Biomedical subjects

R Rudolph

Publications and source records attributed to R Rudolph.

At least 109 records · Page 6Linked to original sources

PubMed1991

Renaturation, purification and characterization of recombinant Fab-fragments produced in Escherichia coli.

Cytoplasmatic expression of murine antibody chains in Escherichia coli results in the formation of insoluble and inactive protein aggregates (inclusion bodies). By systematic variation of the parameters influencing the folding, formation of disulfide bonds and association of the constituent polypeptide chains, we have designed a renaturation procedure allowing the production of microbially expressed Fab-fragments at yields up to 40 percent of the total amount of recombinant protein. The strategy of optimization is generally applicable for disulfide containing proteins produced as inclusion bodies in bacteria. The purified recombinant antibody fragments obtained are identical with the native murine Fab in all functional and physicochemical parameters tested.

Animals↗

PubMed1991

Protein aggregation in vitro and in vivo: a quantitative model of the kinetic competition between folding and aggregation.

Protein aggregation is frequently observed as a major side-reaction of protein folding. We present quantitative models explaining the formation of aggregates during protein folding in vitro and in vivo on the basis of a kinetic competition between correct folding and aggregation reactions. Both models are in good agreement with experimental data. The model implies that, in vitro, the yield of native protein obtained upon refolding is determined by the rates of the competing first order folding and second order aggregation reactions. Therefore, a high protein concentrations aggregation dominates over folding and leads to the formation of insoluble protein. For in vivo protein synthesis, the model shows that the yield of native protein is only dependent on the rate of folding, on the rate of aggregation and on the rate of protein synthesis. In the cell, several mechanisms, including "folding helpers" seem to have evolved, which influence these processes and thereby prevent unproductive side reactions.

Kinetics↗

PubMed1991

Coronary thrombolytic properties of a novel recombinant plasminogen activator (BM 06.022) in a canine model.

We studied the thrombolytic dose-response relationship of a recombinant plasminogen activator (rPA) (BM 06.022) compared with alteplase in a canine model of coronary artery thrombosis. BM 06.022 consists of the kringle 2 and protease domains of human tissue PA (tPA) and lacks oligosaccharide side chains because of its expression in Escherichia coli. Thrombus formation in anesthetized, open-chest dogs was induced by electrical injury to the intimal surface of the left circumflex coronary artery in the presence of a critical stenosis. Intravenous bolus injection of BM 06.022 (50, 100, 140, and 200 kU/kg) or of alteplase (200, 800, 1,130, and 1,600 kU/kg) 30 min after coronary occlusion to six heparinized dogs per group achieved a dose-dependent increase in reperfusion rate and decrease in residual thrombus wet weight. Vehicle-treated dogs did not reperfuse. Semilogarithmic regression analysis showed that the effective dose that produced 50% reperfusion of BM 06.022 (83 kU/kg) was 11.6-fold lower than that of alteplase (951 kU/kg). Comparison with infusion experiments showed that intravenous bolus injection of 140 kU/kg of BM 06.022 was equieffective to a 90-min infusion of 800 kU/kg (= 1 mg/kg) of alteplase as a standard treatment regarding reperfusion rate (66%) and time to reperfusion (15 +/- 6 vs. 18 +/- 8 min). Pharmacokinetic analysis for functionally active BM 06.022 or alteplase in plasma revealed a total plasma clearance of 4.1-6.6 ml/min/kg for BM 06.022 and of 12.6-42.3 ml/min/kg for alteplase.(ABSTRACT TRUNCATED AT 250 WORDS)

Anesthesia↗

PubMed1991

Analysis of protein folding by fast protein liquid chromatography. Modular domain folding of gamma-II-crystallin from calf eye-lens.

Fast protein liquid chromatography was effectively applied to analyse the folding mechanism of gamma-II-crystallin from calf eye-lens. The protein undergoes a bimodal folding/unfolding transition, according to a three-state model: N in equilibrium I in equilibrium D where N, I, and D stand for the native, intermediate and denatured states (R. Rudolph, R. Siebendritt, G. Nesslauer, A.K. Sharma & R. Jaenicke (1990) Proc. Natl. Acad. Sci. USA 87, 4625-4629). Using Superose 12 HR 10/30, the intermediate with the N-terminal domain intact, and the C-terminal domain unfolded, could be separated from the native protein. The N----I transition is sufficiently slow to allow kinetic measurements, following the variation of the respective peak-heights during denaturation/renaturation. The corresponding relaxation times are in agreement with kinetic data based on the change in fluorescence emission accompanying the N in equilibrium I transition.

Animals↗

PubMed1991

The myofibroblast in Dupuytren's contracture.

Dupuytren's contracture nodules, but not cords, contain myofibroblasts. These cells, which combine many electron microscopic, physiologic, and immunohistochemical characteristics of fibroblasts and smooth muscle cells, are probably the active force of contraction. Prominent myofibroblasts and intracellular microtubules correlate with increased likelihood of clinical recurrence after surgery. Tissue culture of cells derived from Dupuytren's contracture myofibroblasts show consistently slower cell replication than from fibroblasts and show persistence of electron microscopic characteristics in early passages. Research in Dupuytren's contracture myofibroblasts has been done on human tissue and so has clinical correlation. Myofibroblast presence may help to predict recurrence of disease and suggests that palmar skin should be excised when adherent to disease nodules. The theory of myofibroblasts helps explain why the open technique often succeeds, and why full thickness skin grafts inhibit recurrent contracture.

Dupuytren Contracture↗

PubMed1991

Venlafaxine in depressed outpatients.

Venlafaxine is a structurally novel compound with a biochemical and pharmacological profile suggesting antidepressant properties. We report the results of a Phase II, double-blind, placebo-controlled clinical trial assessing the efficacy and safety of venlafaxine in a sample of 93 depressed outpatients. Venlafaxine doses of 25 mg t.i.d., 75 mg t.i.d., and 125 mg t.i.d. were compared to placebo. Patients receiving venlafaxine showed a significantly greater improvement in their mood symptoms compared to those receiving placebo. Venlafaxine was well tolerated and the most common side effect was nausea. There was some evidence to suggest that venlafaxine may have antidepressant activity within the first 2 weeks of treatment.

Adult↗

PubMed1991

Platelet-derived growth factor-BB and transforming growth factor beta 1 selectively modulate glycosaminoglycans, collagen, and myofibroblasts in excisional wounds.

Recombinant platelet-derived growth factor (PDGF) and transforming growth factor beta 1 (TGF-beta 1) influence the rate of extracellular matrix formed in treated incisional wounds. Because incisional healing processes are difficult to quantify, a full-thickness excisional wound model in the rabbit ear was developed to permit detailed analyses of growth-factor-mediated tissue repair. In the present studies, quantitative and qualitative differences in acute inflammatory cell influx, glycosaminoglycan (GAG) deposition, collagen formation, and myofibroblast generation in PDGF-BB (BB homodimer)- and TGF-beta 1-treated wounds were detected when analyzed histochemically and ultrastructurally. Although both growth factors significantly augmented extracellular matrix formation and healing in 10-day wounds compared with controls (P less than 0.002). PDGF-BB markedly increased macrophage influx and GAG deposition, whereas TGF-beta 1 selectively induced significantly more mature collagen bundles at the leading edge of new granulation tissue (P = 0.007). Transforming growth factor-beta 1-treated wound fibroblasts demonstrated active collagen fibrillogenesis and accretion of subfibrils at the ultrastructural level. Myofibroblasts, phenotypically modified fibroblasts considered responsible for wound contraction, were observed in control, but were absent in early growth-factor-treated granulating wounds. These results provide important insights into the mechanisms of soft tissue repair and indicate that 1) PDGF-BB induces an inflammatory response and provisional matrix synthesis within wounds that is qualitatively similar but quantitatively increased compared with normal wounds; 2) TGF-beta 1 preferentially triggers synthesis and more rapid maturation of collagen within early wounds; and 3) both growth factors inhibit the differentiation of fibroblasts into myofibroblasts, perhaps because wound contraction is not required, due to increased extracellular matrix synthesis.

Animals↗

PubMed1990

Limited proteolysis of gamma II-crystallin from calf eye lens. Physicochemical studies on the N-terminal domain and the intact two-domain protein.

Gamma II-crystallin from calf eye lens consists of two homologous domains, connected by a six-residue linker peptide. In order to study the intrinsic properties of the domains and their mutual stabilization, limited proteolysis was applied. Optimum conditions providing a homogeneous 10-kDa fragment at high yield were pepsin cleavage in 0.1 M NaCl/HCl pH 2.0, in the presence of 3.0 M urea. Determination of the N-terminus and the C-terminal sequence showed that cleavage occurred at the Phe88-Arg89 peptide bond, giving rise to the complete N-terminal domain including the connecting hexapeptide. The C-terminal part of the polypeptide chain is cleaved to small fragments. Comparing the spectral properties of the isolated N-terminal domain and intact gamma II-crystallin proved the structure of the fragment to be closely similar to that of the native domain. Small differences in absorbance, fluorescence emission and circular dichroism point to alterations caused by the increase in surface area as a consequence of domain separation. The resistance of the 10-kDa fragment toward thermal and alkaline denaturation, as well as unfolding in the presence of urea or guanidine . HCl is decreased, due to the lack of domain interactions stabilizing the intact protein. Unfolding/folding kinetics of the 10-kDa fragment coincide with the second phase of the bimodal transition of intact gamma II-crystallin, in agreement with independent sequential folding and modular assembly of the domains within the native molecule.

Animals↗

PubMed1990

Structural and catalytic properties of oxidized and reduced chloroplast NADP-malate dehydrogenase upon denaturation and renaturation.

Chloroplast NADP-dependent malate dehydrogenase exists in two interconvertible forms: the inactive disulfide-containing form and the active dithiol form. No major difference in secondary structure or conformation was found between the oxidized and the reduced enzyme as determined by circular dichroism and intrinsic protein fluorescence. The guanidine/HCl-dependent unfolding of the enzyme is characterized by two transition midpoints: those of the reduced enzyme are lower by about 0.2 M guanidine/HCl compared to the oxidized enzyme. As shown by analytical ultracentrifugation, there was no effect of guanidine/HCl concentrations up to 0.25 M on the quaternary structure of the enzyme in its oxidized and reduced forms: both sedimentation coefficient (S20,w = 4.9 +/- 0.1 S) and sedimentation equilibrium (75 +/- 3 kDa) yield the dimer. In the oxidized state the enzyme undergoes guanidine-dependent dissociation to the monomer with a midpoint of transition at 0.5 M. The kinetics of unfolding were found to be significantly faster for the reduced than for the oxidized enzyme. Renaturation and reactivation of reduced enzyme was more rapid and occurred with higher yields (100%) than for the oxidized enzyme (60-80% yield). Furthermore, the effect of denaturants on catalytic activity, and reductive activation of the oxidized form, were studied. Both increase in protein fluorescence and a stimulatory effect on the activities at low guanidine/HCl concentrations were observed for the oxidized and the reduced form of the enzyme. Denaturants increase the rate of reductive activation of NADP-malate dehydrogenase.

Catalysis↗

PubMed1990

Folding of an all-beta protein: independent domain folding in gamma II-crystallin from calf eye lens.

gamma II-crystallin from calf eye lens consists of two homologous domains, each composed of two similar "Greek key" motifs. As a consequence of the bilobal structure, a biphasic transition is seen upon unfolding by urea at low pH (monitored by circular dichroism, fluorescence emission, and ultracentrifugal analysis). In 3.3 +/- 0.5 M urea, a stable intermediate is formed at equilibrium, whereas 5.5 M urea causes maximum denaturation. Unfolding/folding kinetics display a complex pattern characterized by two kinetic phases. Both reactions exhibit strong dependence on the urea concentration; in the range of the respective transition, their rates are extremely slow (k approximately 1 x 10(-4)s-1). The kinetic mechanism of unfolding and refolding may be described by a three-state model: native in equilibrium intermediate in equilibrium denatured. The rate-determining steps are domain folding rather than domain pairing or proline isomerization. Kinetic analysis of the unfolding/folding of the intermediate populated in 3.0 M urea, pH 2.0, reveals that the kinetic and the equilibrium intermediates have similar structures. Limited proteolysis of gamma II-crystallin by pepsin in 3 M urea, pH 2, allows the NH2-terminal domain of the protein to be isolated. Unfolding/refolding of the fragment parallels the second transition in the above scheme, thus proving that the intermediate contains the COOH-terminal domain in its random state, whereas the NH2-terminal domain is still in its native conformation. In conclusion, folding of gamma II-crystallin proceeds through the independent sequential structuring of the domains.

Animals↗

PubMed1990

Depth of the facial nerve in face lift dissections.

Facial nerve depth was measured in 12 cadaver face halves after bilateral face lift dissections. The main nerve trunk emerged anterior to the midearlobe and was 20.1 +/- 3.1 mm deep. Nerve exit from the parotid edge also was deep, averaging 9.1 +/- 2.8 mm for temporal, 9.2 +/- 2.2 mm for zygomatic, 9.6 +/- 2.0 mm for buccal, and 10.6 +/- 2.7 mm for mandibular branches. Distal to the parotid gland, danger areas where nerve branches became superficial were distal temporal, lower buccal, and upper mandibular branches over the masseter muscle and marginal mandibular as it crossed the facial artery. Some protection in these danger areas was provided by fascia, especially superficial temporal and masseteric, while platysma provided some protection for the mandibular branch. Fascial and muscle protection was less in thin cadavers. Face lift dissection can be rapid in areas where facial nerve branches are deep or absent, such as postauricular, inferior to the zygomatic prominence, and near the earlobe.

Facial Nerve↗

PubMed1990

Cell adhesion mechanisms and their potential impact on wound healing and tumor control.

Cells attach to surrounding extracellular protein matrix via structural receptors called integrins. Many of the integrins attach to a specific amino acid sequence in structural proteins, that of arginine-glycine-aspartic acid (RGD). These structural receptors are composed of alpha and beta subunits, which vary depending on which cells are involved and on which specific protein is to be bound. The integrin-matrix bond can be inhibited by monoclonal antibodies directed against receptor subunits, or by synthetic proteins (peptides) that contain the RGD sequence. This inhibition of cell adhesion has important potential for cellular control of all phases of wound healing, as well as of malignant cell growth and metastasis.

Blood Proteins↗

PubMed1990

Properties of a novel plasminogen activator (BM 06.022) produced in Escherichia coli.

Since currently available thrombolytics still show disadvantages, such as administration by infusion, occurrence of intracranial hemorrhage, major hemorrhagic complications, allergic reactions, and high price, a novel tissue plasminogen activator has been developed. BM 06.022 is a t-PA mutant produced in Escherichia coli by DNA technology. It has no oligosaccharide side-chains and comprises the kringle 2- and protease domains of t-PA. Like t-PA, the enzymatic activity of BM 06.022 can be stimulated by fibrin. However, BM 06.022 binds to neither endothelial cells nor fibrin. Despite this, BM 06.022 demonstrates the same fibrin selectivity in vivo as t-PA. Investigation of the pharmacokinetic properties in rats, rabbits, dogs, and primates reveals, depending on species, a 4.5- - 10.4-fold longer dominant half-life and 3- - 8.4-fold slower plasma clearance than t-PA (alteplase). In spite of its lower specific activity in vitro, BM 06.022 has a thrombolytic potency which is 4.6 - 11.5 times higher in vivo than that of alteplase in the rabbit model of venous thrombosis and the canine model of coronary artery thrombosis. BM 06.022 achieves reperfusion significantly more rapid than long-acting anistreplase. Therefore, because of its improved pharmacokinetic properties, BM 06.022 might be administered to infarct patients by i.v. injection in the pre-hospital phase to achieve rapid lysis. Due to its lack of fibrin- and endothelial-cell-binding, combined with retained fibrin selectivity, BM 06.022 further promises to induce fewer hemorrhagic complications than either t-PA or streptokinase.

Animals↗

PubMed1990

Healing in the irradiated wound.

Poor or nonhealing of irradiated wounds has been attributed to progressive obliterative endarteritis. Permanently damaged fibroblasts may also play an important part in poor healing. Regardless of the cause, the key to management of irradiated skin is careful attention to prevent its breakdown and conservative, but adequate, treatment when wounds are minor. When wounds become larger and are painful, complete excision of the wound or ulcer is called for and coverage should be provided by a well-vascularized nonparasitic distant flap.

Humans↗

PubMed1990

Massive transfusion: complications and their management.

Massive transfusion, or the rapid administration of a quantity of blood products that approximates an individual's blood volume, is associated with many potentially lethal complications. If the need for transfusion is immediate, ie, before adequate typing and crossmatching procedures can be completed, O negative RBCs can be given safely in the interim. Hypothermia caused by cold banked blood is aggravated by multiple environmental factors and should be aggressively avoided through the use of heat lamps, warming coils, blankets, and other warming devices. The coagulopathy seen in massive transfusion probably has a mixed etiology involving dilution and consumption of clotting factors and platelets. Although fresh frozen plasma and platelets both play a critical role in blood replacement, deficiencies should be treated with appropriate component therapy dictated by coagulation studies rather than by protocol. Transfusion reactions, the most serious type of which is the hemolytic reaction, may go unrecognized in the bleeding patient in critical condition. Hemolytic reactions can usually be prevented by careful attention to administrative and clerical accuracy. Although the overwhelming majority of the 10 million units of blood transfused annually are uncontaminated, transmission of hepatitis and the human immunodeficiency virus through blood products remains a significant screening problem. Posttransfusion hyperkalemia and acidosis are more likely to be related to inadequate resuscitation from shock than to administration of blood. Citrate toxicity and hypocalcemia are usually self-limiting disturbances. Prophylactic use of calcium chloride is dangerous and unnecessary. The complexity of the conditions necessitating massive transfusion demands frequent reevaluation of multiple laboratory and clinical factors for effective resuscitation and for safe administration of blood.

Blood Coagulation Disorders↗

PubMed1989

A carboxypeptidase Y pulse method to study the accessibility of the C-terminal end during the refolding of ribonuclease A.

Carboxypeptidase Y pulses, applied after various times of refolding, were employed to probe the accessibility of the C-terminus of RNAase A during the refolding process. The increase in resistance against proteolytic cleavage was measured by determination of the amount of liberated C-terminal amino acids and by activity assays. The results indicate that the C-terminus of RNAase becomes inaccessible early in the course of refolding, if folding is carried out at low temperatures under conditions that effectively stabilize the native state. At higher temperatures (25 degrees C) or under conditions of marginal stability, intermediates are not populated and protection against proteolytic cleavage is not detectable before the formation of the native state. The method described may be used to monitor the accessibility of the C-terminus of various proteins during refolding. However, intermediates on the folding pathway can only be observed if the native state is stable against carboxypeptidase attack.

Carboxypeptidases↗

PubMed1989

Usefulness of data on albumin and prealbumin concentrations in determining effectiveness of nutritional support.

In this ongoing study, albumin and prealbumin (transthyretin) changes were compared in 40 patients managed with enteral and (or) parental support with attainment of caloric/protein goals. The concentration of prealbumin in serum changed rapidly and more accurately reflected current nutritional status of these patients than did that of albumin. We determined concentrations of albumin and prealbumin that reflected significant improvement in nutritional status, using Rudolph's approach based on Shannon information measures. Reference values for albumin and prealbumin in the treatment populations were 25 g/L and 107 mg/L, respectively. A prealbumin concentration of 135 mg/L or greater reflected a return to stable status.

Enteral Nutrition↗

PubMed1989

[The frequency and pathogenesis of feline infectious peritonitis (FIP)].

Between 1980 and 1987 about 10% of the cats which underwent a post mortem examination at the Institute of Veterinary Pathology of the Freie Universität Berlin were infected with feline infectious peritonitis (FIP). The exudative, granulomatous or mixed form of the FIP are all symptoms of the same disease whose clinical picture is dependent on the state of the cellular immunity. Statistically there is no significant relationship between the form of the FIP and the age of the cats. A breed or a sex disposition is also not apparent. 42 organs and/or tissue samples were taken from a total of 30 cats and were examined both histologically and immunohistochemically. The antigen is located, above all, in the mesothelium and the cells of the mononuclear phagocyte system, whereas the parenchymatous inflammation foci show little evidence of the FIP-virus-antigen. The antigen is, however, also found in the nerve cells. Within the framework of a second viraemia, occurring after infection of the mesothelium (polyserositis), a perivasculitis non purulenta generalisata occurs. Possible excretory organs of the antigen are the respiratory, digestive and urinary tract.

Animals↗