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Biomedical subjects

Stefan Fischer

Publications and source records attributed to Stefan Fischer.

At least 37 records · Page 2Linked to original sources
PubMed2005

Tuning of retinal twisting in bacteriorhodopsin controls the directionality of the early photocycle steps.

Productive proton pumping by bacteriorhodopsin requires that, after the all-trans to 13-cis photoisomerization of the retinal chromophore, the photocycle proceeds with proton transfer and not with thermal back-isomerization. The question of how the protein controls these events in the active site is addressed here using quantum mechanical/molecular mechanical reaction-path calculations. The results indicate that, while retinal twisting significantly contributes to lowering the barrier for the thermal cis-trans back-isomerization, the rate-limiting barrier for this isomerization is still 5-6 kcal/mol larger than that for the first proton-transfer step. In this way, the retinal twisting is finely tuned so as to store energy to drive the subsequent photocycle while preventing wasteful back-isomerization.

Bacteriorhodopsins↗
PubMed2005

Protein/ligand binding free energies calculated with quantum mechanics/molecular mechanics.

The calculation of binding affinities for flexible ligands has hitherto required the availability of reliable molecular mechanics parameters for the ligands, a restriction that can in principle be lifted by using a mixed quantum mechanics/molecular mechanics (QM/MM) representation in which the ligand is treated quantum mechanically. The feasibility of this approach is evaluated here, combining QM/MM with the Poisson-Boltzmann/surface area model of continuum solvation and testing the method on a set of 47 benzamidine derivatives binding to trypsin. The experimental range of the absolute binding energy (DeltaG = -3.9 to -7.6 kcal/mol) is reproduced well, with a root-mean-square (RMS) error of 1.2 kcal/mol. When QM/MM is applied without reoptimization to the very different ligands of FK506 binding protein the RMS error is only 0.7 kcal/mol. The results show that QM/MM is a promising new avenue for automated docking and scoring of flexible ligands. Suggestions are made for further improvements in accuracy.

Ligands↗
PubMed2005

Automated computation of low-energy pathways for complex rearrangements in proteins: application to the conformational switch of Ras p21.

The computation of minimum energy paths (MEPs) is an approach for gaining insight into protein conformational transitions that are too slow to be observed with unconstrained molecular dynamics simulations. MEPs have the advantage of providing the energy barrier of the rate-limiting step(s), allowing discrimination among different paths. Finding low-energy MEPs for complex transitions, such as those involving rearrangements of the backbone fold or repacking of buried side chains, has hitherto been unfeasible in a reliable, automated manner, the MEP often displaying unphysical behavior, such as the crossing of bonds. Here, this problem is addressed by combining a counterintuitive procedure for generating an initial guess of the path, in which all side chains are shrunk, with the conjugate peak refinement (CPR) method. The effectiveness of the approach is tested on the conformational switch in Ras p21. This conformational transition involves some partial unfolding and re-folding, a process for which a multitude of pathways are likely to exist and for which a single MEP does not provide a complete description. However, this transition requires some sterically demanding rearrangements, thus testing the ability of a method to find low-energy pathways free of structurally unphysical events. This is achieved by the present approach, which finds a path whose rate-limiting barrier is compatible with experiment. This demonstrates that the method can be used to compute plausible pathways for complex rearrangements in proteins in an automated manner that is unbiased by external driving constraints.

Automation↗
PubMed2005

Structural mechanism of the recovery stroke in the myosin molecular motor.

The power stroke pulling myosin along actin filaments during muscle contraction is achieved by a large rotation ( approximately 60 degrees ) of the myosin lever arm after ATP hydrolysis. Upon binding the next ATP, myosin dissociates from actin, but its ATPase site is still partially open and catalytically off. Myosin must then close and activate its ATPase site while returning the lever arm for the next power stroke. A mechanism for this coupling between the ATPase site and the distant lever arm is determined here by generating a continuous series of optimized intermediates between the crystallographic end-states of the recovery stroke. This yields a detailed structural model for communication between the catalytic and the force-generating regions that is consistent with experimental observations. The coupling is achieved by an amplifying cascade of conformational changes along the relay helix lying between the ATPase and the domain carrying the lever arm.

Actins↗
PubMed2005

Heterocyclic alpha-alkylidene cyclopentenones obtained via a Pauson-Khand reaction of amino acid derived allenynes. A scope and limitation study directed toward the preparation of a tricyclic pyrrole library.

The synthesis of a novel class of tricyclic pyrroles has been accomplished by using a Pauson-Khand/Stetter/Paal-Knorr reaction sequence. Full details of the Pauson-Khand reaction of amino acid tethered allenynes 4a-e and 9a-d are disclosed. The study of this reaction led to the discovery of an unprecedented substituent effect on the diastereoselectivity of the Mo(CO)6 mediated allenic Pauson-Khand reaction. It was found that amino acid tethered allenynes with aromatic side chains afford alpha-alkylidene cyclopentenones with the opposite diastereoselectivity compared to those with aliphatic side chains. This effect has been attributed to complexation of the metal mediator to the aromatic ring in the substrate. Furthermore, an isomerization of one of the diastereomers of the alpha-alkylidene cyclopentenones was encountered, leading to eventual decomposition. The stable diastereomers were found to react well in the Stetter reaction leading to 1,4-diketones that were converted to pyrroles. The observation that the first generation of 2-alkyl-substituted pyrroles was unstable led to a second generation of 2-carboxamide pyrroles with sufficient stability for biological tests which are in progress.

Alkynes↗
PubMed2005

Protein stability and ligand binding: new paradigms from in-silico experiments.

Computer simulations are used to investigate two features of proteins: ligand binding and ligand entry/exit. Both reveal surprising new insights into the physics of such complex systems and suggest at possible interpretations that depart from the usual paradigms. A ligand binding study using normal mode analysis suggests that, contrary to the perceived notion that ligand binding induces a tightening of the protein (as would be evidenced by a blue shift in its vibrational spectrum), there seem to be cases where ligand binding causes an increase in the entropy through a red-shift in the vibrational spectrum of the protein; this occurs in the part of the spectrum that is associated with large-scale low-frequency delocalized motions of proteins. Moreover, this increase seems to be dependent on the ability of the ligand to form hydrogen bonds within the polar cavity of the protein. This suggests an additional driving force for stabilizing complex formation. In parallel, pathways of ligand access to cavities in two proteins are mapped and it is found that, in agreement with recent interpretations of experimental data emerging from NMR studies, these pathways are characterized by a ruggedness of the energy landscape, which leads to a picture that has a physically more appealing basis than the traditional two-state paradigm normally invoked for ligand binding.

Animals↗
PubMed2005

Nonuniform charge scaling (NUCS): a practical approximation of solvent electrostatic screening in proteins.

In molecular mechanics calculations, electrostatic interactions between chemical groups are usually represented by a Coulomb potential between the partial atomic charges of the groups. In aqueous solution these interactions are modified by the polarizable solvent. Although the electrostatic effects of the polarized solvent on the protein are well described by the Poisson--Boltzmann equation, its numerical solution is computationally expensive for large molecules such as proteins. The procedure of nonuniform charge scaling (NUCS) is a pragmatic approach to implicit solvation that approximates the solvent screening effect by individually scaling the partial charges on the explicit atoms of the macromolecule so as to reproduce electrostatic interaction energies obtained from an initial Poisson--Boltzmann analysis. Once the screening factors have been determined for a protein the scaled charges can be easily used in any molecular mechanics program that implements a Coulomb term. The approach is particularly suitable for minimization-based simulations, such as normal mode analysis, certain conformational reaction path or ligand binding techniques for which bulk solvent cannot be included explicitly, and for combined quantum mechanical/molecular mechanical calculations when the interface to more elaborate continuum solvent models is lacking. The method is illustrated using reaction path calculations of the Tyr 35 ring flip in the bovine pancreatic trypsin inhibitor.

Algorithms↗
PubMed2005

Ex vivo repair and renal autotransplantation for complex renal artery aneurysms in a solitary kidney.

Renal artery aneurysm (RAA) is a rare clinical entity with an incidence of 0.015-1%. Indications for interventional or surgical repair of RAAs are expanding aneurysms, diameter >2.5 cm, intractable renovascular hypertension, dissecting RAA, hematuria, and renal infarction after distal embolization. Interventional insertion of a stent graft as well as aortorenal bypass implantation are both low-risk procedures in simple aneurysms of the proximal renal artery. However, complex distal renal aneurysms involving several renal artery branches require not only an excellent result of vascular reconstruction, but also a surgical technique offering maximal protection for the kidney during the ischemic period. Here, we present a case of a solitary kidney with two consecutive RAAs of segmental renal artery branches (type 2 RAA). A surgical strategy including renal explantation, ex vivo renal preservation, ex vivo reconstruction of the renal artery, and renal heterotopic autotransplantation was successfully applied. The technique of ex vivo repair is a safe and effective surgical procedure in this clinical setting.

Aneurysm↗
PubMed2005

Iloprost to improve surfactant function in porcine pulmonary grafts stored for twenty-four hours in low-potassium dextran solution.

OBJECTIVES: The optimal strategy for pulmonary graft preservation remains elusive. Experimental work and initial clinical experience support low-potassium dextran solutions as lung perfusates. We have previously shown a protective effect of prostaglandin E 1 on ischemia-reperfusion injury in lung transplantation by a shift from proinflammatory to anti-inflammatory cytokines in a rat lung transplantation model. In this study, we tested the hypothesis that the addition of a prostacyclin analog (iloprost) to low-potassium dextran might lead to improved surfactant and ultimately graft function. METHODS: In a randomized, blinded study with a porcine left single-lung transplantation model, donor lungs were flushed with 1 L of either low-potassium dextran solution or low-potassium dextran solution modified by the addition of 250 microg iloprost (n = 6 in each group). Grafts were stored at 4 degrees C for 24 hours. After transplantation, the right bronchus and pulmonary artery were clamped, and the animals remained dependent on the graft. Posttransplantation graft function was assessed throughout a 7-hour observation period by measuring oxygenation (30-minute intervals), different pulmonary and systemic hemodynamic parameters, and wet/dry lung weight ratios. Bronchoalveolar lavage fluid was obtained before and 2 hours after reperfusion. Surfactant function was measured from bronchoalveolar lavage fluid with a pulsating bubble surfactometer. Neutrophil sequestration was assessed by a myeloperoxidase assay performed on lung tissue specimens taken at the end of the observation period. RESULTS: Pulmonary vascular resistance remained lower in the iloprost group than in the control group (P < .05). Tissue water content after 7 hours of reperfusion remained lower in the iloprost group (P < .05). In addition, significantly reduced myeloperoxidase tissue activity was observed in the iloprost group (P < .05). Although there was no difference in degradation of surface active surfactant large aggregates to small aggregates, the surface tension measured at minimal bubble diameter was lower in the iloprost group (P < .05). CONCLUSIONS: Modification of low-potassium dextran solution with the prostacyclin analog iloprost resulted in a significant amelioration of ischemia-reperfusion injury and improved preservation of surfactant function in transplanted lungs. This intriguing approach merits further evaluation with respect to the mechanisms involved and, ultimately, potential introduction into clinical lung transplantation.

Animals↗
PubMed2005

Lung transplantation with lungs from donors fifty years of age and older.

BACKGROUND: A shortage of donors has led to the progressive expansion of criteria for donor selection in lung transplantation. The outcome of recipients of lungs from donors aged 50 years or older is analyzed systematically. METHODS: From March 1998 to June 2003, 49 recipients received lungs from donors aged 50 years or older (range 50-64 years, mean 54 +/- 3 years). This group of recipients was compared with 244 patients receiving lungs from donors aged less than 50 years (range 7-49 years, mean 32 +/- 11 years). This study was undertaken on all 293 patients at our institution who received Perfadex-preserved lungs (Vitrolife, Goteborg, Sweden). RESULTS: Recipient age, sex, and indications for transplant did not differ significantly between groups. Also, the percentage of the different types of transplants (bilateral or single lung transplantation) performed was equal in both cohorts. Donor Pa(O2) /F(IO2) ratios before lung retrieval (415 +/- 91 vs 439 +/- 113, respectively) and length of ischemic time (347 +/- 67 minutes vs 351 +/- 84 minutes, respectively) did not differ significantly between the older and younger donor groups. The following posttransplant parameters were also not statistically different: first Pa(O2)/F(IO2) at intensive care unit arrival (274 +/- 125 in the older donor group vs 253 +/- 119 in the younger donor group, respectively), mechanical ventilation time (328 +/- 427 hours vs 269 +/- 425 hours, respectively), and length of stay in the intensive care unit (16 +/- 18 days vs 14 +/- 18 days, respectively). Recipient survival in the older and younger donor groups at 30 days, 3, 6, 12, 24, and 60 months was 77% +/- 6%, 75% +/- 6%, 73% +/- 7%, 73% +/- 7%, 68% +/- 5%, and 68% +/- 4% versus 86% +/- 2%, 83% +/- 3%, 80% +/- 3%, 78% +/- 3%, 71% +/- 4%, and 66% +/- 4%, respectively. CONCLUSIONS: Lung grafts from elderly donors have been considered as marginal organs for transplantation. However, this study indicates that transplantation of lungs from carefully selected donors aged 50 years or more may lead to similar short- and long-term outcomes compared with lungs from younger donors. The use of lungs from elderly donors may help to increase the number of donor organs in lung transplantation.

Adolescent↗
PubMed2005

Glutathione improves the function of porcine pulmonary grafts stored for twenty-four hours in low-potassium dextran solution.

BACKGROUND: Flush perfusion with low-potassium dextran is the standard strategy in clinical lung preservation. Despite improved outcome, endothelial cell injury and surfactant dysfunction remain a significant problem after lung transplantation. The radical scavenger glutathione has been shown to be responsible for the efficacy of Celsior solution in lung preservation. We tested the hypothesis that the addition of glutathione to low-potassium dextran might further improve graft function by ameliorating ischemia-reperfusion injury. METHODS: In 12 domestic pigs, lungs were flush preserved with either low-potassium dextran (n = 6) or low-potassium dextran supplemented by 5 mmol glutathione (n = 6). Left single lung transplantation was performed after 24-hour storage in low-potassium dextran at 8 degrees C. After 15 minutes of reperfusion the right main bronchus and pulmonary artery were crossclamped. Hemodynamic and respiratory measures were recorded in 30-minute intervals for a total observation period of 7 hours. Bronchoalveolar lavage fluid was obtained from the native lung and 2 hours after reperfusion from the graft. Bronchoalveolar lavage fluid and surfactant composition, and surfactant function analyses were performed. Neutrophil sequestration was assessed by myeloperoxidase activity assay. Tissue water content was calculated from wet/dry weight ratios at the end of the experiment. RESULTS: In the low-potassium dextran group, 2 animals died during reperfusion. After reperfusion, pulmonary vascular resistance (P = .01) and pulmonary artery pressure remained lower in the glutathione/low-potassium dextran group, which was associated with a higher cardiac output (P = .05) in this group. Also, the oxygenation index at the end of the observation period was higher in the glutathione/low-potassium dextran group compared with the low-potassium dextran group (430 +/- 130 vs 338 +/- 184, respectively; P < .05). The graft water content representing postreperfusion lung edema was not different between the 2 study groups. Alteration of surfactant was less in the glutathione/low-potassium dextran group with a significantly decreased small to large aggregate ratio (P = .03) versus low-potassium dextran group. Myeloperoxidase activity was twice as high in the low-potassium dextran group when compared with the glutathione/low-potassium dextran group (glutathione/low-potassium dextran: 134 +/- 110 mU/g vs low-potassium dextran: 274 +/- 168 mU/g, P = .07). CONCLUSION: The addition of glutathione to low-potassium dextran preservation solution reveals beneficial effects on vascular function and surfactant composition in transplanted lungs. Therefore, glutathione ameliorates ischemia-reperfusion injury in a preclinical model of lung transplantation. Future studies are needed to evaluate this promising modification in clinical lung transplantation.

Animals↗
PubMed2005

Mechanism of a molecular valve in the halorhodopsin chloride pump.

Halorhodopsin is a light-driven chloride anion pump in which the trans-->cis photoisomerization of a retinal chromophore triggers a photocycle resulting in the translocation of chloride across the plasma membrane. The mechanism of chloride transfer past the cis retinal is determined here by computing multiple pathways for this process. The calculations reveal two conditions of the valve mechanism. First, a lumen absent in the ground state structure is transiently opened by chloride passage. Second, this activated opening, which is achieved by flexible deformation of the surrounding protein, is shown to significantly raise the chloride translocation barrier between photocycles, thus preventing chloride backflow. Unlike macroscopic valve designs, the protein allows differential ion flows in the pumping and resting states that are tuned to match the physiological timescales of the cell, thus creating a "kinetic" valve.

Chlorides↗
PubMed2004

Key role of electrostatic interactions in bacteriorhodopsin proton transfer.

The first proton transport step following photon absorption in bacteriorhodopsin is from the 13-cis retinal Schiff base to Asp85. Configurational and energetic determinants of this step are investigated here by performing quantum mechanical/molecular mechanical minimum-energy reaction-path calculations. The results suggest that retinal can pump protons when in the 13-cis, 15-anti conformation but not when 13-cis, 15-syn. Decomposition of the proton transfer energy profiles for various possible pathways reveals a conflict between the effect of the intrinsic proton affinities of the Schiff base and Asp85, which favors the neutral, product state (i.e., with Asp85 protonated), with the mainly electrostatic interaction between the protein environment with the reacting partners, which favors the ion pair reactant state (i.e., with retinal protonated). The rate-limiting proton-transfer barrier depends both on the relative orientations of the proton donor and acceptor groups and on the pathway followed by the proton; depending on these factors, the barrier may arise from breaking and forming of hydrogen bonds involving the Schiff base, Asp85, Asp212, and water w402, and from nonbonded interactions involving protein groups that respond to the charge rearrangements in the Schiff base region.

Bacteriorhodopsins↗
PubMed2004

Structure, dynamics and reactions of protein hydration water.

The apparent simplicity of the water molecule belies the wide range of fascinating protein phenomena in which it participates. We review recent computer simulation work on buried, internal water molecules, discussing the thermodynamics of water molecule binding and the participation of water in proton transfer reactions. Surface water molecules are also considered, with emphasis on the modification of average solvent structure on a protein surface, the role of water in the protein dynamical 'glass' transition and a simplified description of the protein motions thereby activated.

Computer Simulation↗
PubMed2004

Replacement of the aortic root for acute prosthetic valve endocarditis: prosthetic composite versus aortic allograft root replacement.

OBJECTIVE: Aortic root replacement for prosthetic aortic valve endocarditis with accompanying destruction of the aortic root is a well-established surgical intervention. However, there is still no consensus whether prosthetic material or allogeneic material should be used. Here we report on our experience with prosthetic composite and aortic allograft root replacement in such patients during a 10-year interval. METHODS: From 1991 through 2001, 29 patients with prosthetic aortic valve endocarditis combined with aortic root destruction underwent reoperation at our institution. Sixteen patients received aortic root replacement with a cryopreserved aortic root allograft (group A) and 13 with a prosthetic composite graft (group B). The interval between the initial operation and reoperation was 29 months (range, 5-168 months) in group A and 55 months (range, 7-248 months) in group B. RESULTS: Hospital mortality was 18.5% (n = 5 patients, 3 in group A and 2 in group B). Median follow-up was 21 months (range, 1-48 months) for group A and 34 months (range, 1-152 months) for group B (P >.2). Survival at 1 and 5 years was 81% +/- 10% and 81% +/- 10% in group A and 85% +/- 10% and 85% +/- 10% in group B, respectively. No patient underwent reoperation for recurrent prosthetic aortic valve endocarditis. CONCLUSIONS: Our results indicate that excellent long-term results can be achieved regardless of the material used for aortic root replacement in patients with prosthetic aortic valve endocarditis.

Acute Disease↗
PubMed2004

Mechanism of primary proton transfer in bacteriorhodopsin.

Recent structures of putative intermediates in the bacteriorhodopsin photocycle have provided valuable snapshots of the mechanism by which protons are pumped across the membrane. However, key steps remain highly controversial, particularly the proton transfer occurring immediately after retinal trans-->cis photoisomerization. The gradual release of stored energy is inherently nonequilibrium: which photocycle intermediates are populated depends not only on their energy but also on their interconversion rates. To understand why the photocycle follows a productive (i.e., pumping), rather than some unproductive, relaxation pathway, it is necessary to know the relative energy barriers of individual steps. To discriminate between the many proposed scenarios of this process, we computed all its possible minimum-energy paths. This reveals that not one, but three very different pathways have energy barriers consistent with experiment. This result reconciles the conflicting views held on the mechanism and suggests a strategy by which the protein renders this essential step resilient.

Bacteriorhodopsins↗
PubMed2004

Risk quantification of early outcome after lung transplantation: donor, recipient, operative, and post-transplant parameters.

BACKGROUND: Because there is no reliable evaluation system of recipient acuity after lung transplantation, comparing patients among centers is difficult. The purpose of our study was to identify risk factors for 30-day mortality and prolonged intensive care unit stay and to develop a scoring system to evaluate the severity of impairment and to predict surgical outcomes. METHODS: We prospectively collected data from 122 lung transplant recipients and from 119 donors from January 1997 to June 2000. We assessed donor, recipient, and operative factors; ischemic time; and immediate post-operative physiologic parameters to identify risk factors for 30-day mortality and prolonged intensive care unit stay. Furthermore, we sub-classified these factors into grades to develop a scoring system for predicting surgical outcomes. RESULTS: Cardiopulmonary bypass use, body mass index >25 kg/m2, immediate post-operative systolic pulmonary arterial pressure, trend of oxygenation index from 12 to 24 hours after transplantation, and the Acute Physiology and Chronic Health Evaluation II score correlated significantly with outcomes, and the sum of these 5 scores correlated strongly with outcomes (p < 0.0001). CONCLUSIONS: We conclude that the total score of these 5 risk factors could be used to predict 30-day mortality and prolonged intensive care unit stay. This scoring system also will facilitate standardization among transplant centers in evaluating post-transplant severity of illness.

Female↗