosine (rG), and deoxythymidine (dT), and the nucleoside triphosphates ATP and GTP in neutral aqueous solution. The nucleosides and NTPs each quench the fluorescence of d2AP by a combination of static (affecting only the quantum yield) and dynamic (affecting both the quantum yield and the lifetime, proportionately) mechanisms. The peak wavelength and shape of the emission spectrum are not altered by either of these effects. The static quenching is saturable and has half-maximal effect at approximately 20 mM nucleoside or NTP, consistent with an aromatic stacking interaction. The rate constant for dynamic quenching is near the diffusion limit for collisional interaction (k(q) approximately 2 x 10(9) M(-1) s(-1)). Neither of these effects varies significantly between the various nucleosides and NTPs studied. In contrast, hydrogen bonding with water was observed to have a negligible effect on the emission wavelength, fluorescence quantum yield, or lifetime of 2AP in either dioxane or DMF. In nonpolar solvents, the fluorescence lifetime and quantum yield decrease dramatically, accompanied by significant shifts in the emission spectrum to shorter wavelengths. However, these effects of polarity do not coincide with the observed emission wavelength-independent quenching of 2AP fluorescence in DNA. Therefore, we conclude that the fluorescence quenching of 2AP in DNA arises from base stacking and collisions with neighboring bases only but is insensitive to base-pairing or other hydrogen bonding interactions. These results implicate both structural and dynamic properties of DNA in quenching of 2AP and constitute a simple model within which the fluorescence changes induced by protein-DNA binding or other perturbations may be interpreted.
- Published
- 2001
- Full Text
- View/download PDF
9. Kinetic models and data analysis methods for fluorescence anisotropy decay.
- Author
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Rachofsky EL and Laws WR
- Subjects
- Algorithms, Least-Squares Analysis, Models, Statistical, Time Factors, Fluorescence Polarization methods, Kinetics, Statistics as Topic
- Published
- 2000
- Full Text
- View/download PDF
10. Dynamics of biomolecules: assignment of local motions by fluorescence anisotropy decay.
- Author
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Bialik CN, Wolf B, Rachofsky EL, Ross JB, and Laws WR
- Subjects
- Algorithms, Animals, Computer Simulation, Horses, Kinetics, Liver enzymology, Macromolecular Substances, Monte Carlo Method, Spectrometry, Fluorescence, Tryptophan chemistry, Alcohol Dehydrogenase chemistry, Fluorescence Polarization
- Abstract
Many biological systems have multiple fluorophores that experience multiple depolarizing motions, requiring multiple lifetimes and correlation times to define the fluorescence intensity and anisotropy decays, respectively. To simplify analyses, an assumption often made is that all fluorophores experience all depolarizing motions. However, this assumption usually is invalid, because each lifetime is not necessarily associated with each correlation time. To help establish the correct associations and recover accurate kinetic parameters, a general kinetic scheme that can examine all possible associations is presented. Using synthetic data sets, the ability of the scheme to discriminate among all nine association models possible for two lifetimes and two correlation times has been evaluated. Correct determination of the association model, and accurate recovery of the decay parameters, required the global analysis of related data sets. This general kinetic scheme was then used for global analyses of liver alcohol dehydrogenase anisotropy data sets. The results indicate that only one of the two tryptophan residues in each subunit is depolarized by process(es) independent of the enzyme's rotations. By applying the proper kinetic scheme and appropriate analysis procedures to time-resolved fluorescence anisotropy data, it is therefore possible to examine the dynamics of specific portions of a macromolecule in solution.
- Published
- 1998
- Full Text
- View/download PDF
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