Biomedical subjects

R Rudolph

Publications and source records attributed to R Rudolph.

At least 55 records · Page 3Linked to original sources

PubMed1996

Enzyme flexibility, solvent and 'weak' interactions characterize thrombin-ligand interactions: implications for drug design.

BACKGROUND: The explosive growth in the rate of X-ray determination of protein structures is fuelled largely by the expectation that structural information will be useful for pharmacological and biotechnological applications. For example, there have been intensive efforts to develop orally administrable antithrombotic drugs using information about the crystal structures of blood coagulation factors, including thrombin. Most of the low molecular weight thrombin inhibitors studied so far are based on arginine and benzamidine. We sought to expand the database of information on thrombin-inhibitor binding by studying new classes of inhibitors. RESULTS: We report the structures of three new inhibitors complexed with thrombin, two based on 4-aminopyridine and one based on naphthamidine. We observe several geometry changes in the protein main chain and side chains which accompany inhibitor binding. The two inhibitors based on 4-aminopyridine bind in notably different ways: one forms a water-mediated hydrogen bond to the active site Ser195, the other induces a rotation of the Ser214-Trp215 peptide plane that is unprecedented in thrombin structures. These binding modes also differ in their 'weak' interactions, including CH-O hydrogen bonds and interactions between water molecules and aromatic pi-clouds. Induced-fit structural changes were also seen in the structure of the naphthamidine inhibitor complex. CONCLUSIONS: Protein flexibility and variable water structures are essential elements in protein-ligand interactions. Ligand design strategies that fail to take this into account may overlook or underestimate the potential of lead structures. Further, the significance of 'weak' interactions must be considered both in crystallographic refinement and in analysis of binding mechanisms.

4-Aminopyridine↗

PubMed1996

The 2.3 A crystal structure of the catalytic domain of recombinant two-chain human tissue-type plasminogen activator.

Tissue-type plasminogen activator (t-PA), a multidomainal serine proteinase of the trypsin-family, catalyses the rate-limiting step in fibrinolysis, the activation of plasminogen to the fibrin-degrading proteinase plasmin. Trigonal crystals have been obtained of the recombinant catalytic domain of human-two-chain t-PA, consisting of a 17 residue A chain and the 252 residue B chain. Its X-ray crystal structure has been solved applying Patterson and isomorphous replacement methods, and has been crystallographically refined to an R-value of 0.184 at 2.3 A resolution. The chain fold, active-site geometry and Ile276-Asp477 salt bridge are similar to that observed for trypsin. A few surface-located insertion loops differ significantly, however. The disulfide bridge Cys315-Cys384, practically unique to the plasminogen activators, is incorporated without drastic conformational changes as the insertion loop preceding Cys384 makes a bulge on the molecular surface. The unique basic insertion loop Lys296-Arg304 flanking the primed subsites, which has been shown to be of importance for PAI-1 binding and for fibrin specificity, is partially disordered; it can therefore freely adapt to proteins docking to the active site. The S1 pocket of t-PA is almost identical to that of trypsin, whereas the S2 site is considerably reduced in size by the imposing Tyr368 side-chain, in agreement with the measured preference for P1 Arg and P2 Gly residues. The neighbouring S3-S4 hydrophobic groove is mainly hydrophobic in nature. The structure of the proteinase domain of two-chain t-PA suggests that the formation of a salt bridge between Lys429 and Asp477 may contribute to the unusually high catalytic activity of single-chain t-PA, thus stabilizing the catalytically active conformation without unmasking the Ile276 amino terminus. Modeling studies show that the covalently bound kringle 2 domain in full-length t-PA could interact with an extended hydrophobic groove in the catalytic domain; in such a docking geometry its "lysine binding site" and the "fibrin binding patch" of the catalytic domain are in close proximity.

Amino Acid Sequence↗

PubMed1996

Improved refolding of an immobilized fusion protein.

Fusion proteins of monomeric alpha-glucosidase from Saccharomyces cerevisiae containing N- or C-terminal hexa-arginie peptides were expressed in the cytosol of Escherichia coli in soluble form. The polycationic peptide moieties allow noncovalent binding of the denatured fusion proteins to a polyanionic solid support. Upon removal of the denaturant, refolding of the matrix-bound protein can proceed without perturbation by aggregation. However, nonspecific interactions of the denatured polypeptide, or of folding intermediates, with the matrix cause a drastic decrease in renaturation under suboptimal folding conditions. At low salt concentrations, ionic interactions of the refolding polypeptide with the matrix result in lower yields of renaturation. At higher salt concentrations, renaturation is prevented by hydrophobic interactions with the matrix. Apart from ionic strength, renaturation of the denatured matrix-bound fusion protein must be optimized with respect to pH, temperature, cosolvents, and matrix material used. Under optimum conditions, immobilized alpha-glucosidase can be renatured with a high yield at protein concentrations up to 5 mg/ml, whereas folding of the wild-type enzyme in solution is feasible only at an extremely low protein concentration (15 micrograms/ml). Thus, folding of the immobilized alpha-glucosidase allows an extremely high yield of the renaturated model protein. The technology should be applicable to other proteins that tend to aggregate during refolding.

Biotechnology↗

PubMed1996

A fusion protein designed for noncovalent immobilization: stability, enzymatic activity, and use in an enzyme reactor.

We have designed a new method for enzyme immobilization using a fusion protein of yeast alpha-glucosidase containing at its C-terminus a polycationic hexa-arginine fusion peptide. This fusion protein can be directly adsorbed from crude cell extracts on polyanionic matrices in a specific, oriented fashion. Upon noncovalent immobilization by polyionic interactions, the stability of the fusion protein is not affected by pH-, urea-, or thermal-denaturation. Furthermore, the enzymatic properties (specific activity at increasing enzyme concentration, Michaelis constant, or activation energy of the enzymatic reaction) are not influenced by this noncovalent coupling. The operational stability of the coupled enzyme under conditions of continuous substrate conversion is, however, increased significantly compared to the soluble form. Fusion proteins containing polyionic peptide sequences are proposed as versatile tools for the production of immobilized enzyme catalysts.

Bioreactors↗

PubMed1996

Systemic, metastatic, eu- and heterotope tumours of the heart in necropsied dogs.

10,090 dissections of dogs exhibited 2631 neoplastic processes of which 309 involved the heart in the form of eu- and heteropic tumours. The most common primary and/or secondary heart tumour type was hemangiosarcoma (n = 187), followed by paraganglioma (n = 46), carcinoma (n = 33), malignant lymphoma (n = 12), thyroid heart base tumour (n = 9), melanoma (n = 7), mast cell tumour (n = 3) and blastoma (n = 2). The tumour diagnoses were immunohistochemically proved by various antibodies to cytokeratins, vimentin, GFAP, NSE, von Willebrand factor, CD3, CD45RA, S100, thyroglobulin as well as histochemically with argyrophilic, Fontana-Masson and heterochromatic reactions. The odds ratio (OR) for breed and tumour prevalences were determined: German shepherds showed the highest OR for hemangiosarcomas and boxers for paragangliomas.

Animals↗

PubMed1996

In vitro folding of inclusion body proteins.

Insoluble, inactive inclusion bodies are frequently formed upon recombinant protein production in transformed microorganisms. These inclusion bodies, which contain the recombinant protein in an highly enriched form, can be isolated by solid/liquid separation. After solubilization, native proteins can be generated from the inactive material by using in vitro folding techniques. New folding procedures have been developed for efficient in vitro reconstitution of complex hydrophobic, multidomain, oligomeric, or highly disulfide-bonded proteins. These protocols take into account process parameters such as protein concentration, catalysis of disulfide bond formation, temperature, pH, and ionic strength, as well as specific solvent ingredients that reduce unproductive side reactions. Modification of the protein sequence has been exploited to improve in vitro folding.

Inclusion Bodies↗

PubMed1996

Venlafaxine for panic disorder: results from a double-blind, placebo-controlled study.

Venlafaxine has demonstrated efficacy for depression, and recent reports and clinical experience suggest that it may be effective for the treatment of anxiety disorders as well. We present what we believe are the first data from a controlled study designed to test the efficacy of venlafaxine for the treatment of panic disorder. There were 25 patients enrolled at one site of a five-center study; 13 received venlafaxine and 12 received placebo. There were more dropouts for placebo than for venlafaxine (8/12 vs. 2/13) in this 8-week acute trial. Patients treated with venlafaxine experienced significantly greater global improvement than those on placebo and exhibited trends toward greater improvement on anxiety and depression symptoms as assessed by the Hamilton rating scales. These data encourage further evaluation of venlafaxine for the treatment of panic disorder.

Adult↗

PubMed1995

Association of antibody chains at different stages of folding: prolyl isomerization occurs after formation of quaternary structure.

The folding pathways of multi-domain proteins are still poorly understood due to the complexity of the reaction involving domain folding, association and, in many cases, prolyl cis/trans isomerization. Here, we have established a kinetic model for the folding of the Fab fragment of the antibody MAK 33 with intact disulfide bonds. Folding of the hetero-dimeric protein from the completely denatured, oxidized state comprises the pairwise association of the two domains of each chain with those of the partner protein. Both the reactivation of the Fab fragment in which the two constituent polypeptide chains were covalently linked via a cystine bond (Fab) and that of a mutant lacking this covalent linkage (Fab/-cys) were monitored by ELISA. Folding of the Fab fragment is a slow process, which can be described by a single exponential term. The kinetic phase reflects a folding step after the association of the two chains. The same reaction was detected in the folding of Fab/-cys but an additional rate-limiting step is involved that is due to a unimolecular step in the folding of the isolated light chain. This implies that, during Fab reactivation, Fd associates with the light chain at the stage of an earlier folding intermediate, thus eliminating the additional slow folding step of the light chain observed with Fab/-cys. Both in Fab and Fab/-cys renaturation, the folding reaction after association is determined by prolyl isomerization. Therefore, at least four different association-competent folding intermediates have to be postulated according to the folding stage of light chain and the configuration of at least one prolyl-peptide bond. Using the different substrate specificities of cyclophilin and FK506 binding protein, we have obtained evidence that Pro159 within the Fd fragment may be responsible for the observed slow folding phase after association, although three other proline residues adopt a cis configuration in the native protein. Furthermore, the data suggest that in the case of the Fab fragment, association is a prerequisite for cis/trans isomerization of prolyl peptide bonds, implying that the quaternary but not the tertiary structure determines the cis-configuration of the prolyl residue in Fd involved in the rate-limiting folding reaction.

Amino Acid Sequence↗

PubMed1995

Pressure (decubitus) ulcer: variation in histopathology--a light and electron microscope study.

The histopathology of pressure (decubitus) ulcers from 20 patients was examined by light and electron microscopy. Surgical biopsy specimens from the ulcer edge, ulcer margin, and adjacent normal, nonulcerated skin showed four general types of ulcer histopathology. Morphological features common to all ulcer groups were a dense fibrin matrix, occluded blood vessels, numerous inflammatory cells, and a range of cellular viability from functionally normal appearing cells to cells in all stages of necrosis. These data present structural reference points for comparing pressure ulcer pathology and evaluating the effects of growth factors on wound healing.

Humans↗

PubMed1995

"Metastatic breast carcinoma" that is neither metastatic nor breast.

Histologically, primary sweat gland neoplasms can mimic metastatic breast adenocarcinoma. The clinical history of a chronic, solitary nodule helps in distinguishing these two tumors. Understanding the differential diagnosis is essential in managing nodular skin tumors.

Adenocarcinoma↗

PubMed1995

Detection of platelet-derived growth factor (PDGF)-AA in actively healing human wounds treated with recombinant PDGF-BB and absence of PDGF in chronic nonhealing wounds.

Some human chronic dermal wounds treated with recombinant platelet-derived growth factor-BB (rPDGF-BB) show increased healing coupled with fibroblast activation and granulation tissue formation. To determine whether endogenous PDGF is associated with healing and nonhealing dermal ulcer phenotypes, we developed monoclonal antibodies capable of recognizing the three isoforms of PDGF, AA, AB, and BB dimers, and capable of discriminating between two alternatively spliced A chain transcripts. We detected little PDGF isoform expression in normal skin and in nonhealing dermal ulcers. In contrast, in surgically created acute wounds and chronic ulcers treated with rPDGF-BB, markedly upregulated levels of PDGF-AA (long form) were found. In both types of wounds, increased PDGF-AA was detected primarily in capillaries and fibroblasts, although in rPDGF-BB-treated chronic wounds, widespread expression of PDGF-AA was somewhat delayed. With continued treatment, the long form of PDGF-AA, which can preferentially bind extracellular matrix, was expressed only in capillaries, while fibroblasts began synthesizing the short form of PDGF-AA. Within capillaries, all endothelial cells and varying numbers of pericytes and smooth muscle cells contained PDGF-AA. In all wounds, macrophages and keratinocytes were not a major contributor. While PDGF-BB and PDGF-AB were present in a minority of healing wounds, they were usually present at lower levels than PDGF-AA. PDGF-beta receptors, which bind only PDGF-BB and not other isoforms, were found in normal skin and granulation tissue, providing a molecular basis for treating human chronic wounds with exogenous rPDGF-BB.

Adult↗

PubMed1995

[Mammary carcinoma of the female dog: clinical relevance of the immunohistochemical demonstration of micrometastases in the regional lymph nodes].

77 formalin-fixed and paraffin-embedded mammary carcinomas and the regional lymph nodes of bitches were examined by immunocytochemical technique, all of them free of metastases in routine HE-staining. To compare the two methods, two serial sections were cut of three parts of the lymph nodes. One was used for incubation with the antibody AE1, which reacts with cytokeratin subtype I/A, the other for staining with HE. From 60 of the 77 bitches we received information about the operation and the follow up study period (surgical method, new tumours, survival rate and causes of death). These data were compared with the immunocytochemical results of the lymph nodes. In 84.4% of all 77 lymph nodes tumour cell embolism and/or micrometastases were detected. Comparing the two methods, we found that with HE-staining it was only possible to detect two thirds of all micrometastases containing more than 50 tumor cells. Smaller micrometastases were suspicious in a few cases, the majority could not be detected at all. In the follow up study there was evidence of a better prognosis for bitches with tumours detected in an early stage of growth and treated with radical mastectomy. This was independent of a positive or negative result of tumour cells in lymph nodes. The presence of cancer cells in lymph nodes was only important, if either the tumour was treated in an advanced stage or only single complexes or tumour nodes were extirpated. These dogs often showed metastases in the lung.

Animals↗

PubMed1995

Snakebite treatment at a southeastern regional referral center.

Our objective was to determine the prevalence of poisonous snakebite victims admitted to a regional trauma center in Southeastern Georgia over a 10-year period, as well as the type of snake, grade of envenomation, treatment administered, morbidity and mortality, and outcome. Records of patients admitted to the center for snakebite from a 24-county catchment area during the 10-year period (January 1984 to January 1994) were retroactively reviewed. Sixty-three (63) bites in 62 victims of venomous snakebites were treated. The snake distribution was rattlesnake: 19 (30%), copperhead: 18 (29%), cottonmouth moccasin: 8 (12%), unknown: 18 (29%). Envenomation grades were Grade I: 20 (32%), Grade II: 24 (38%), Grade III: 10 (16%), and Grade IV: 9 (14%). Fourteen of 19 (74%) Grades III and IV envenomations were from rattlesnakes. Antivenin was used in all Grade IV and half of the Grade III envenomations. Antivenin was administered within 3 hours of injury in all but one case. Five patients had surgery. Two patients (both Grade I) developed anaphylaxes from antivenin given before hospitalization. All patients recovered. An average of 6 snakebites were treated each year. Expeditious transport, attention to the type of snake inflicting the bite, and judicious use of antivenin will result in a favorable outcome for the snakebite victim.

Adult↗

PubMed1995

[Mammary carcinoma in female dogs: a new routine diagnosis by the detection of occult micrometastases in the regional lymph nodes].

It was here to find an antibody staining either the epithelial tissues of the canine mammary or the tumor cell embolisms and metastases in their regional lymph nodes. 77 formalin-fixed and paraffin-embedded mammary carcinomas and their regional lymph nodes were examined by immunocytochemical technique, all of them free of metastases in routine haematoxylin and eosin (HE) staining. To compare the two methods two serial sections were cut of three parts of the lymph nodes. One was stained with the antibody AE1, which reacts with cytokeratin subtype I/A, in the SEQUENZA by APAAP-method, the other with HE. In 84.4% of all 77 lymph nodes tumour cell embolism and/or micrometastases were detected. Comparing the two methods we found that in HE-staining it was only possible to detect two thirds of all micrometastases containing more than 50 tumor cells. Smaller micrometastases were suspicious in a few cases, the majority could not be detected at all.

Adenocarcinoma↗

PubMed1995

Venlafaxine in depressed geriatric outpatients: an open-label clinical study.

A 12-month open-label clinical trial was conducted to evaluate patient acceptance and safety of venlafaxine, a novel antidepressant, in ambulatory geriatric depressed patients. The sample consisted of 58 depressed patients aged 65 years and older who needed long-term antidepressant treatment. The setting was multiple study sites in California, Florida, New York, Utah, and Washington. All patients took venlafaxine; 52 qualified for the intent-to-treat analysis, and 24 completed 12 months of treatment. Repeated-measures analysis of variance within subjects showed significant improvements in Clinical Global Impressions severity and improvement, Modified Symptom Checklist, and Quality of Life Questionnaire scores. One patient developed a rash that was judged to be a serious drug-related side effect. The most common side effects were headache (n = 25), nausea (n = 21), insomnia (n = 18), dry mouth (n = 18), and sweating (n = 18). The results demonstrate the safety and patient acceptance of venlafaxine in depressed geriatric outpatients for acute and maintenance treatment.

Aged↗

PubMed1994

Normal transcutaneous oxygen pressure in skin after radiation therapy for cancer.

BACKGROUND: Chronic deleterious changes in human skin after radiation therapy often have been ascribed to progressive ischemia (decreased blood supply and oxygenation). Recent studies suggest, however, that damaged irradiated skin is not ischemic. Transcutaneous oxygen pressure (TCPO2), that accurately reflects skin oxygenation, was studied in 100 patients who had undergone prior extensive radiation therapy for cancer. METHODS: In the 100 patients, the mean time since radiation was 7.86 +/- 10.56 years (mean, +/- SD) (range, 1-58 years). Radiation skin effects were graded (0-4+), and TCPO2 was measured in irradiated and control nonirradiated sites, with patients first breathing room air, then 100% O2 6 l/min for 10 minutes. Data were stratified according to skin grades, sex, time since irradiation, site, type, and dose of radiation. RESULTS: The mean TCPO2 in patients breathing room air was 52.0 17.8 mm Hg (mean +/- SD) for all irradiated skin, compared with 131.8 +/- 51.1 at the same irradiated sites in response to oxygen breathing (P < 0.0001); the mean TCPO2 for normal, nonirradiated skin was 56.5 +/- 12.6 when patients were breathing room air, compared with 151.5 +/- 48.1 when breathing 100% oxygen (P < 0.0001). Higher skin damage grades correlated with increasing time after radiation therapy. However, neither increasing time after irradiation nor grade of skin damage correlated with TCPO2, which was normal in 88% of the patients. CONCLUSIONS: Human skin, even many decades after radiation therapy, retains normal tissue oxygenation and TCPO2 response to inspired oxygen. Postradiation scarring, poor healing, and rare ulceration are not solely due to ischemia and may be caused by other radiation effects, such as permanent changes in fibroblasts.

Adult↗

PubMed1994

Structure and dynamics of the human granulocyte colony-stimulating factor determined by NMR spectroscopy. Loop mobility in a four-helix-bundle protein.

Recombinant 15N- and 13C-labeled human granulocyte colony-stimulating factor (rh-metG-CSF) has been studied by 2D and 3D NMR using uniformly labeled protein, as well as residue-specific 15N-labeled samples. Assignment of 90% of the backbone resonances and 85% of side-chain resonances has enabled the determination of both the secondary and tertiary structures of the protein. The fold is similar to those of the human growth hormone and other growth factors. Four stretches of helices were identified between residues 11 and 41 (helix A), 71 and 95 (helix B), 102 and 125 (helix C), and 145 and 170 (helix D), which form a left-handed four-helix bundle with helices A and B aligned parallel to one another (up-up) and antiparallel to helices C and D (down-down). An additional short fifth helix (E) is part of the AB loop connecting helices A and B. Examination of the protein's relaxation behavior, based on the model-free approach of Lipari and Szabo, shows that the G-CSF backbone has a well-defined structure of limited conformational flexibility in helices. In contrast, the long loop connecting helices C and D exhibits substantial fast internal motion compared to the overall rotational correlation time of the whole molecule, which is on the order of 13 ns.

Chemical Phenomena↗

PubMed1994

The refined structures of a stabilized mutant and of wild-type pyruvate oxidase from Lactobacillus plantarum.

The crystal structure of pyruvate oxidase (EC 1.2.3.3) from Lactobacillus plantarum stabilized by three point mutations has been refined at 2.1 A resolution using the simulated annealing method. Based on 87,775 independent reflections in the resolution range 10 to 2.1 A, a final R-factor of 16.2% was obtained at good model geometry. The wild-type enzyme crystallizes isomorphously with the stabilized enzyme and has been analyzed at 2.5 A resolution. Pyruvate oxidase is a homotetramer with point group symmetry D2. One 2-fold axis is crystallographic, the others are local. The crystallographic asymmetric unit contains two subunits, and the model consists of the two polypeptide chains (residues 9 through 593), two FAD, two ThDP*Mg2+ and 739 water molecules. Each subunit has three domains; the CORE domain, the FAD domain and the ThDP domain. The FAD-binding chain fold is different from those of other known flavoproteins, whereas the ThDP-binding chain fold resembles the corresponding folds of the two other ThDP enzymes whose structure is known, transketolase and pyruvate decarboxylase. The peptide environment most likely forces the pyrimidine ring of ThDP into an unusual tautomeric form, which is required for catalysis. The structural differences between the wild-type and the stabilized enzyme are small. All three point mutations are at or near to the subunit interfaces, indicating that they stabilize the quarternary structure as had been deduced from reconstitution experiments.

Amino Acid Sequence↗