[Therapy of pulmonary embolism].
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Humans↗
Biomedical subjects
R Zahn
Publications and source records attributed to R Zahn.
[Diagnosis of pulmonary embolism].
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Angiography↗
Effect of single point mutations in citrate synthase on binding to GroEL.
Six single point mutants of yeast citrate synthase were analyzed for binding to the molecular chaperone GroEL. In contrast to the wild-type and G276S, all other G276-mutants were able to displace pre-beta-lactamase from GroEL. The off-rate constant for pre-beta-lactamase must be at least partially rate-limiting, leading to an equilibrium dissociation constant between 10(-10) M and 10(-12)M. Direct evidence for binding of citrate synthase was obtained from gel filtration experiments. The results suggest that thermodynamic rather than structural features of the mutants determine the degree of binding to the chaperone.
Binding, Competitive↗
Catalysis of amide proton exchange by the molecular chaperones GroEL and SecB.
Hydrogen-deuterium exchange of 39 amide protons of Bacillus amyloliquefaciens ribonuclease (barnase) was analyzed by two-dimensional nuclear magnetic resonance in the presence of micromolar concentrations of the molecular chaperones GroEL and SecB. Both chaperones bound to native barnase under physiological conditions and catalyzed exchange of deeply buried amide protons with solvent. Such exchange required complete unfolding of barnase, which occurred in the complex with the chaperones. Subsequent collapse of unfolded barnase to the exchange-protected folding intermediate was markedly slowed in the presence of GroEL or SecB. Thus, both chaperones have the potential to correct misfolding in proteins by annealing.
Adenosine Diphosphate↗
[Feasibility and safety of dobutamine stress echocardiography: experiences with 1,000 studies].
The use of dobutamine stress echocardiography for the evaluation of coronary artery disease is rapidly expanding. Despite its widespread use, the feasibility and safety of dobutamine stress echocardiography has not been sufficiently documented. Between November 1992 and June 1995, we performed 1000 dobutamine stress echocardiographies. There were 744 men and 256 women with a mean age of 59 +/- 11 years. Anti anginal medication was not routinely withdrawn before the test. The mean maximal dobutamine dose was 41,4 +/- 10 mu g/kg center dot min(-1). Atropine was given additionally in 440 patients, with a mean dose of 0.5 mg. In patients receiving beta-blockers additional atropine was more often necessary as compared to those not receiving beta-blockers (278/457 = 61% versus 162/543 = 30 %, p < 0.0001). Reasons for discontinuing dobutamine infusion were achievement of target heart rate (64 % of cases) and maximal dose (12 % of cases). In 791 (79,1 %) patients no side-effects of dobutamine stress echocardiography were noticed. Termination of the study because of adverse side-effects occurred in 6.6 %. A total of 103 (10,3 %) noncardiac side-effects were observed: dizziness or nausea 6.4 %, headache 1.7 %. In one patient a focal cerebral seizure occurred. 156 cardiac side-effects occurred: blood pressure decrease of more than 20 mm Hg in 25 patients, extreme palpitations in 16 patients and pulmonary edema in one case. Most common cardiac side-effects consisted of arrhythmias (11.4 %): 9.1 % ventricular and 2.3 % supraventricular arrhythmias. Most ventricular arrhythmias were less severe (uniform and multiform premature ventricular beats, ventricular bigeminy or couplets in 71 patients). Nonsustained ventricular tachycardia, with a maximum duration of 20 s, occurred in 18 patients. In one patient sustained ventricular tachycardia developed and progressed towards ventricular fibrillation. This patient could be successfully defibrillated. Supraventricular arrhythmias presented as new atrial fibrillation in 10 patients, supraventricular tachycardia in three patients, junctional rhythm with a short decline in heart rate in nine patients and a second-degree AV block in another case. Dobutamine stress echocardiography has proven to be a safe and feasible method in the diagnosis of coronary heart disease. Minor side-effects are common and sometimes unpleasant for the patient, but do not often require termination of the study. Severe side-effects are seldom (< 1 %), but nevertheless, adequate medical and technical (defibrillator) support should be rapidly available.
Adult↗
Do 5-F-catheters reduce the incidence of a pseudoaneurysm?
Femoral artery pseudoaneurysm is a major problem in patients undergoing cardiac catheterization. This study describes our experience with 5 French (5 F) and 7 French (7 F) introduction sheaths and 7 investigators at our institution regarding the incidence of pseudoaneurysms. During 54 months (1/1990-6/1994) 8715 consecutive patients after diagnostic cardiac catheterization were first clinically checked for pseudoaneurysm and in case of suspicion a duplex sonography was performed. In 86 (1%) patients, 44 (52%) women and 42 (48%) men, mean age 63 +/- 9.7 years we observed this complication by duplex ultrasound. 54 (62%) patients had arterial hypertension, 18 (20%) diabetes and only 3 (3.6%) had peripheral arteriosclerosis. An antithrombotic medication was used in 60% (52 patients). As compared to a control group of 450 consecutive patients a pseudoaneurysm was significantly more likely to occur in patients with a history of hypertension (63% vs 25%, p < 0.0001). Women are also at higher risk representing 51% of all pseudoaneurysms as compared to 29% in the control group (p < 0.0001). Using 7-F catheters more pseudoaneurysms occurred (82/7183; 1%) than using 5 F (4/1532; 0.2%) introduction sheaths (p = 0.0005). There were also significantly more pseudoaneurysms caused by investigator 1 (21/787; 2.7%) as compared to the other investigators (65/7829; 0.8%), (p = 0.0002). Investigator 1 had a more distal puncture technique than the others. Pseudoaneurysms complicating cardiac catheterization occur 5-times more frequent using 7 F (1%) as compared to 5 F catheters (0.2%). Moreover distal puncture site is associated with a higher frequency of pseudoaneurysms.
Aneurysm, False↗
A major allergen of lymphatic filarial nematodes is a parasite homolog of the gamma-glutamyl transpeptidase.
BACKGROUND: Bm2325, a major IgE-inducing antigen of the filarial parasite Brugia malayi has been implicated in the pathology of tropical pulmonary eosinophilia (TPE), a pulmonary syndrome thought to result from hypersensitivity to microfilariae. MATERIALS AND METHODS: Affinity-purified IgE to Bm2325 from patients with TPE was used to identify a complementary DNA (cDNA) from a B. malayi expression library. Sequence analysis of the cDNA revealed a hitherto unknown parasite protein. Immunoblotting of the recombinant filarial protein using sera of patients with TPE determined its IgE-binding capacity. Reactivity to human lung epithelial cell proteins was analyzed using murine anti-Bm2325 antibodies and serum from patients with TPE. RESULTS: The predicted protein is a homolog of the entire precursor of the gamma-glutamyl transpeptidase (gamma-GT), a key enzyme in the synthesis and degradation of glutathione. The filarial precursor encodes both the heavy (H) and the light (L) chain subunits and shares structural similarities with the mammalian enzymes. The Bm2325 allergen was identified as the homolog of the enzyme light chain subunit. Murine antibodies against the recombinant parasite gamma-GT cross-reacted with the human enzyme present in human airway epithelial cells, and human gamma-GT is a target of antibodies present in the serum of patients with TPE. CONCLUSION: Molecular mimicry between the parasite gamma-GT homolog and the host membrane-bound gamma-GT present in lung epithelial cells likely contributes to the pathogenesis observed in tropical pulmonary eosinophilia.
Amino Acid Sequence↗
[Renaissance of beta blocker therapy in myocardial infarct].
Beta-adrenergic blocking drugs have been evaluated for the treatment of arrhythmias and for the prevention of sudden cardia death, particularly in post-myocardial infarction patients. Betablockers have been demonstrated to reduce mortality, reinfarction, ventricular fibrillation and cardiac rupture in acute infarction. Therefore, in patients with suspected myocardial infarction and without contraindications, treatment with betablockers should be initiated early and continued for at least 2 years. Side-effects are mild and occur in approximately 10% of patients. Patients who have contraindications for betablockers use early in myocardial infarction should be reevaluated before discharge from the hospital and considered for such therapy. Because betablockers prevent some of the adverse arrhythmogenic mechanisms seen in chronic heart failure, it may be reasonable to expect that these drugs could have a role in preventing sudden cardiac death in these patients. Analysis of some of the betablocker post-infarction trials indicate that betablockers reduced the risk of sudden death in patients with heart failure at baseline. Some studies demonstrated also the symptomatic improvement following therapy with betablockers in patients with heart failure. But the currently available data are too limited to provide conclusive information.
Adrenergic beta-Antagonists↗
Tetravalent miniantibodies with high avidity assembling in Escherichia coli.
We have designed tetravelent miniantibodies assembling in the periplasm of Escherichia coli. They are based on single-chain Fv fragments, connected via a flexible hinge to an amphipathic helix which tetramerizes the molecule. The amphipathic helix is derived from the coiled coil helix of the transcription factor GCN4, in which all hydrophobic a positions of every heptad repeat have been exchanged to leucine and all d positions to isoleucine. Gel filtration shows tetramer assembly of the miniantibody even at low concentrations. As expected, the functional affinity (avidity) of the tetravalent miniantibody is higher in ELISA and BIAcore measurements than that of the bivalent construct and the gain is dependent on surface epitope density.
Amino Acid Sequence↗
[Takayasu arteritis--a rare differential diagnosis in aortic dissection. A case report].
A 36-year-old woman was delivered to our hospital with suspected aortic dissection from an outlying hospital in May 1994. She reported a history of acute and persistent thoracic and epigastric pain. The physical examination revealed a minor senso-motorical palsy of the left side as residuum after minor strokes occurring 12/93 and 4/94. Also, a marked hypertension (170/100 mm Hg) was present. The hematologic and blood chemical values were normal with a white cell count of 12,000, an erythrocyte sedimentation rate (ESR) of 19 mm/h and a c-reactive protein (CRP) of 2.2 mg/dl. The electrocardiogram was normal. Transthoracic- and transesophageal-echocardiography (TTE, TEE) revealed an eccentric thickening of the whole wall of the descending aorta up to the bifurcation with a stenosis at the side of the diaphragma. Those findings were confirmed by computed tomography. Because of the acute onset of symptoms and the results of the imaging procedures aortic dissection de Bakey type III was diagnosed and the patient was treated with beta-blockers. Symptoms were relieved over the following days. After 2 days a pleuric effusion developed and all inflammatory tests rose (fibrinogen 780 mg/dl, ESR 80 mm/hr, CRP 16 mg/dl). At this time the differential diagnosis of an arteritis was considered. A new TEE-study demonstrated no change, but now more attention was given to the fact that no dissection membrane could be visualized and all wall structures were thickened. In combination with the history of cerebral infarction due to carotid obstruction and the elevated laboratory values the diagnosis of Takayasu-aortitis was established and corticoid therapy was started. Within a few weeks the arterial changes diminshed markedly and the woman became free of symptoms. This case demonstrates the rare situation of an arteritis mimicking aortic dissection, in which the clinical course revealed the true diagnosis.
Adult↗
Thermodynamic partitioning model for hydrophobic binding of polypeptides by GroEL. I. GroEL recognizes the signal sequences of beta-lactamase precursor.
From equilibrium measurements with urea we found a three-state thermodynamic and kinetic folding behavior for the precursor and mature form of Escherichia coli beta-lactamase TEM2. The thermodynamic intermediate H of Escherichia coli beta-lactamase and its precursor had no enzymatic activity, and a quenched tryptophan fluorescence intensity, but a native-like wavelength of maximum intensity. State H of mature beta-lactamase was 8.7 kcal mol-1 less stable than the native state N and about 4.2 kcal mol-1 more stable than the unfolded state U, extrapolated to absence of urea. In contrast, state H of precursor beta-lactamase was even more stable than N by about 0.5 kcal mol-1 and about 6.9 kcal mol-1 more stable than U. Native pre-beta-lactamase could be stabilized by lowering the pH value from 7.0 to 5.5, probably by protonating a histidine residue leading to an improved solubility of the signal sequence. Synthetic peptides, containing 23 or 38 N-terminal amino acid residues of pre-beta-lactamase, were unable to compete with pre-beta-lactamase for binding to GroEL. However, GroEL prevented the inactivation of mature beta-lactamase by p38, consistent with competition between GroEL and mature beta-lactamase for binding to p38. The equilibrium constant for dissociation KD of the complex between GroEL and p23, a peptide containing exclusively the signal sequence of pre-beta-lactamase, was measured with the BIAcore instrument to be in the range 10(-7) to 10(-8) M. Our results are consistent with co-operative binding of GroEL to the mature part and to the signal sequence of pre-beta-lactamase. We suggest a thermodynamic partitioning model for hydrophobic binding of polypeptides by GroEL.
Amino Acid Sequence↗
Thermodynamic partitioning model for hydrophobic binding of polypeptides by GroEL. II. GroEL recognizes thermally unfolded mature beta-lactamase.
By thermal equilibrium measurements we found a three-state folding behavior of mature Escherichia coli beta-lactamase TEM2. The thermodynamically stable intermediate H had no enzymatic activity, but a native-like secondary structure. State H was 9 kcal mol-1 less stable than the native state N and 4 kcal mol-1 more stable than the totally unfolded state U, which is consistent with urea equilibrium measurements of mature beta-lactamase measured under similar conditions. Between 38 degrees C and 50 degrees C there was a decrease in the apparent equilibrium constant for dissociation K'D of the complex between GroEL and mature beta-lactamase, at least partially caused by a decrease in the thermodynamic stability of the native form of mature beta-lactamase. GroEL-bound beta-lactamase was released either after addition of ATP, or in the presence of a competing substrate (i.e. a single-chain antibody), or after lowering the temperature. Whereas at 10 degrees C the folding reaction of mature beta-lactamase was rate limiting, at 37 degrees C the release reaction was the rate-determining step for the regain of beta-lactamase activity, consistent with a decrease of the equilibrium constant for dissociation KD of the complex with temperature. A temperature dependent behavior of GroEL was also observed, when measuring the anilinonaphthalene sulfonic acid (ANS) fluorescence of the chaperone. Similar to all other substrate proteins studied so far, the maximal tryptohan fluorescence of GroEL-bound beta-lactamase was observed at 342 nm. Our results are compatible with a hydrophobic binding pocket of GroEL and confirm the suggested thermodynamic partitioning model for hydrophobic binding of polypeptides by GroEL.
Bacterial Proteins↗
Destabilization of the complete protein secondary structure on binding to the chaperone GroEL.
Protein folding in vivo is mediated by helper proteins, the molecular chaperones, of which Hsp60 and its Escherichia coli variant GroEL are some of the best characterized. GroEL is an oligomeric protein with 14 subunits each of M(r) 60K, which possesses weak, co-operative ATPase activity and high plasticity. GroEL seems to interact with non-native proteins, binding one or two molecules per 14-mer in a 'central cavity', but little is known about the conformational state of the bound polypeptides. Here we use nuclear magnetic resonance techniques to show that the interaction of the small protein cyclophilin with GroEL is reversible by temperature changes, and all amide protons in GroEL-bound cyclophilin are exchanged with the solvent, although this exchange does not occur in free cyclophilin. The complete secondary structure of cyclophilin must be disrupted when bound to GroEL.
Amino Acid Isomerases↗
Two-dimensional crystals of the molecular chaperone GroEL reveal structural plasticity.
For two-dimensional (2-D) crystallization we have purified the molecular chaperone GroEL from Escherichia coli to homogeneity. The final and important step for crystallization in the purification procedure was an ATP-agarose column, on which the spacer between ATP and agarose was attached to C8 of adenine. Using the mica spreading "negative staining-carbon film" procedure and polyethylene glycol as a precipitant, we obtained four different 2-D periodic arrays. Two of them turned out to be true crystals. One crystal has P2 symmetry and lattice constants of a = 24.3 nm and b = 16.9 nm, the other has essentially P4 symmetry and shows smoothly varying local changes in the lattice parameters (a = b = 23 (+/- 1.3) nm). Very striking in the P4 crystal is the departure within each individual GroEL particle from the GroEL-typical seven-fold symmetry, which seems to be required for GroEL to accommodate to a crystal symmetry.
Bacterial Proteins↗
GroE prevents the accumulation of early folding intermediates of pre-beta-lactamase without changing the folding pathway.
In folding studies of pre-beta-lactamase in the presence of GroE, we investigated the pH dependence of the folding reaction. Two critical intermediates in the folding pathway were defined kinetically. I1 is an early folding intermediate recognized by GroE; the misfolding of I1 leads to aggregation, and this is prevented by GroE. A second intermediate I2 is released from GroE after ATP hydrolysis. Its pH-dependent misfolding to a nonnative form, which is not an aggregate, is not prevented by GroE. From these results, a model is proposed, in which the crucial role of GroE consists of allowing the change from I1 to I2 to take place in the complex. Fluorescence spectra of the pre-beta-lactamase complexed to GroE are very similar to those of the native state. The pathway of pre-beta-lactamase folding is not changed by GroE as evidenced by the same half-time and pH dependence of the folding reaction. GroE probably recognizes the signal sequence and some portion of the mature protein since mature beta-lactamase does not interact with GroE even under conditions of slow folding.
Bacterial Proteins↗
Role of glycine-82 as a pivot point during the transition from the inactive to the active form of the yeast Ras2 protein.
Ras proteins bind either GDP or GTP with high affinity. However, only the GTP-bound form of the yeast Ras2 protein is able to stimulate adenylyl cyclase. To identify amino acid residues that play a role in the conversion from the GDP-bound to the GTP-bound state of Ras proteins, we have searched for single amino acid substitutions that selectively affected the binding of one of the two nucleotides. We have found that the replacement of glycine-82 of the Ras2 protein by serine resulted in an increased rate of dissociation of Gpp(NH)p, a nonhydrolysable analog of GTP, while the GDP dissociation rate was not significantly modified. Glycine-82 resides in a region that is highly conserved between the yeast and human proteins. However, this residue is structurally distant from residues that participate in the binding of the nucleotide, as determined from the crystal structure of the human H-ras gene product. Therefore, the ability of the nucleotide binding site to discriminate between GDP and GTP is dependent not only on residues that are spatially close to the nucleotide, but also on distant amino acids. This is in agreement with the role of glycine-82 as a pivot point during the transition from the GDP- to the GTP-bound form of the Ras proteins.
Amino Acid Sequence↗
Identification of regulatory residues of the yeast adenylyl cyclase.
We have attempted to identify amino acid residues of the yeast adenylyl cyclase that are involved in the regulation of its activity, by isolating adenylyl cyclase-linked spontaneous mutations capable of suppressing the temperature-sensitive phenotype of ras1- ras2-ts1 strains. We previously identified a mutated adenylyl cyclase in which a single point mutation, called CR14, led to the replacement of threonine 1651 with isoleucine. We have now investigated the biological effects of CR14, and of other mutations that cause the replacement of threonine 1651 by distinct amino acids. We have observed that the response of adenylyl cyclase to Ras can be either enhanced or attenuated, without significant effects on the steady-state level of the former enzyme in vivo, depending on the amino acid side chain at position 1651. Therefore, this residue identifies a regulatory region on the adenylyl cyclase molecule. We have also taken advantage of the attenuation of adenylyl cyclase function caused by the replacement of threonine 1651 with aspartic acid to isolate intragenic suppressor mutations. We have identified several point mutations, leading to single amino acid substitutions, individually capable of reactivating the attenuated adenylyl cyclase. The corresponding amino acid changes are located within a relatively small region, including residues 1331, 1345, 1348 and 1374. This region could be physiologically involved in the negative control of the carboxy-terminal catalytic domain.
Adenylyl Cyclases↗
Antibodies against vertebrate microfilament proteins in the analysis of cellular motility and adhesion.
Microinjection of specific antibodies can be an alternative and a supplement to genetic engineering in dissecting the function of individual cytoskeletal components. In this report, we describe some of the requirements for using this technique, its potential application in conjunction with morphological and biochemical analyses, and its limitations. Examples are given for the injection of antibodies to alpha-actinin, vinculin and myosin, and the effects of such treatment on adhesion, motility and cytokinesis of the recipient cells.
Animals↗