Biomedical subjects

F X Schmid

Publications and source records attributed to F X Schmid.

At least 73 records · Page 4Linked to original sources

PubMed1996

Kinetic analysis of the unfolding and refolding of ribonuclease T1 by a stopped-flow double-mixing technique.

Often protein folding reactions show complex kinetics, because multiple unfolded species are present, which refold simultaneously. After conformational unfolding, these species are formed by the slow cis/trans equilibrations at Xaa-Pro peptide bonds. To dissect the roles of individual prolines for unfolding and refolding, we used ribonuclease T1, a protein with two cis prolyl peptide bonds, preceding Pro39 and Pro55, and two variants with substitutions at these positions. A stopped-flow double-mixing technique was employed (i) to measure the rates of the individual prolyl isomerizations in the unfolded proteins and (ii) to study the refolding of transient species that are not well populated at equilibrium. In particular, the elusive species with correct prolyl isomers could be produced by short unfolding pulses, and its refolding kinetics could be measured. The two isomerizations in unfolded ribonuclease T1 could be assigned to Pro39 and Pro55, because they occurred with almost identical rates in the wild-type protein, in the single-cis proline variants, and in tetrapeptide-4-nitroanilides, which contained prolines in the same sequential context at Pro39 and Pro55 or ribonuclease T1. The direct refolding reaction of the unfolded molecules with correct prolyl isomers shows a time constant of 180 ms (at 25 degrees C, pH 4.6). This reaction is almost unaffected by the proline substitutions. It depends nonlinearly on temperature with a maximum near 25 degrees C, which suggest that the activated state for this reaction resembles the native rather than the unfolded state in heat capacity. The formation of a transient intermediate with incorrect prolyl isomers could be studied as well. Surprisingly, this reaction is only about 5-fold slower than direct folding, and it is also accompanied by a strong decrease in the apparent heat capacity.

Isomerism↗

PubMed1996

Preferential binding of an unfolded protein to DsbA.

The oxidoreductase DsbA from the periplasm of escherichia coli introduces disulfide bonds into proteins at an extremely high rate. During oxidation, a mixed disulfide is formed between DsbA and the folding protein chain, and this covalent intermediate reacts very rapidly either to form the oxidized protein or to revert back to oxidized DsbA. To investigate its properties, a stable form of the intermediate was produced by reacting the C33A variant of DsbA with a variant of RNase T1. We find that in this stable mixed disulfide the conformational stability of the substrate protein is decreased by 5 kJ/mol, whereas the conformational stability of DsbA is increased by 5 kJ/mol. This reciprocal effect suggests strongly that DsbA interacts with the unfolded substrate protein not only by the covalent disulfide bond, but also by preferential non-covalent interactions. The existence of a polypeptide binding site explains why DsbA oxidizes protein substrates much more rapidly than small thiol compounds. Such a very fast reaction is probably important for protein folding in the periplasm, because the accessibility of the thiol groups for DsbA can decrease rapidly when newly exported polypeptide chains begin to fold.

Disulfides↗

PubMed1996

[Thromboendarterectomy in chronic thromboembolic pulmonary hypertension. Hemodynamics and right-heart function over the long term].

OBJECTIVE: To find out whether pulmonary thromboendarterectomy (PTE) can achieve lasting reduction of pulmonary vascular resistance in patients with pulmonary arterial hypertension due to chronic thromboembolism. PATIENTS AND METHODS: 45 patients (25 women, 20 men; mean age 45 +/- 24 [19-67] years) were re-investigated a mean of 21 (13-32) months after successful PTE. Two patients had then been in New York Heart Association (NYHA) stage II, 26 in stage III, and 17 in stage IV. In addition to clinical examination and chest radiogram 36 patients had right heart catheterization, 28 pulmonary angiography and 44 echocardiography. RESULTS: Definite improvement of symptoms had occurred in all. 34 were now in NYHA stage I, nine in stage II, and two in stage III. The pulmonary vascular resistance was significantly lower than before and immediately after PTE (pre-PTE: 1052 +/- 472 dyn.s.cm-5; post-PTE: 293 +/- 175 dyn.s.cm-5; at follow-up: 187 +/- 92 dyn.s.cm-5; P < 0.001 for follow-up vs pre-PTE; P < 0.05 for follow-up vs post-PTE). Correspondingly, cardiac index had significantly increased (3.0 +/- 0.5 vs 2.0 +/- 0.7 l/min.m2; P < 0.001). Radiological and echocardiographic examinations showed a definite decrease in right ventricular dimensions and improvement in right ventricular function. CONCLUSION: In patients with pulmonary arterial hypertension due to chronic pulmonary thromboembolism PTE can achieve a reduction in pulmonary vascular resistance with lasting improvement in right heart function and clinical symptoms.

Adult↗

PubMed1996

Gradual angioplasty and stent implantation to treat complete superior vena cava occlusion after Mustard procedure.

A 16-year-old male was admitted with complete occlusion of the superior vena cava pathway 14 years after Mustard procedure for transposition of the great arteries. From a left subclavian vein approach, the atretic vein segment was perforated using a straight guidewire, and was followed by sequential balloon dilation. Implantation of a 30 mm Palmaz-stent through a femoral vein approach resulted in a widely patent channel of the vena cava superior into the systemic venous atrium.

Adolescent↗

PubMed1996

Mid-term results of pulmonary thromboendarterectomy for chronic thromboembolic pulmonary hypertension.

BACKGROUND: In patients with chronic thromboembolic pulmonary hypertension, acute and striking decreases of pulmonary artery pressures and vascular resistance can be achieved by pulmonary thromboendarterectomy. In this study, the long-term effects of pulmonary thromboendarterectomy on hemodynamic indices and right ventricular function were investigated. METHODS: Sixty-five patients (31 women and 34 men; mean age, 47 +/- 17 years; range, 19 to 69 years; New York Heart Association [NYHA] functional class II, n = 3; class III, n = 38; class IV, n = 24) were reassessed 13 to 48 months (mean, 27 months) after pulmonary thromboendarterectomy. Measurements are reported as mean +/- standard deviation. RESULTS: All patients reported a significant improvement of symptoms: 46 patients were in NYHA functional class I, 16 patients in class II, and 3 patients in class III. Mean pulmonary vascular resistance was significantly reduced compared with preoperative and postoperative values (preoperative: 1,015 +/- 454 dynes.s.cm-5; postoperative: 322 +/- 154 dynes.s.cm-5; follow-up: 198 +/- 72 dynes.s.cm-5; p < 0.001 versus preoperative; p < 0.025 versus postoperative). Concomitantly, cardiac index was significantly increased compared with preoperative values (preoperative: 2.0 +/- 0.7 L.min-1.m-2; follow-up: 2.9 +/- 0.5 L.min-1.m-2; p < 0.001). Significant reductions of right ventricular dimensions and recovery of right ventricular function could be demonstrated radiologically and echocardiographically. In 3 patients (preoperative NYHA class IV, NYHA class III at follow-up) with proven coagulation abnormalities, pulmonary vascular resistance was moderately increased at follow-up compared with postoperative measurements. CONCLUSIONS: In patients with chronic thromboembolic pulmonary hypertension, a persistent decrease of pulmonary vascular resistance and improvement of right ventricular function and NYHA functional status can be achieved by pulmonary thromboendarterectomy.

Adult↗

PubMed1996

The extended transseptal approach in complex mitral valve surgery--evaluation of risks and benefits.

The extended transseptal approach to the mitral valve was used in 32 patients undergoing isolated or combined mitral valve surgery. In all cases exposure of the entire mitral valvular apparatus was excellent. Two patients died of low output within 30 days of surgery. No cause of death was related to the extended transseptal approach. In one early patient reexploration revealed arterial bleeding from the right atrial suture line which was caused by damage to the sinus nodal artery. In 7 patients temporary atrial conduction disturbances occurred which completely resolved within 10 days after responding well to dual-chamber pacing. Temporary ventricular pacing was necessary in two patients with preoperative bradyarrhythmia. In two patients undergoing mitral re-do surgery a permanent ventricular pacer was implanted. The extended transseptal approach offers an excellent exposure of the entire mitral valve both in primary isolated or combined mitral surgery particularly in re-do surgery where the primary standard vertical left atriotomy is impeded or the conventional transseptal approach gives only limited access. Temporary atrial dysrhythmia is not crucial and is easily controlled by short-term dual-chamber pacing.

Arrhythmias, Cardiac↗

PubMed1996

Extrathoracic arteriosclerotic vascular changes preclude the use of the internal thoracic artery for coronary artery bypass grafting.

During a two-years period we have treated 6 patients where use of the internal thoracic artery for coronary artery grafting was precluded because of extrathoracic arteriosclerotic vascular lesions. In four patients with severe aorto-iliac occlusive disease preoperative digital angiography demonstrated collateralisation of the lower extremity by either the left, right, or both internal thoracic arteries (ITA). In these cases use of the ITA was excluded in order to preserve the collateral supply and coronary bypass grafting was performed using only saphenous vein. In two patients with proximal occlusion of the left subclavian artery the right ITA was used as in-situ bypass to graft the left anterior descending artery. All patients survived the operation without development of a perioperative myocardial infarction, neurological deficit, or peripheral ischemia. Although they rarely do, extrathoracic vascular disorders can exclude the use of the ITA for grafting. Especially in the case of aorto-iliac occlusive disease or proximal arteriosclerotic subclavian lesions angiographic evaluation is mandatory to prevent the development of life-threatening peripheral ischemia by harvesting an ITA and to avoid the use of an inadequate ITA graft with in-flow occlusion.

Aged↗

PubMed1996

[Intravascular stents for treatment of systemic venous obstructions].

Two young adults, 16 and 18 years of age were admitted with complete occlusion of the superior vena cava pathway following previous Mustard procedure. In both patients, perforation of complete occluded vein segment using a straight guidewire and subsequent sequential enlargement by balloon dilation were successfully performed by a left subclavian vein approach. Implantation of a 30 mm Palmaz-stent resulted in a widely patent channel of the vena cava superior into the systemic venous atrium. In short-term follow-up of 3 months both stents remained patent. Repeat dilation of the stents was performed after 3 months. A third patient with systemic venous obstruction was a 3-year old child who suffered low cardiac output following modified Fontan operation caused by a narrowing of the superior vena cava to right pulmonary artery connection and stenosis of distal left pulmonary artery. The severe hemodynamic problem was resolved by implantation of Palmaz-stents in the two separate obstructions. At recatheterization 6 months later, both stents were patent without any residual pressure gradient.

Adolescent↗

PubMed1996

[Surgical closure of atrial septum defects via right-sided thoracotomy. Value with reference to the development of interventricular closure techniques].

Surgical closure of atrial septal defect is a safe and effective procedure with low morbidity and mortality. A right anterior thoracotomy approach is a suitable alternative to that through a median sternotomy and provides superior cosmetic results. Thirty patients at the age of 1 year, 3 months to 49 years underwent repair of atrial septal defects through a right thoracotomy. Twenty-four patients had secundum, three ostium primum, two sinus venosus defect, and one patient had Scimitar's syndrome. Details of the surgical procedure on cardiopulmonary bypass are presented. There was no operative or late mortality, and no morbidity directly related to the alternative approach. All patients or their parents considered the cosmetic result fair or satisfying. The following paper reflects our experience with the thoracotomy approach for repair of atrial septal defects, as well as a critical review of new developments in interventional ASD occlusion techniques.

Adolescent↗

PubMed1995

A ribosome-associated peptidyl-prolyl cis/trans isomerase identified as the trigger factor.

Peptidyl-prolyl cis/trans isomerases (PPIases) are enzymes that catalyse protein folding both in vitro and in vivo. We isolated a peptidyl-prolyl cis/trans isomerase (PPIase) which is specifically associated with the 50S subunit of the Escherichia coli ribosome. This association was abolished by adding at least 1.5 M LiCl. Sequencing the N-terminal amino acids in addition to three proteolytic fragments totalling 62 amino acids revealed that this PPIase is identical to the E.coli trigger factor. A comparison of the amino acid sequence of trigger factor with those of other PPIase families shows little similarities, suggesting that trigger factor may represent an additional family of PPIases. Trigger factor was purified to homogeneity on a preparative scale from E.coli and its enzymatic properties were studied. In its activity towards oligopeptide substrates, the trigger factor resembles the FK506-binding proteins (FKBPs). Additionally, the pattern of subsite specificities with respect to the amino acid preceding proline in Suc-Ala-Xaa-Pro-Phe-4-nitroanilides is reminiscent of FKBPs. However, the PPIase activity of the trigger factor was not inhibited by either FK506 or by cyclosporin A at concentrations up to 100 microM. In vitro, the trigger factor catalysed the proline-limited refolding of a variant of RNase T1 much better than all other PPIases that have been examined so far.

Amino Acid Isomerases↗

PubMed1995

Destabilization of a protein helix by electrostatic interactions.

Electrostatic interactions between charged residues and the helix dipole in a protein were investigated by protein engineering methods. In ribonuclease T1, two surface-exposed acidic residues (Glu28 and Asp29) are located near the carboxyl terminus of the alpha-helix between residues 13 and 29. They were replaced, individually and in concert, by the uncharged amides Gln28 and Asn29, and the stabilities of the wild-type protein and its variants were determined as a function of pH. The effects of the two mutations are additive. Either one leads to a marginal destabilization by 0.7 kJ/mol at pH 2 but to a strong stabilization by about 3.2 kJ/mol at pH 7. This suggests that the deprotonations of Glu28 and Asp29 reduce the free energy of stabilization of folded ribonuclease T1 by about 4 kJ/mol each. This destabilization is probably caused by unfavorable electrostatic interactions of Glu28 and Asp29 with the negative end of the helix dipole. The activation energies for the unfolding of the different variants of ribonuclease T1 change in parallel with the differences in the thermodynamic stability when the pH is varied. This indicates that the unfavorable electrostatic interactions of Glu28 and Asp29 are lost very early in unfolding, and are not present in the activated state of unfolding.

Asparagine↗

PubMed1995

Protein folding. Prolyl isomerases join the fold.

Cyclophilins have prolyl isomerase activity, but evidence for their suggested role in protein folding in cells has been scarce; now they have been found to accelerate the folding of mitochondrial precursor proteins.

Amino Acid Isomerases↗

PubMed1995

Influence of protein conformation on disulfide bond formation in the oxidative folding of ribonuclease T1.

In oxidative protein folding the interdependence between the acquisition of an ordered native-like conformation and disulfide bond formation was investigated by using the C2S/C10N variant of ribonuclease T1 as a model. This protein of 104 residues has a single disulfide bond between Cys6 and Cys103. In the reduced form it is unfolded in the presence of urea, but native-like folded when > or = 1.5 M NaCl is present. The influence of a folded conformation on the individual thiol/disulfide exchange reactions between the protein and glutathione could thus be studied in oxidative folding by varying the urea and NaCl concentrations. When the reduced protein was folded native-like the initial formation of the mixed disulfide between the protein and glutathione was decelerated about fourfold. The attachment of a glutathionyl moiety in this step destabilizes the protein by about 5 kJ mol-1 and led to a local unfolding near the two Cys residues. The reacting thiol groups still remained in close proximity for the subsequent intramolecular thiol/disulfide exchange reaction, but an increase in the energy of the transition state (e.g. by a hydrophobic environment or by steric strain) could be avoided. As a consequence the formation of the protein disulfide in this reaction was 100-fold faster when the mixed-disulfide species was in this ordered conformation. These results illustrate the importance of a low stability and a high flexibility of folding intermediates.

Aspergillus oryzae↗

PubMed1995

Structure of human parathyroid hormone 1-37 in solution.

Human parathyroid hormone (hPTH), amino acids Ser1 to Leu37, is biologically active with respect to both receptor binding and activation of adenylate cyclase to influence the serum calcium concentration. It induces DNA synthesis via an unknown signal pathway. We investigated the structure of hPTH(1-37) in H2O/buffer solution under near physiological conditions, that is pH 6.0 and 270 mM salt, by circular dichroism, ultracentrifugation, nuclear magnetic resonance spectroscopy, and molecular dynamics calculations. Complete sequence specific assignments of all 1H resonances were performed by using 1H two-dimensional NMR measurements (double quantum-filtered correlated spectroscopy, nuclear Overhauser effect spectroscopy (NOESY), and total correlation spectroscopy with suppression of NOESY-type cross-peaks spectra). hPTH(1-37) obtained helical structure and showed hydrophobic interactions defining a tertiary structure. The NH2-terminal four amino acids of hPTH(1-37) did not show a stable conformation. Evidence for an alpha-helical region between Ile5 and Asn10 was found. This region was followed by a flexible link (Gly12, Lys13) and a well defined turn region, His14 to Ser17. The latter was stabilized by hydrophobic interactions between Trp23 and Leu15. Ser17 through at least Leu28 formed an alpha-helix. Arg20 and Lys27 were involved in the core built by His14 to Ser17. Unrestrained molecular dynamics simulations indicated that the structure was stable on the 200 ps time scale.

Amino Acid Sequence↗

PubMed1995

DsbA-mediated disulfide bond formation and catalyzed prolyl isomerization in oxidative protein folding.

The interrelationship between the acquisition of ordered structure, prolyl isomerization, and the formation of the disulfide bonds in assisted protein folding was investigated by using a variant of ribonuclease T1 (C2S/C10N-RNase T1) with a single disulfide bond and two cis-prolyl bonds as a model protein. The thiol-disulfide oxidoreductase DsbA served as the oxidant for forming the disulfide bond and prolyl isomerase A as the catalyst of prolyl isomerization. Both enzymes are from the periplasm of Escherichia coli. Reduced C2S/C10N-RNase T1 is unfolded in 0 M NaCl, but native-like folded in > or = 2 M NaCl. Oxidation of 5 microM C2S/C10N-RNase T1 by 8 microM DsbA (at pH 7.0, 25 degrees C) is very rapid with a t1/2 of about 10 s (the second-order rate constant is 7 x 10(3) s-1 M-1), irrespective of whether the reduced molecules are unfolded or folded. When they are folded, the product of oxidation is the native protein. When they are denatured, first the disulfide bond is formed in the unfolded protein chains and then the native structure is acquired. This slow reaction is limited in rate by prolyl isomerization and catalyzed by prolyl isomerase. The efficiency of this catalysis is strongly decreased by the presence of the disulfide bond. Apparently, the rank order of chain folding, prolyl isomerization, and disulfide bond formation can vary in the oxidative folding of ribonuclease T1. Such a degeneracy could generally be an advantage for protein folding both in vitro and in vivo.

Amino Acid Sequence↗

PubMed1995

A kinetic analysis of the folding of human carbonic anhydrase II and its catalysis by cyclophilin.

The kinetics of unfolding and refolding of human carbonic anhydrase II (HCAII) and its catalysis by the peptidyl-prolyl-cis/trans-isomerase cyclophilin were investigated. HCAII contains 15 trans- and 2 cis-prolyl peptide bonds, and, when long-term denatured, virtually all unfolded molecules contain non-native prolyl isomers. In unfolding these molecules (Us) are produced slowly in a biphasic process reflecting the isomerization of several trans-prolines and of one cis-proline. In refolding, the rapid formation of an intermediate of the molten globule type is followed by several slow prolyl isomerizations, which determine the rate of reactivation. By a short 10-s incubation in 5.0 M guanidinium chloride at 2 degrees C, unfolded HCAII species with all prolines still in the native conformation (Uf) could be produced. Surprisingly, only a fraction of Uf refolds rapidly, but the other molecules refold slowly. Evidently, some prolyl peptide bonds isomerize early in refolding, at the stage of the molten globule and as a consequence, molecules with incorrect prolyl isomers are formed in competition with the productive folding of Uf. This fraction of slow-folding molecules is strongly increased when cyclophilin is present, because it accelerates the formation of non-native prolyl isomers as long as the molecules remain in the molten globule state. Later cyclophilin catalyzes the isomerization of these prolyl peptide bonds toward the native state, which are stabilized in their conformation by further folding to the native state. This catalysis is very efficient, because only prolines that are accessible in the molten globule are involved in this sequence of isomerization and reisomerization.

Amino Acid Isomerases↗

PubMed1995

Non-prolyl cis-trans peptide bond isomerization as a rate-determining step in protein unfolding and refolding.

In wild-type ribonuclease T1 the peptide bond between Tyr38 and Pro39 is in the cis conformation. When Pro39 is replaced by an alanine this cis conformation is retained, and a non-prolyl cis Tyr38-Ala39 peptide bond is generated. We employed a stopped-flow double-mixing technique to investigate the kinetics of the cis-->trans isomerization of this peptide bond in the unfolding and the trans-->cis isomerization in the refolding of Pro39Ala-ribonuclease T1. In 6.0 M GdmCl (pH 1.6) and 25 degrees C the protein unfolds rapidly with a time constant of 20 ms, followed by Tyr38-Ala39 cis-->trans isomerization. This reaction shows a time constant of 730 ms and is about 60-fold faster than the isomerization of the Tyr38-Pro39 bond in the wild-type protein. Unfolded molecules with the Tyr38-Ala39 bond still in the native-like cis conformation accumulate transiently for a short time after unfolding is initiated, and they can refold very rapidly to the native state with a time constant of 290 ms (in 1.0 M GdmCl, pH 4.6, 25 degrees C). After more than three seconds of unfolding virtually all protein molecules contain an incorrect trans Tyr38-Ala39 bond and refolding is decelerated approximately 1000-fold, because Tyr38-Ala39 trans-->cis re-isomerization is very slow and, with its time constant of 480 s, determines the overall rate of refolding. Due to the coupling of the cis-trans equilibrium with protein folding it was possible to measure the kinetic parameters of the isomerization of a non-prolyl peptide bond in a protein. Previously this could not be accomplished, because the trans isomer is strongly preferred for unsubstituted peptide bonds in oligopeptides under virtually all conditions. Our data indicate that the kinetics of Tyr38-Pro39 and of Tyr38-Ala39 isomerization differ predominantly in the rate of the cis-->trans, rather than of the trans-->cis reaction. The rate of the trans-->cis reaction is, however, measured during refolding and may be influenced by the formation of ordered protein structure.

Escherichia coli↗

PubMed1995

Left ventricular outflow tract obstruction in TGA: treatment with LV-to-PA valved conduit.

Progressive or recurrent left ventricular outflow tract obstruction after a previous Mustard or Senning operation represents a rare but challenging problem. The obstruction can be resected in some patients, but abnormal attachment of the mitral valve or a long fibromuscular tunnel represents a difficult surgical problem. Between 1979 and 1993, we encountered this type of left ventricular outflow tract obstruction in 10 patients, 4 to 13 years after the atrial repair. They ranged in age from 5 to 15 years (mean, 8.8 years) and weighed between 11.5 and 47 kg (mean, 25.3 kg). Operations were performed through a left thoracotomy with the patient on hypothermic cardiopulmonary bypass but without aortic cross-clamping. The left atrial appendage and descending aorta were cannulated. Good relief of the gradient was obtained in all patients (mean residual gradient, 14.8 mm Hg). All patients survived the operation. One patient died suddenly at home 6 months later; 2 patients required conduit replacement. All 9 long-term survivors are asymptomatic as of 6 months to 8 years after their conduit placement or replacement. We recommend the placement of a left ventricle-to-pulmonary artery valved conduit for the relief of severe left ventricular outflow tract obstruction arising after a Senning or Mustard operation that cannot be managed by other means.

Adolescent↗