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Biomedical subjects

R Rudolph

Publications and source records attributed to R Rudolph.

At least 37 records · Page 2Linked to original sources
PubMed1999

The HU protein from Thermotoga maritima: recombinant expression, purification and physicochemical characterization of an extremely hyperthermophilic DNA-binding protein.

The histone-like protein TmHU from the hyperthermophilic eubacterium Thermotoga maritima was cloned, expressed to high levels in Escherichia coli, and purified to homogeneity by heat precipitation and cation exchange chromatography. CD spectroscopical studies with secondary structure analysis as well as comparative modeling demonstrate that the dimeric TmHU has a tertiary structure similar to other homologous HU proteins. The Tm of the protein was determined to be 96 degrees C, and thermal unfolding is nearly completely reversible. Surface plasmon resonance measurements for TmHU show that the protein binds to DNA in a highly cooperative manner, with a KD of 73 nM and a Hill coefficient of 7.6 for a 56 bp DNA fragment. It is demonstrated that TmHU is capable to increase the melting point of a synthetic, double-stranded DNA (poly[d(A-T)]) by 47 degrees C, thus suggesting that DNA stabilization may be a major function of this protein in hyperthermophiles. The significant in vitro protection of double-helical DNA may be useful for biotechnological applications.

Allosteric Site↗
PubMed1999

Maintenance treatment with medroxyprogesterone acetate in patients with advanced breast cancer responding to chemotherapy: results of a randomized trial. Essen Breast Cancer Study Group.

The purpose of this randomized phase III trial was to study whether medroxyprogesterone acetate (MPA) maintenance treatment prolongs the time to progression in advanced breast cancer patients responding to an induction chemotherapy. Patients with progressive advanced breast cancer previously untreated with anthracylines and progestins were given epirubicin (30 mg/m2) and ifosfamide (2 g/m2) on days 1 and 8 at 3-weekly intervals. Patients without disease progression after 6 cycles of chemotherapy were randomly assigned to receive, until progression, either no treatment or MPA at a daily total dose of 500 mg. Ninety patients were randomized: 46 to the MPA arm and 44 to the observation arm. Median time to progression was longer in the MPA arm: 4.9 months versus 3.7 months in the intent-to-treat analysis (p = 0.02), and 4.9 months versus 3.0 months in the secondary efficacy analysis (p = 0.012). Seven patients were removed from MPA due to side effects. The changes in patient-rated quality of life scores were similar in both groups. The median length of survival from randomization was 17.4 months for patients receiving MPA and 18.3 months for patients randomized to observation (p = 0.39). In conclusion, in patients with advanced breast cancer achieving remission or non-progression with 6 cycles of epirubicin and ifosfamide chemotherapy, MPA maintenance treatment led to a significant, though modest, prolongation of the time to progression without affecting overall survival of the study patients.

Aged↗
PubMed1999

A gene for inherited cutaneous venous anomalies ("glomangiomas") localizes to chromosome 1p21-22.

Venous malformations (VMs) are localized defects of vascular morphogenesis. They can occur in every organ system, most commonly in skin and muscle. They can cause pain and bleeding, and in some critical locations they can be life threatening. Usually venous anomalies occur sporadically, but families with dominant inheritance have been identified. Using linkage analysis, we have established in earlier reports that some families with inherited VMs show linkage to chromosome 9p21; the mutation causes ligand-independent activation of an endothelial cell-specific receptor tyrosine kinase, TIE-2. Here we show that VMs with glomus cells (known as "glomangiomas"), inherited as an autosomal dominant trait in five families, are not linked to 9p21 but, instead, link to a new locus, on 1p21-p22, called "VMGLOM" (LOD score 12.70 at recombination fraction.00). We exclude three known positional candidate genes, DR1 (depressor of transcription 1), TGFBR3 (transforming growth factor-beta receptor, type 3), and TFA (tissue factor). We hypothesize that cutaneous venous anomalies (i.e., glomangiomas) are caused by mutations in a novel gene that may act to regulate angiogenesis, in concert with the TIE-2 signaling pathway.

Chromosomes, Human, Pair 1↗
PubMed1999

Correlation of haemostatic abnormalities with tumour stage and characteristics in dogs with mammary carcinoma.

Sixty female dogs with untreated mammary carcinoma, comprising equal numbers of dogs in tumour stages I to IV, were evaluated for haemostatic abnormalities using the following tests: platelet count, prothrombin time, activated partial thromboplastin time, thrombin time, plasma activity of factor V, VIII and X, plasma concentration of fibrinogen, fibrin monomers and fibrinogen degradation products, and plasma antithrombin III activity. Two-thirds of all dogs had one or more haemostatic test abnormality of which the likelihood and frequency was increased in those with stage III and IV neoplasia. Haemostatic abnormalities were more frequently observed in dogs which had mammary tumours with distant metastases, extended tumour necrosis, inflammatory carcinomas, tumours fixed to underlying structures, or tumours in which there was penetration of the tumour capsule by tumour cells. As in humans with mammary carcinoma, these haemostatic abnormalities might be used as prognostic indicators, but their clinical importance remains unknown.

Adenocarcinoma↗
PubMed1999

Site-specific fluorescence labelling of recombinant polyomavirus-like particles.

For the development of gene therapy protocols based on polyomavirus-like particles, we describe a method for fluorescence labelling of virions in order to study virus-cell interactions preceding gene delivery. Site-specific fluorescence labelling of polyomavirus-like particles is achieved via a single cysteine residue and maleimide conjugates of fluorescence dyes (fluorescein, Texas Red). Polyomavirus-like particles can be assembled in vitro from recombinant capsomers produced in E. coli. Since the assembly process is independent of disulfide bond formation, all cysteine residues of the wild-type protein were replaced by serines, and a new unique cysteine residue was introduced for the attachment of the fluorescence marker.

Animals↗
PubMed1998

Expression and renaturation of the N-terminal extracellular domain of torpedo nicotinic acetylcholine receptor alpha-subunit.

The N-terminal extracellular region (amino acids 1-209) of the alpha-subunit of the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue was expressed as inclusion bodies in Escherichia coli using the pET 3a vector. Employing a novel protocol of unfolding and refolding, in the absence of detergent, a water-soluble globular protein of 25 kDa was obtained displaying approximately 15% alpha-helical and 45% beta-structure. The fragment bound alpha-[3H]bungarotoxin in 1:1 stoichiometry with a KD value of 0.5 nM as determined from kinetic measurements (4 nM from equilibrium binding). The kinetics of association of toxin and fragment were of second order, with a similar rate constant (8.2 x 10(5) M-1 s-1) as observed previously for the membrane-bound heteropentameric nAChR. Binding of small ligands was demonstrated by competition with alpha-[3H]bungarotoxin yielding the following KI values: acetylcholine, 69 microM; nicotine, 0.42 microM; anatoxin-a, 3 miroM; tubocurarine, 400 microM; and methyllycaconitine, 0.12 microM. The results demonstrate that the N-terminal extracellular region of the nAChR alpha-subunit forms a self-assembling domain that functionally expresses major elements of the ligand binding sites of the receptor.

Amino Acid Sequence↗
PubMed1998

Folding and association of beta-Galactosidase.

beta-D-Galactosidase from Escherichia coli is one of the largest tetrameric enzymes known at present. Although its physiological importance, the regulation of its synthesis, its enzymatic properties and its structure are well established, little is known about the stability and the folding pathway of this enzyme. Here we show that the overall folding mechanism of chemically denatured beta-galactosidase consists of three stages: (i) formation of elements of secondary structure; (ii) collapse to subdomains and structured monomers; (iii) association to the native quaternary structure via dimeric intermediates. The first rate-limiting step is the association of structured monomers to form dimers in a bi-molecular reaction, with a rate constant of 4.3x10(3) M-1 s-1 at 20 degreesC. The second rate-limiting uni-molecular folding step leads to dimers which are competent for further association, with a rate constant of 0.5x10(-3) s-1 at 20 degreesC. Tetramers form from these dimers in a fast reaction. By determining a similar mechanism for alpha-complementation of beta-galactosidase fragments it could be confirmed that beta-galactosidase follows a consecutive bi-uni-molecular mechanism of folding and association.

Dimerization↗
PubMed1998

Activation of thiamin diphosphate and FAD in the phosphatedependent pyruvate oxidase from Lactobacillus plantarum.

The phosphate- and oxygen-dependent pyruvate oxidase from Lactobacillus plantarum is a homotetrameric enzyme that binds 1 FAD and 1 thiamine diphosphate per subunit. A kinetic analysis of the partial reactions in the overall oxidative conversion of pyruvate to acetyl phosphate and CO2 shows an indirect activation of the thiamine diphosphate by FAD that is mediated by the protein moiety. The rate constant of the initial step, the deprotonation of C2-H of thiamine diphosphate, increases 10-fold in the binary apoenzyme-thiamine diphosphate complex to 10(-2) s-1. Acceleration of this step beyond the observed overall catalytic rate constant to 20 s-1 requires enzyme-bound FAD. FAD appears to bind in a two-step mechanism. The primarily bound form allows formation of hydroxyethylthiamine diphosphate but not the transfer of electrons from this intermediate to O2. This intermediate form can be mimicked using 5-deaza-FAD, which is inactive toward O2 but active in an assay using 2,6-dichlorophenolindophenol as electron acceptor. This analogue also promotes the rate constant of C2-H dissociation of thiamine diphosphate in pyruvate oxidase beyond the overall enzyme turnover. Formation of the catalytically competent FAD-thiamine-pyruvate oxidase ternary complex requires a second step, which was detected at low temperature.

Catalysis↗
PubMed1998

The influence of cimetidine on the disposition kinetics of the antidepressant venlafaxine.

The influence of cimetidine on the disposition pharmacokinetics of the antidepressant drug, venlafaxine, and its active metabolite, O-desmethylvenlafaxine, was examined in 18 healthy young men and women. The steady-state pharmacokinetic profiles of venlafaxine and O-desmethylvenlafaxine were evaluated during a 24-hour period after 5 days of treatment with venlafaxine (50 mg three times a day) and during a second 24-hour period after 5 days of combination treatment with venlafaxine (50 mg three times a day) and cimetidine (800 mg once a day). The apparent oral clearance of venlafaxine decreased significantly in the presence of cimetidine and the average steady-state plasma concentration of venlafaxine increased significantly in the presence of cimetidine, but there were no changes in the corresponding concentrations of the active metabolite. However, O-desmethylvenlafaxine exhibits pharmacologic activity that is approximately equimolar to that of venlafaxine, and the sum of venlafaxine plus O-desmethylvenlafaxine plasma concentrations was increased by an average of only 13%. Therefore, the effect of cimetidine coadministration is not expected to result in clinically important alterations in the response to venlafaxine in patients with depression. This may not be true, however, for patients with compromised hepatic metabolic function.

Adolescent↗
PubMed1998

Advances in refolding of proteins produced in E. coli.

Inclusion body production is a common theme in recombinant protein technology. Hence, renaturation of these inclusion body proteins is a field of increasing interest for gaining large amounts of proteins. Recent developments of renaturation procedures include the inhibition of aggregation during refolding by the application of low molecular weight additives and matrix-bound renaturation techniques.

Animals↗
PubMed1998

Fibroblast variability in photoaged and nonphotoaged facelift skin.

Pre- and postauricular skin was harvested from 25 facelift patients to study fibroblast replicative ability (RA). In tissue cultures, considerable heterogeneity was found in the ability of facial skin fibroblasts to replicate, both from patient to patient, and in the relationship of preauricular and postauricular skin. Fibroblast RA ranged from 8.11 to 67.89 population doublings until senescence. Continued cellular ability to divide did not appear to be related to patient age. Fibroblasts from some older patients were able to go through as many population doublings as cells from younger patients. Fibroblasts in facial skin exposed to solar radiation were often able to replicate as well as or better than fibroblasts from protected skin. Fibroblast RA differences ranged from +45% (anterior > posterior) to -49% (posterior > anterior). Fibroblast replication in solar-exposed skin may be influenced by topical tretinoin and by female hormone replacement therapy. Further study is needed. This wide range of fibroblast RA must be taken into account when planning and evaluating any facial skin treatment.

Adult↗
PubMed1997

Immunohistological assessment of fibrin deposition and thrombus formation in canine mammary neoplasia.

A commercially available monoclonal antibody against human fibrin was used to detect fibrin in canine formalin-fixed, paraffin wax-embedded tissue by applying a slightly modified alkaline phosphatase anti-alkaline phosphatase (APAAP) technique. Twenty-eight mammary tumours from six bitches were examined for the presence of fibrin. Thrombi and extravascular fibrin deposits were detected in 15 tumours (12 complex adenocarcinomas, one adenocarcinoma, two solid carcinomas), and a single thrombus was detected in one adenoma; 12 tumours (three adenomas, one complex adenoma, four complex adenocarcinomas and four adenocarcinomas) did not show any staining reaction.

Adenocarcinoma↗
PubMed1997

Epithelial neoplasms of the skin, the cutaneous mucosa and the transitional epithelium in dogs: an immunolocalization study for papillomavirus antigen.

In order to study the prevalence of papillomavirus antigen in canine epithelial neoplasms, 535 neoplastic and hyperplastic specimens of the skin, the cutaneous mucosa and the transitional epithelium were immunohistochemically stained with a polyclonal antiserum against papillomavirus antigen. A positive staining result occurred in 44.2% in a total of 95 papillomas and in 27% of 100 diagnosed squamous cell carcinomas, other tumours did not react with the applied antiserum. Papillomavirus antigen was detectable in 54.2% of all oral and ocular papillomas and in 37.0% of all cutaneous papillomas. The majority of the squamous cell carcinomas with detectable papillomavirus antigen were considered positive but not without restrictions. The average age of dogs with viral oral and ocular papillomas was 2.3 years, with viral cutaneous papillomas it was 3.2 years. The average age of dogs with virus-positive squamous cell carcinomas was nearly 11 years. Papillomavirus-like particles were demonstrated by means of transmission electron microscopy in three positive oral papillomas, in the positive squamous cell carcinomas virion detection failed.

Animals↗
PubMed1997

Methods for the differentiation of giant cells in canine and feline neoplasias in paraffin sections.

In the following study cells with at least two cell nuclei are addressed as giant cells. In 47 biopsies of feline neoplasias (fibrosarcoma, haemangioendothelsarcoma, mammary adenocarcinoma, osteoidsarcoma, complex sarcoma), and 25 biopsies of canine neoplasias (malignant seminoma, mammary adenocarcinoma, haemangioendothelsarcoma, fibrosarcoma, osteoblastic sarcoma, complex sarcoma) giant cells are distinguished either as neoplastic giant cells or as reactive (non-neoplastic) giant cells. Cell nuclei of neoplastic giant cells which are labelled with the monoclonal antibody MIB 1 are mitotic active; cell nuclei are polymorph and can show atypical mitosis; the cytoplasmic reaction with tartrate resistant acid phosphatase (TRAP) is negative. Negative reactions with MIB 1, positive TRAP staining and homogeneous cell nuclei are distinctive for osteoclast-like glant cells. Other non-neoplastic giant cells (e.g. foreign body cells, Langhans-giant cells) are negative with both MIB 1 and TRAP. Double staining of paraffin sections is possible. Routine formalin-fixation, embedding in paraffin and decalcifying tissue samples do not interfere with MIB 1 immunoreactions or TRAP reactions. Methodological modifications that were necessary for the preparation of paraffin sections from canine and feline tissue samples are discussed. As the presence of neoplastic giant tumour cells is an index for a poor prognosis in human medicine, not only the entity of the tumour must be named, but also the exact significance of the giant cell type:, e.g. fibrosarcoma with osteoclast-like giant cells, hepatic carcinoma with reactive giant cells, malignant seminoma with neoplastic giant cells, angiosarcoma with both neoplastic giant cells and osteoclast-like giant cells. This would enable the classification of further neoplasias dealing with clinical courses of the diseases. Over the past years our stains have remained stable. It is possible to carry out retrospective investigations with archived tissue samples and make permanent preparations. A reclassification and a refined form of diagnosis (tumour and giant cell type) would be recommended.

Animals↗
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↗