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Biomedical subjects

R Rudolph

Publications and source records attributed to R Rudolph.

At least 91 records · Page 5Linked to original sources
PubMed1992

Stability and reconstitution of pyruvate oxidase from Lactobacillus plantarum: dissection of the stabilizing effects of coenzyme binding and subunit interaction.

Pyruvate oxidase from Lactobacillus plantarum is a homotetrameric flavoprotein with strong binding sites for FAD, TPP, and a divalent cation. Treatment with acid ammonium sulfate in the presence of 1.5 M KBr leads to the release of the cofactors, yielding the stable apoenzyme. In the present study, the effects of FAD, TPP, and Mn2+ on the structural properties of the apoenzyme and the reconstitution of the active holoenzyme from its constituents have been investigated. As shown by circular dichroism and fluorescence emission, as well as by Nile red binding, the secondary and tertiary structures of the apoenzyme and the holoenzyme do not exhibit marked differences. The quaternary structure is stabilized significantly in the presence of the cofactors. Size-exclusion high-performance liquid chromatography and analytical ultracentrifugation demonstrate that the holoenzyme retains its tetrameric state down to 20 micrograms/mL, whereas the apoenzyme shows stepwise tetramer-dimer-monomer dissociation, with the monomer as the major component, at a protein concentration of < 20 micrograms/mL. In the presence of divalent cations, the coenzymes FAD and TPP bind to the apoenzyme, forming the inactive binary FAD or TPP complexes. Both FAD and TPP affect the quaternary structure by shifting the equilibrium of association toward the dimer or tetramer. High FAD concentrations exert significant stabilization against urea and heat denaturation, whereas excess TPP has no effect. Reconstitution of the holoenzyme from its components yields full reactivation. The kinetic analysis reveals a compulsory sequential mechanism of cofactor binding and quaternary structure formation, with TPP binding as the first step. The binary TPP complex (in the presence of 1 mM Mn2+/TPP) is characterized by a dimer-tetramer equilibrium transition with an association constant of Ka = 2 x 10(7) M-1. The apoenzyme TPP complex dimer associates with the tetrameric holoenzyme in the presence of 10 microM FAD. This association step obeys second-order kinetics with an association rate constant k = 7.4 x 10(3) M-1 s-1 at 20 degrees C. FAD binding to the tetrameric binary TPP complex is too fast to be resolved by manual mixing.

Apoenzymes↗
PubMed1992

Characterization of the stabilizing effect of point mutations of pyruvate oxidase from Lactobacillus plantarum: protection of the native state by modulating coenzyme binding and subunit interaction.

Point mutations in the gene of pyruvate oxidase from Lactobacillus plantarum, with proline residue 178 changed to serine, serine 188 to asparagine, and alanine 458 to valine, as well as a combination of the three single point mutations, lead to a significant functional stabilization of the protein. The enzyme is a tetrameric flavoprotein with tightly bound cofactors, FAD, TPP, and divalent metal ions. Thus, stabilization may be achieved either at the level of tertiary or quaternary interactions, or by enhanced cofactor binding. In order to discriminate between these alternatives, unfolding, dissociation, and cofactor binding of the mutant proteins were analyzed. The point mutations do not affect the secondary and tertiary structure, as determined by circular dichroism and protein fluorescence. Similarly, the amino acid substitutions neither modulate the enzymatic properties of the mutant proteins nor do they stabilize the structural stability of the apoenzymes. This holds true for both the local and the global structure with unfolding transitions around 2.5 M and 5 M urea, respectively. On the other hand, deactivation of the holoenzyme (by urea or temperature) is significantly decreased. The most important stabilizing effect is caused by the Ala-Val exchange in the C-terminal domain of the molecule. Its contribution is close to the value observed for the triple mutant, which exhibits maximum stability, with a shift in the thermal transition of ca. 10 degrees C. The effects of the point mutations on FAD binding and subunit association are interconnected. Because FAD binding is linked to oligomerization, the stability of the mutant apoenzyme-FAD complexes is increased. Accordingly, mutants with maximum apparent FAD binding exhibit maximum stability. Analysis of the quaternary structure of the mutant enzymes in the absence and in the presence of coenzymes gives clear evidence that both improved ligand binding and subunit interactions contribute to the observed thermal stabilization.

Alanine↗
PubMed1992

Pharmacokinetic and thrombolytic properties of unglycosylated recombinant tissue-type plasminogen activator (BM 06.021) produced in Escherichia coli.

Recombinant tissue-type plasminogen activator (rt-PA) was produced in Escherichia coli cells in order to obtain an unglycosylated rt-PA (BM 06.021) with increased thrombolytic potency due to altered pharmacokinetic properties. The pharmacokinetics were studied in rabbits upon intravenous infusion of 200 kU/kg over 30 min. The thrombolytic dose-response effects were evaluated in a rabbit model with 125I-labeled venous thrombi upon intravenous infusion over 4 h. The thrombolytic effects after intravenous bolus injection of 200 kU/kg BM 06.021 were investigated in a canine model of coronary artery thrombosis. All studies were performed comparing BM 06.021 with glycosylated rt-PA (alteplase). BM 06.021 demonstrated a longer (p less than 0.05) half-life (5.6 +/- 2.6 vs. 2.1 +/- 0.3 min) and a lower (p less than 0.05) clearance rate (7.5 +/- 0.8 vs. 22.2 +/- 3.1 ml.min-1.kg-1) than alteplase in rabbits upon intravenous infusion. The dose-response curve of BM 06.021 for thrombolysis in a rabbit model of jugular vein thrombosis was located to the left of that for alteplase with a 2.1-fold lower effective dose of 50% thrombolysis (ED50) of BM 06.021 (207 vs. 436 kU/kg). Intravenous bolus injection of 200 kU/kg BM 06.021 induced the same reperfusion rate (4/6) as intravenous infusion of 800 kU/kg alteplase over 90 min in a canine model of coronary artery thrombosis. The residual thrombus wet weight did not significantly differ between BM 06.021 and alteplase (5.7 +/- 1.8 vs. 6.3 +/- 1.1 mg). The results indicate that unglycosylated rt-PA (BM 06.021) has a higher in vivo thrombolytic potency than glycosylated rt-PA (alteplase).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Do forehead lifts lift or unfurl?

Forehead lifts are becoming increasingly more popular as an adjunct to facial rejuvenation. Considerable confusion exists as to how much the eyebrow should be elevated and how much scalp should be removed to achieve this goal. In an effort to evaluate this question, we have reviewed our recent forehead lift experience. These were done with forehead flap dissection down to glabella and supraorbital ridge, partial resection of corrugator supercilii, procerus, and frontalis muscles, and limited skin resection. We suggest that a natural-appearing, rejuvenating forehead lift should unfurl facial wrinkles without excessive eyebrow elevation. Technical maneuvers to obtain this goal are discussed.

Forehead↗
PubMed1992

Biochemical properties of the kringle 2 and protease domains are maintained in the refolded t-PA deletion variant BM 06.022.

BM 06.022 is a t-PA deletion variant which comprises the kringle 2 and the protease domain. Production of BM 06.022 in Escherichia coli leads to the formation of inactive inclusion bodies, which have to be refolded by an in vitro refolding process to achieve activity and proper structure of the domains. We analysed the biochemical properties of BM 06.022 to obtain some information about the structure of kringle 2 and the protease as compared with the structure of these domains in the intact t-PA molecule. The kinetic analysis of the amidolytic activity of BM 06.022 and CHO-t-PA yielded similar values for kcat (13.9 s-1 and 11.4 s-1 for the single chain forms and 33.9 s-1 and 27.1 s-1 for the two chain forms of BM 06.022 and CHO-t-PA, respectively) and for Km (2.5 mM and 2.1 mM for the single chains forms and 0.5 mM and 0.3 mM for the two chain forms of BM 06.022 and CHO-t-PA, respectively). BM 06.022 and CHO-t-PA have the same plasminogenolytic activity in the absence of CNBr fragments of fibrinogen. However, BM 06.022 has a lower plasminogenolytic activity in the presence of CNBr fragments of fibrinogen and a lower affinity to fibrin as compared with CHO-t-PA. The affinity of BM 06.022 for fibrin is completely suppressed by 0.3 mM epsilon-aminocaproic acid, while the intact t-PA has a residual affinity of approximately 30%. The dissociation constants for the interaction with the lysine analogue epsilon-aminocaproic acid are 0.10 mM and 0.09 mM for BM 06.022 and the intact t-PA, respectively. Furthermore, BM 06.022 and CHO-t-PA are inhibited by PAI-1 in a similar manner.

Amidohydrolases↗
PubMed1992

Cholecystectomy in patients with sickle cell disease: experience at a regional hospital in southeast Georgia.

The treatment of patients with sickle cell disease and cholelithiasis is controversial. This retrospective study assesses the outcome of preoperative transfusion and timely cholecystectomy in symptomatic sickle cell disease patients. Fourteen patients who had undergone cholecystectomy were determined to have sickle cell disease. The patients' mean age was 17.9 years. Eleven patients were female. Thirteen patients had complained of abdominal pain. Ultrasound confirmed the diagnosis of cholelithiasis in 12 of 13 patients tested. Hemoglobin before treatment averaged 7.7 g/dL. Transfusion or exchange transfusion was given to 12 patients, raising the average hemoglobin to 10.3 g/dL. Postoperative morbidity was 14%: one patient had a urinary tract infection and another a left-lower-lobe pneumonia. No sickle cell crises or deaths occurred. Postoperative hospital stay averaged 4.4 days. With judicious use of preoperative transfusion, early cholecystectomy for symptomatic gallstones was well tolerated by sickle cell disease patients and is advisable to avoid the morbid sequelae of acute cholecystitis and peroperative sickle cell crisis.

Adolescent↗
PubMed1992

[Self-attention of psychosomatic patients].

In the present study, based on the theory of objective self-attentiveness, a sample of psychosomatic patients was compared with persons in good health in a control group. It was established that psychosomatic patients had a significantly lower habitual tendency then healthy persons to reflect upon relevant, in particular emotional, self aspects; that they had a considerably higher habitual inclination to reflect upon social appraisals and the fulfilling of social standards; furthermore, they showed a distinctly higher social anxiety and lower self-acceptance. The findings have been discussed on the background of previous findings relating to alexithymia, and to enable the findings to be interpreted a model of a self stabilizing system was proposed.

Adult↗
PubMed1991

Reconstitution of a heat shock effect in vitro: influence of GroE on the thermal aggregation of alpha-glucosidase from yeast.

alpha-Glucosidase from yeast is inactivated rapidly at temperatures above 42 degrees C. The thermal inactivation is accompanied by aggregation. The molecular chaperone GroEL suppresses the formation of aggregates by binding the thermally inactivated alpha-glucosidase. Spectroscopic studies suggest that GroEL binds alpha-glucosidase in an intermediately folded state. The complex between alpha-glucosidase and GroEL can be dissolved by MgATP. GroES accelerates the MgATP-dependent dissociation of the alpha-glucosidase-GroEL complex. At elevated temperatures this release leads to the formation of aggregates, while at lower temperatures native, enzymatically active molecules are formed.

Adenosine Triphosphate↗
PubMed1991

[Hematology and cytodiagnosis of leukosis of the horse (review)].

In a review supplementary to prior evaluations further hematological or cytodiagnostic aspects respectively of equine leucosis are discussed. Leukemic nature of the disease is characterized by increased count of normal blood cells or by the presence of morphologically non-differentiated cells in circulating blood or by disorder or loss of function of blood cells respectively. Thrombocytopenia in the most cases is accompanied by anemia, leukemia and tumor cell infiltration in bone marrow. Tumor cells in circulating blood indicate their presence in bone marrow too. In cases of equine leucosis with effusions in thoracic and/or in abdominal cavities tumor cells seem to be present more frequently in hydrothorax than in ascites.

Anemia↗
PubMed1991

[Clinical diagnostic keys and special manifestations in equine leukosis].

The literature contains about 500 cases of equine leucosis, though the reports are deposited in a great number of journals and vary considerably concerning particular topics. During the last years there has been a remarkable increase of publications about this syndrome in the equine. The clinical leucosis key recommended by us has been confirmed in principle considering the latest literature. In about 70 individual symptoms which can be clinically observed in equine with leucosis 11 can be considered as main symptoms because of their frequency; they are again classified in primary (lymph node tumours including splenomegaly--loss of condition, weakness--cachexia, weight loss, periphery oedema), secondary (anorexia, inappetence--fever--paleness of mucous membrane--anaemia--tachycardia) and accessory (incoordination--tachypnoea, dyspnoea--apathy, lethargy) main symptoms. Furthermore in future it will be necessary to take into more consideration the symptoms "recurrent colic" and "hydrothorax" within differential diagnosis. The main symptom "incoordination" (ataxia, asynergy, paresis, paralysis) is used by us more precisely only in case of impairment of nervous system by neoplastic infiltrations and does not signify as possible symptoms of general physical weakness, for example faltering, staggering, tumbling or lameness. The morphological classification follows further on our previous recommendation. There exist generalized forms with tumour infiltrations in abdominal and in thoracic cavity as well as especially in peripheral lymph nodes. On the other hand there are characteristic manifestations in certain regions of the body, which establish distinctly the clinical symptomatology. They are marked as regional multicentric forms with the main localizations "mediastinal", "splenic", "mesenteric" or "intestinal".(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Alternatively folded states of an immunoglobulin.

Well-defined, non-native protein structures of low stability have been increasingly observed as intermediates in protein folding or as equilibrium structures populated under specific solvent conditions. These intermediate structures, frequently referred to as molten globule states, are characterized by the presence of secondary structure, a lack of significant tertiary contacts, increased hydrophobicity and partial specific volume as compared to native structures, and low cooperativity in thermal unfolding. The present study demonstrates that under acidic conditions (pH less than 3) the antibody MAK33 can assume a folded stable conformation. This A-state is characterized by a high degree of secondary structure, increased hydrophobicity, a native-like maximum wavelength of fluorescence emission, and a tendency toward slow aggregation. A prominent feature of this low-pH conformation is the stability against denaturant and thermal unfolding that is manifested in highly cooperative reversible phase transitions indicative of the existence of well-defined tertiary contacts. These thermodynamic results are corroborated by the kinetics of folding from the completely unfolded chain to the alternatively folded state at pH 2. The given data suggest that MAK33 at pH 2 adopts a cooperative structure that differs from the native immunoglobulin fold at pH 7. This alternatively folded state exhibits certain characteristics of the molten globule but differs distinctly from it by its extraordinary structural stability that is characteristic for native protein structures.

Amino Acids↗
PubMed1991

Thrombolysis with an Escherichia coli-produced recombinant plasminogen activator (BM 06.022) in the rabbit model of jugular vein thrombosis.

The recombinant plasminogen activator BM 06.022 consists of the kringle 2 and the protease domains of human t-PA and is unglycosylated because of the expression in Escherichia coli. The thrombolytic and pharmacokinetic properties as well as the hemostasis effects of BM 06.022 were investigated in the rabbit model of jugular vein thrombosis. The thrombi were 125I-fibrin labeled. Intravenous bolus injection of 50, 100, 200, and 400 kU/kg BM 06.022 or 400, 800, and 1600 kU/kg alteplase over 15 s to six rabbits/dose produced a dose-dependent increase of thrombolysis determined 2 h post injection. The dose-response curve of BM 06.022 was located left compared with that of alteplase. The effective dose of 50% thrombolysis (ED50) obtained by half-logarithmic regression analysis was 163 kU/kg (= 0.28 mg/kg) for BM 06.022 and 871 kU/kg (= 1.09 mg/kg) for alteplase. At equipotent doses (50% thrombolysis), the residual concentration of fibrinogen was 74.2% and 76.5%, that of plasminogen 66.7% and 69.4%, and that of alpha 2-antiplasmin 47.3% and 46% for BM 06.022 and alteplase, respectively. Pharmacokinetic analysis for plasma activity at a dose of 400 kU/kg revealed a half-life of 18.9 +/- 1.5 min for BM 06.022, whereas alteplase was distributed with a half-life of 2.1 +/- 0.1 min, accounting for 86.7 +/- 1.9% of the total AUC, followed by a beta-phase with a half-life of 13.8 +/- 0.9 min. Plasma clearance of BM 06.022 was 4.7 +/- 0.7 ml min-1 kg-1 compared with 20 +/- 1.2 ml min-1 kg-1 for alteplase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Pharmacokinetic properties of an Escherichia-coli-produced recombinant plasminogen activator (BM 06.022) in rabbits.

The recombinant plasminogen activator BM 06.022 consists of the kringle 2 and the protease domains of human t-PA and is unglycosylated because of its expression in Escherichia coli. The pharmacokinetic properties of BM 06.022 following intravenous injection over 1 min were characterized in anesthetized male New Zealand white rabbits. BM 06.022 was injected at doses of 50, 100, 200, and 400 kU/kg bw (n = 5-6/dose). Activity concentrations in plasma were determined using an indirect spectrophotometric assay. The maximum plasma concentration and the area under the plasma concentration vs. time curve (AUC0-00) of BM 06.022 increased linearly with dose. The systemic clearance ranged from 2.5 to 3.0 ml.min-1.kg-1 and did not show dose-dependency, in contrast to alteplase which was studied at doses of 200, 400, 800, and 1600 kU/kg. A direct comparison of clearance rates of BM 06.022 and alteplase at doses of 200 and 400 kU/kg each revealed a 8.5-fold slower clearance rate of BM 06.022. The majority (18/23) of rabbits with BM 06.022 injection showed a pharmacokinetic profile which was best characterized by a one-compartment model in contrast to alteplase (10/23). The dose-groups of BM 06.022 showed an average dominant half-life ranging from 11.6 to 15.4 min, which was about five-times longer than the dominant half-life values of alteplase (2.3 to 4.5 min). Assuming a two-compartment model in the remaining animals, the initial alpha-phase of BM 06.022 accounted for 40.1 +/- 13.2% (n = 5) of the total AUC, whereas the alpha-phase of alteplase accounted for 82.7 +/- 3% (n = 13) of the total AUC.

Animals↗
PubMed1991

A kinetic study of the competition between renaturation and aggregation during the refolding of denatured-reduced egg white lysozyme.

The recovery of proteins following denaturation is optimal at low protein concentrations. The decrease in yield at high concentrations has been explained by the kinetic competition of folding and "wrong aggregation". In the present study, the renaturation-reoxidation of hen and turkey egg white lysozyme was used as a model system to analyze the committed step in aggregate formation. The yield of renatured protein for both enzymes decreased with increasing concentration in the folding process. In addition, the yield decreased with increasing concentrations of the enzyme in the denatured state (i.e., prior to its dilution in the renaturation buffer). The kinetics of renaturation of turkey lysozyme were shown to be very similar to those of hen lysozyme, with a half-time of about 4.5 min at 20 degrees C. The rate of formation of molecular species that lead to formation of aggregates (and therefore fail to renature) was shown to be rapid. Most of the reaction occurred in less than 5 s after the transfer to renaturation buffer, and after 1 min, the reaction was essentially completed. Yet, by observing the effects of the delayed addition of denatured hen lysozyme to refolding turkey lysozyme, it was shown that folding intermediates become resistant to aggregation only much more slowly, with kinetics indistinguishable from those observed for the appearance of native molecules. The interactions leading to the formation of aggregates were nonspecific and do not involve disulfide bonds. These observations are discussed in terms of possible kinetic and structural aspects of the folding pathway.

Animals↗
PubMed1991

GroE facilitates refolding of citrate synthase by suppressing aggregation.

The molecular chaperone GroE facilitates correct protein folding in vivo and in vitro. The mode of action of GroE was investigated by using refolding of citrate synthase as a model system. In vitro denaturation of this dimeric protein is almost irreversible, since the refolding polypeptide chains aggregate rapidly, as shown directly by a strong, concentration-dependent increase in light scattering. The yields of reactivated citrate synthase were strongly increased upon addition of GroE and MgATP. GroE inhibits aggregation reactions that compete with correct protein folding, as indicated by specific suppression of light scattering. GroEL rapidly forms a complex with unfolded or partially folded citrate synthase molecules. In this complex the refolding protein is protected from aggregation. Addition of GroES and ATP hydrolysis is required to release the polypeptide chain bound to GroEL and to allow further folding to its final, active state.

Animals↗
PubMed1991

Routes to active proteins from transformed microorganisms.

Over-expression of recombinant proteins in microbial hosts results in the formation of active soluble protein or of insoluble aggregates (inclusion bodies). Efficient in vitro refolding strategies have been developed to reactivate inactive proteins from inclusion bodies. Co-expression of molecular chaperones may provide a tool to promote correct structure formation of recombinant proteins in vivo.

Cloning, Molecular↗