Molecular mechanisms of cholestasis: causes and consequences of impaired bile formation.
Explore the source record for details and available documents.
Bile Acids and Salts↗
Biomedical subjects
M Müller
Publications and source records attributed to M Müller.
Microsatellite analysis of plasma DNA from patients with clear cell renal carcinoma.
Deletions of DNA sequences on chromosome 3p [loss of heterozygosity (LOH)] are characteristic of clear cell renal carcinoma, which accounts for about 80% of all renal malignancies. Comparing tumor DNA to DNA from normal cells, LOH analysis of microsatellite sequences has aided in molecular diagnosis of renal carcinoma. Because clinically useful tumor markers do not exist for this cancer entity, the aim of the present study was to detect chromosome 3p microsatellite alterations (LOH and microsatellite instability) in plasma DNA from patients with clear cell renal carcinoma. Four chromosome 3p microsatellites (D3S1307, D3S1560, D3S1289, and D3S1300) were amplified by fluorescent PCR using DNA isolated from normal blood cells and plasma of 40 patients. Corresponding tumor DNA was available from 21 patients. Analyzing PCR products on an automated DNA sequencer, we found LOH in at least one locus in 25 plasma samples (63%), and 14 plasma samples (35%) exhibited LOH at more than one locus. Microsatellite instability of plasma DNA was detectable in one patient (3%). No significant association of advanced (>T2N0M0) tumor stages with LOH in plasma DNA could be demonstrated. If present, modifications of plasma DNA and tumor DNA were identical. No alterations of plasma DNA were found in healthy controls. Analysis of plasma DNA from patients with clear cell renal carcinoma reveals tumor-specific microsatellite alterations and may therefore have diagnostic potential as a molecular tumor marker.
Adenocarcinoma, Clear Cell↗
Antisense phosphorothioate oligodeoxynucleotide down-regulation of the insulin-like growth factor I receptor in ovarian cancer cells.
The insulin-like growth factors (IGF-I and IGF-II) play a key role in cellular proliferation and are involved in cellular transformation. The expression of the IGF-I receptor has been demonstrated in a variety of human tumor cell lines including ovarian cancer cells. Phosphorothioate antisense oligodeoxynucleotides (S-ODNs) were analyzed for their potential to suppress the IGF-I receptor in the NIH: OVCAR-3 ovarian cancer cell line. The application of the antisense S-ODN reduced potently the cell growth of unstimulated NIH:OVCAR-3 cells, whereas sense and mismatch S-ODNs were without any effect. This effect resembled that of the monoclonal antibody (MAb) alphaIR-3. In contrast to the antisense compound, this MAb only partially inhibited the IGF-I-induced proliferation of ovarian cancer cells. The concentration of the antisense S-ODN to exhibit an identical inhibition of cell proliferation was reduced to 50 nM when the oligonucleotides were delivered by the cationic lipid formulation lipofectin. The specificity of the antisense S-ODN action was confirmed by reduction of the receptor protein and of the receptor mRNA, as assayed by flow cytometry and by Northern blot hybridizations. Our data demonstrate the potency of antisense S-ODNs to target the IGF-I receptor message and show that antisense strategies against the IGF-I receptor may provide new strategies for the therapy of ovarian cancer.
Cell Division↗
Detection of infectious adeno-associated virus particles in human cervical biopsies.
Recently we reported that DNA of the human oncogenic papillomaviruses (HPV) and the tumor suppressive human helper virus-dependent parvoviruses, adeno-associated viruses type 2 (AAV-2), colocalize in cervical epithelium. To analyze whether infectious AAV particles are present in cervical tissue, we examined cervical biopsies from 36 patients with HPV-related lesions (squamous intraepithelial lesions) for the presence of AAV DNA and of infectious AAV. From each patient specimens from the lesion and from adjacent normal epithelium were analyzed. After PCR analysis AAV DNA-containing samples were purified by CsCl gradient centrifugation. The presence of AAV virions in CsCl gradients was analyzed and infectivity of AAV was determined. In addition, the biopsies were tested for the presence of HPV DNA. AAV DNA could be detected in biopsies from 23 of 36 patients. AAV particles were found in 11 AAV DNA-positive biopsies from 7 patients (lesions and/or normal tissue, respectively). AAV particles were found to be infectious virions in 10 of the 11 cases. These results demonstrate for the first time that infectious AAV can be isolated from human cervical biopsies, indicating a possible sexual transmission of AAV.
Animals↗
Re: Micrometastatic breast cancer cells in bone marrow at primary surgery: prognostic value in comparison with nodal status.
Explore the source record for details and available documents.
Antibodies, Monoclonal↗
Interstitial methotrexate kinetics in primary breast cancer lesions.
The transfer of cytotoxic agents across the tumor endothelium into the interstitial tumor space is considered a critical step in clinical resistance of solid tumors to antineoplastic chemotherapy. However, experimental data on drug transfer from the blood into the interstitium of solid tumors are scarce. Therefore, in this study, we used an innovative technique, in vivo microdialysis, for measuring interstitial tumor pharmacokinetics and plasma-to-tumor transfer rates of methotrexate (MTX) in breast cancer patients. Microdialysis probes were inserted into the primary tumor and the periumbilical s.c. adipose layer of nine previously chemotherapy-naive breast cancer patients to monitor interstitial concentrations following i.v. administration of MTX (40 mg/m2) during a three-drug treatment regimen. Mean interstitial MTX load in breast tumors, expressed as area under curve (AUC), was 60 +/- 20% (mean +/- SE; coefficient of variation = 100%) of mean plasma MTX load. There was no correlation between plasma AUC and the AUC in the interstitial space of tumor tissue (P = 0.93). Not one of the parameters plasma, interstitial tumor load, and transfer rate of MTX to the interstitial space was associated with favorable clinical response. In conclusion, plasma levels of MTX are not predictive of intratumor levels. There is a high interindividual variability in transendothelial MTX transfer. Under the present conditions, access of MTX to the interstitial space is not a rate-limiting step for clinical response to chemotherapy.
Antimetabolites, Antineoplastic↗
[Urinary incontinence in the elderly. 1: Forms of urinary incontinence--basic diagnosis--additional diagnosis].
Every geriatric patient should routinely be questioned about possible urinary incontinence. The basic diagnostic work-up comprises the clinical history, wherever indicated a geriatric assessment, a nursing care history, a physical examination and a micturition protocol. Furthermore, determination of postvoid residual urine, laboratory investigations and a urine-analysis should be done in all patients. Ultrasonography of the urinary tract is a simple non-invasive procedure that can exclude the presence of large tumors, stones in the bladder and urinary retention. Only selected patients require a greater diagnostic effort prior to treatment, for example, urologic and/or gynecologic investigations or a urodynamic evaluation.
Aged↗
[Urinary incontinence in the elderly, 2: Therapy--from toilet and pelvic floor training to drug therapy and incontinence aids].
The therapeutic strategy rests on a simple basic diagnostic investigation. As a rule, the greatest importance attaches to toilet training and individual counseling. In this context, the work of a specialist nurse capable of providing both the patient and his/her relatives with competent instruction is of considerable importance. A greater diagnostic effort is needed only for selected prior to the initiation of therapy. Even in the elderly patient urinary incontinence is curable or can at least be improved; it is not an inevitability of old age.
Aged↗
Secondary absence of mitochondria in Giardia lamblia and Trichomonas vaginalis revealed by valyl-tRNA synthetase phylogeny.
Nuclear-coded valyl-tRNA synthetase (ValRS) of eukaryotes is regarded of mitochondrial origin. Complete ValRS sequences obtained by us from two amitochondriate protists, the diplomonad, Giardia lamblia and the parabasalid, Trichomonas vaginalis were of the eukaryotic type, strongly suggesting an identical history of ValRS in all eukaryotes studied so far. The findings indicate that diplomonads are secondarily amitochondriate and give further evidence for such conclusion reached recently concerning parabasalids. Together with similar findings on other amitochondriate groups (microsporidia and entamoebids), this work provides critical support for the emerging notion that no representatives of the premitochondrial stage of eukaryotic phylogenesis exist among the species living today.
Amino Acid Sequence↗
Requirements for the translocation of elongation-arrested, ribosome-associated OmpA across the plasma membrane of Escherichia coli.
An oligodeoxynucleotide-dependent method to generate nascent polypeptide chains was adopted for use in a cell-free translation system prepared from Escherichia coli. In this way, NH2-terminal pOmpA fragments of distinct sizes were synthesized. Because most of these pOmpA fragments could be covalently linked to puromycin, precipitated with cetyltrimethylammonium bromide, and were enriched by sedimentation, they represent a population of elongation-arrested, ribosome-associated nascent chains. Translocation of these nascent pOmpA chains into inside-out membrane vesicles of E. coli required SecA and (depending on size) SecB. Whereas their translocation was strictly dependent on the H+-motive force of the vesicles, no indication for the involvement of the bacterial signal recognition particle was obtained. SecA and SecB, although required for translocation, did not mediate binding of the ribosome-associated pOmpA to membrane vesicles. However, SecA and SecB cotranslationally associated with nascent pOmpA, since they could be co-isolated with the ribosome-associated nascent chains and as such catalyzed translocation subsequent to the release of the ribosome. These results indicate that in E. coli, SecA also functionally interacts with preproteins before they are targeted to the translocase of the plasma membrane.
Adenosine Triphosphatases↗
Genetic variation in functionally important domains of the bovine mtDNA control region.
DNA sequences of the mitochondrial control region (CR) of 32 unrelated Austrian cattle were analysed in order to determine the extent of variability in functionally important domains. Using sequencing of PCR products, allele-specific PCR (AS-PCR) and primer introduced restriction analysis (PIRA), 43 differences were observed. They included 33 transitions, five transversions, one deletion and four differences in the number of consecutive cytosines. Twenty-three of these polymorphisms have not been reported before. In addition, we analysed all available European cattle sequences for this region. The transcriptional start sites, the conserved sequence block CSB 1 and both binding sites for the mitochondrial transcription factor mtTFA were highly conserved. We found a transition in each of the inter-specifically conserved Mt4 and Mt5 elements, three nucleotide substitutions in the termination-associated sequence TAS-A and six polymorphisms in the conserved sequence block CSB 2+3, a region which has been implicated in mitochondrial RNA processing.
Animals↗
Jak2 deficiency defines an essential developmental checkpoint in definitive hematopoiesis.
Janus kinases (Jaks) play an important role in signal transduction via cytokine and growth factor receptors. A targeted inactivation of Jak2 was performed. Jak2-/- embryos are anemic and die around day 12.5 postcoitum. Primitive erythrocytes are found, but definitive erythropoiesis is absent. Compared to erythropoietin receptor-deficient mice, the phenotype of Jak2 deficiency is more severe. Fetal liver BFU-E and CFU-E colonies are completely absent. However, multilineage hematopoietic stem cells (CD34low, c-kit(pos)) can be found, and B lymphopoiesis appears intact. In contrast to IFNalpha stimulation, Jak2-/- cells do not respond to IFNgamma. Jak2-/- embryonic stem cells are competent for LIF signaling. The data provided demonstrate that Jak2 has pivotal functions for signal transduction of a set of cytokine receptors required in definitive erythropoiesis.
Anemia↗
Composition of parental mitochondrial DNA in cloned bovine embryos.
We have investigated parental mitochondrial DNA (mtDNA) in cloned bovine embryos obtained by intraspecific cytoplast-blastomere fusion. Analysis of two-cell to blastocyst stage embryos revealed that in contrast to the exclusion of paternal (sperm) mtDNA during sexual inheritance in the cytoplast-blastomere fusion complexes, there was mixing and co-existence of parental mtDNA. The mixing of mtDNA was non-balanced with the minority deriving from the blastomere. The constant content of mtDNA during embryogenesis until the blastocyst stage suggesting an absence of mtDNA replication was shown for conventional 'in vitro fertilised' (IVF) embryos and for cloned embryos. The ratio of parental mtDNA was in accordance with the estimated quantitative participation of mtDNA from the fusion partners.
Animals↗
Non-balanced mix of mitochondrial DNA in cloned cattle produced by cytoplast-blastomere fusion.
We have investigated the transmission of parental mitochondrial DNA (mtDNA) in three clones of born cattle obtained by intraspecific cytoplast-blastomere fusion. Using allele-specific TaqMan PCR a low level transmission of blastomere mtDNA (DB mtDNA) into the cloned offspring was detected, thereby generating a heteroplasmic population of mtDNA. The amount of DB mtDNA was 13% and 18% in two animals of a clone which derived from a 24-cell morula and 0.6% and 0.4% in two calves of clonal origin derived from a 92-cell morula. These values are in accordance with the tendency expected for neutral mtDNA segregation that the fewer cell divisions that have occurred in the donor embryo, the higher the amount of DB mtDNA. We also found a strong decrease of DB mtDNA which was about three orders of magnitude in the third clone derived from a 52-cell morula stage.
Animals↗
Drug-stimulated nucleotide trapping in the human multidrug transporter MDR1. Cooperation of the nucleotide binding domains.
The human multidrug transporter (MDR1 or P-glycoprotein) is an ATP-dependent cellular drug extrusion pump, and its function involves a drug-stimulated, vanadate-inhibited ATPase activity. In the presence of vanadate and MgATP, a nucleotide (ADP) is trapped in MDR1, which alters the drug binding properties of the protein. Here, we demonstrate that the rate of vanadate-dependent nucleotide trapping by MDR1 is significantly stimulated by the transported drug substrates in a concentration-dependent manner closely resembling the drug stimulation of MDR1-ATPase. Non-MDR1 substrates do not modulate, whereas N-ethylmaleimide, a covalent inhibitor of the ATPase activity, eliminates vanadate-dependent nucleotide trapping. A deletion in MDR1 (Delta amino acids 78-97), which alters the substrate stimulation of its ATPase activity, similarly alters the drug dependence of nucleotide trapping. MDR1 variants with mutations of key lysine residues to methionines in the N-terminal or C-terminal nucleotide binding domains (K433M, K1076M, and K433M/K1076M), which bind but do not hydrolyze ATP, do not show nucleotide trapping either with or without the transported drug substrates. These data indicate that vanadate-dependent nucleotide trapping reflects a drug-stimulated partial reaction of ATP hydrolysis by MDR1, which involves the cooperation of the two nucleotide binding domains. The analysis of this drug-dependent partial reaction may significantly help to characterize the substrate recognition and the ATP-dependent transport mechanism of the MDR1 pump protein.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Misincorporation of nucleotides opposite five-membered exocyclic ring guanine derivatives by escherichia coli polymerases in vitro and in vivo: 1,N2-ethenoguanine, 5,6,7,9-tetrahydro-9-oxoimidazo[1, 2-a]purine, and 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1, 2-a]purine.
A variety of exocyclic modified bases have been shown to be formed in DNA from various procarcinogens (e.g., acrolein, malonaldehyde, vinyl chloride, urethan) and are also found in untreated animals and humans, presumably arising as a result of lipid peroxidation. 1, N2-Ethenoguanine (1,N2-epsilon-Gua), a product known to be formed from several 2-carbon electrophiles, was placed in a known site (6256) in bacteriophage M13MB19 and mutations were analyzed in Escherichia coli, with 2.05% G-->A, 0.74% G-->T, and 0.09% G-->C changes found in uvrA- bacteria. 5,6,7, 9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine (HO-ethanoGua), formally the hydrated derivative of 1,N2-epsilon-Gua, is a stable DNA product also derived from vinyl halides. When this base was placed in the same context, the mutation rate was 0.007-0.19% for G-->A, C, or T changes. The saturated etheno ring derivative of 1, N2-epsilon-Gua, 5,6,7,9-tetrahydro-9-oxoimidazo[1,2-a]purine (ethanoGua) produced G-->A and G-->T mutations (0.71% each). All mutants were SOS-dependent and were attenuated by uvrA activity in E. coli. In vitro studies with four polymerases showed strong blocks to addition beyond the adduct site in the order ethanoGua > HO-ethanoGua > 1,N2-epsilon-Gua. Both E. coli polymerases (pol) I exo- and II exo- and bacteriophage pol T7 exo- showed extensive misincorporation opposite ethanoGua in vitro, with pol I exo- incorporating G and T, pol II exo- incorporating A, and pol T7 exo- incorporating A and G. All modified bases reduced the use of the minus strand bearing the modified guanine in E. coli cells. It is of interest that even though the normal base pairing site of guanine is completely blocked, all of the five-membered ring derivatives incorporate the normal base (C) in >80% of the replication events in E. coli. Major differences in blockage and misincorporation are seen due to what might appear to be relatively modest structural differences, and polymerases can differ dramatically in their selectivities.
DNA Adducts↗
Secretion of heterologous proteins from Schizosaccharomyces pombe using the homologous leader sequence of pho1+ acid phosphatase.
In this study we report the use of the S. pombe leader sequence of pho1+ acid phosphatase (Elliott et al., J. Biol. Chem. 216, 2916-2941, 1986) for the secretion of heterologous proteins into the medium. The green fluorescent protein (GFP) and the Human Papillomavirus (HPV) type 16 E7 protein are normally not secreted; fusion of the S. pombe pho1 leader peptide (SPL) to GFP and HPV 16 E7 resulted in an efficient secretion of these proteins although the latter contains a nuclear targeting sequence. These data suggest that SPL fused constructs could be applied for the production of other recombinant proteins using the S. pombe expression system. Furthermore, since GFP retains its intrinsic fluorescence during the secretion, this system may be useful to study the secretory pathway of fission yeast in vivo.
Acid Phosphatase↗
Transport of glutathione conjugates into secretory vesicles is mediated by the multidrug-resistance protein 1.
Intracellular glutathione-conjugate transport was evaluated in the human small cell lung carcinoma cell line GLC4 with low multidrug resistance protein (MRP1) expression and its 300x doxorubicin-resistant, MRP1-over-expressing, GLC4-Adr subline. Transport of non-toxic concentrations of monochlorobimane and 5-chloro-methyl fluorescein diacetate was evaluated using fluorescence microscopy. After exposure to these compounds, fluorescence was observed especially in intracellular vesicles in GLC4-Adr. Immunotransmission electron microscopy showed that MRP1 was present in the vesicle membranes and plasma membrane, while inside the vesicles the glutathione conjugate of 1-chloro-2,4-dinitrobenzene could be detected. Experiments with brefeldin A, which induces arrest in vesicle release from the Golgi complex, indicated that these vesicles may originate from the trans-Golgi network. In GLC4-Adr cells, doxorubicin also was transported in vesicles, with an arrest in vesicle release from the Golgi complex. Our results indicate that MRP1 functions as a glutathione-conjugate transporter not only at the plasma membrane but also in intracellular secretory vesicles.
ATP-Binding Cassette Transporters↗