Biomedical subjects

M Müller

Publications and source records attributed to M Müller.

At least 325 records · Page 18Linked to original sources

PubMed2000

[Bile formation and cholestasis].

Transport proteins in hepatocytes and bile duct epithelium mediate uptake and secretion of cholephilic compounds in the liver and are involved in bile formation. Many of these proteins have recently been cloned and characterized and appear to belong to large gene families. Apart from the liver these proteins are expressed in the blood-brain barrier, placenta, kidneys, lungs, intestine and seminiferous tubules. Prokaryotes and yeasts contain similar proteins. In cancer cells they are involved in multidrug resistance. Some genetic cholestatic liver diseases, including progressive familial intrahepatic cholestasis, Dubin-Johnson syndrome, benign recurrent intrahepatic cholestasis and intrahepatic cholestasis of pregnancy result from mutations in transport protein genes. These proteins also play a role in drug-induced liver disease and in primary biliary cirrhosis. Cyclosporine and oestradiol (glucuronide) for instance inhibit bile salt export protein (BSEP).

Bile↗

PubMed2000

Potassium-induced enhancement of persistent inward current in hippocampal neurons in isolation and in tissue slices.

Previous work suggested a role for the voltage-dependent persistent sodium current, I(Na,P), in the generation of seizures and spreading depression (SD). Ordinarily, I(Na,P) is small in hippocampal neurons. We investigated the effect of raising external K(+) concentration, [K(+)](o), on whole-cell persistent inward current in freshly isolated hippocampal CA1 pyramidal neurons. I(Na,P) was identified by TTX-sensitivity and dependence on external Na(+) concentration. When none of the ion channels were blocked, I(Na,P) was not usually detectable, probably because competing K(+) current masked it, but after raising [K(+)](o) I(Na,P) appeared, while K(+) currents diminished. With K(+) channels blocked, I(Na,P) could usually be evoked in control solution and raising [K(+)](o) caused its reversible increase in most cells. The increase did not depend on external calcium [Ca(2+)](o). In CA1 pyramidal neurons in hippocampal slices a TTX-sensitive persistent inward current was always recorded and when [K(+)](o) was raised, it was reversibly enhanced. Strong depolarization evoked irregular current fluctuations, which were also augmented in high [K(+)](o). The findings support a role of potassium-mediated positive feedback in the generation of seizures and spreading depression.

Animals↗

PubMed2000

SRP-dependent co-translational targeting and SecA-dependent translocation analyzed as individual steps in the export of a bacterial protein.

Recently it has been recognized that the signal recognition particle (SRP) of Escherichia coli represents a specific targeting device for hydrophobic inner membrane proteins. It has remained unclear, however, whether the bacterial SRP functions in concert with SecA, which is required for the translocation of secretory proteins across the inner membrane. Here, we have analyzed a hybrid protein constructed by fusing the signal anchor sequence of an SRP-dependent inner membrane protein (MtlA) to the mature part of an exclusively SecA-requiring secretory protein (OmpA). We show that the signal anchor sequence of MtlA confers the novel properties onto nascent chains of OmpA of being co-translationally recognized and targeted to SecY by SRP. Once targeted to SecY, ribosome-associated nascent chains of the hybrid protein, however, remain untranslocated unless SecA is present. These results indicate that SRP and SecA cooperate in a sequential, non-overlapping manner in the topogenesis of those membrane proteins which, in addition to a signal anchor sequence, harbor a substantial hydrophilic domain to be translocated into the periplasm.

Adenosine Triphosphatases↗

PubMed2000

Negative regulation of alkylation-induced sister-chromatid exchange by poly(ADP-ribose) polymerase-1 activity.

One of the earliest responses to DNA damage in eukaryotic cells is activation of poly(ADP-ribose) polymerase-1 (PARP-1), a DNA strand break-dependent nuclear enzyme which covalently modifies proteins with poly(ADP-ribose). Here, we show that conditional over-expression of PARP-1 in stably transfected hamster cells, which causes cellular over-accumulation of poly(ADP-ribose) by several-fold, strongly suppresses alkylation-induced sister-chromatid exchange (SCE), while cytotoxicity of alkylation treatment is slightly enhanced. Viewed together with the known potentiation of SCE by abrogation of PARP-1 activity, our results provide evidence that PARP-1 activity is an important regulator of alkylation-induced SCE formation, imposing a control that is strictly negative and commensurate with the level of enzyme activity.

Alkylation↗

PubMed2000

Fluorescent methylation-specific polymerase chain reaction for DNA-based detection of prostate cancer in bodily fluids.

Promoter hypermethylation of the glutathione S-transferase P1 gene (GSTP1) is the most frequent DNA alteration in prostatic carcinoma. Because this epigenetic DNA alteration can be reliably detected by methylation-specific PCR (MSP), we applied this new technique for molecular detection of prostate cancer in various human bodily fluids. We investigated GSTP1 promoter hypermethylation in DNA isolated from plasma, serum, ejaculate, and urine after prostate massage and from prostate carcinoma tissues from 33 patients with prostate cancer and 26 control patients with benign prostatic hyperplasia (BPH). Fluorescently labeled MSP products were analyzed on an automated gene sequencer. Whereas GSTP1 promoter hypermethylation was not detectable by MSP in prostate tissue and bodily fluids from patients with BPH, we found it in 94% of tumors (16 of 17), 72% of plasma or serum samples (23 of 32), 50% of ejaculate (4 of 8) and 36% of urine (4 of 11) from patients with prostate cancer. Additionally, MSP identified circulating tumor cells in 30% (10 of 33) of prostate cancer patients. Analysis of GSTP1 promoter hypermethylation by MSP thus provides a specific tool for molecular diagnosis of prostate cancer in bodily fluids.

Adenocarcinoma↗

PubMed2000

Induction of an HPV 6bL1-specific mucosal IgA response by DNA immunization.

Human papillomavirus (HPV) plays a crucial role in the development of human anogenital dysplasia. To prevent infection, it is important to induce an HPV-specific mucosal immune response. We investigated whether DNA vaccination would induce an intravaginal mucosal antibody response against HPV 6bL1. New Zealand White rabbits were immunized with an HPV 6bL1 DNA vaccine by one of the three routes: muscular, vaginal, or rectal. We found that vaginal immunization of rabbits with HPV 6bL1 DNA induced 6bL1 virus-like particle-specific lgA antibodies in vaginal secretions. They were detectable until at least 14 weeks after the first immunization. The antibodies also showed neutralizing activity in a hemagglutination inhibition assay. No mucosal immune response was detected in vaginal secretions of rabbits immunized intramuscularly or intrarectally. Our data suggest that vaginal immunization with HPV 6bL1 DNA induces long-lasting IgA responses with neutralizing activity in vaginal secretions of rabbits.

Administration, Intravaginal↗

PubMed2000

Multidrug resistance protein MRP1 protects against the toxicity of the major lipid peroxidation product 4-hydroxynonenal.

4-Hydroxynonenal (4HNE) is the most prevalent toxic lipid peroxidation product formed during oxidative stress. It exerts its cytotoxicity mainly by the modification of intracellular proteins. The detection of 4HNE-modified proteins in several degenerative disorders suggests a role for 4HNE in the onset of these diseases. Efficient protection mechanisms are required to prevent the intracellular accumulation of 4HNE. The toxicity of 4HNE was tested with the small cell lung cancer cell lines GLC(4) and the multidrug-resistance-protein (MRP1)-overexpressing counterpart GLC(4)/Adr. In the presence of the MRP1 inhibitor MK571 or the GSH-depleting agent buthionine sulphoximine, both cell lines became more sensitive and showed decreased survival. Transport experiments were performed with the (3)H-labelled glutathione S-conjugate of 4HNE ([(3)H]GS-4HNE) with membrane vesicles from GLC(4)-derived cell lines with different expression levels of MRP1. [(3)H]GS-4HNE was taken up in an ATP-dependent manner and the transport rate was dependent on the amount of MRP1. The MRP1 inhibitor MK571 decreased [(3)H]GS-4HNE uptake. MRP1-specific [(3)H]GS-4HNE transport was demonstrated with membrane vesicles from High Five insect cells overexpressing recombinant MRP1. Kinetic experiments showed an apparent K(m) of 1.6+/-0.21 microM (mean+/-S.D.) for MRP1-mediated [(3)H]GS-4HNE transport. In conclusion, MRP1 has a role in the protection against 4HNE toxicity and GS-4HNE is a novel MRP1 substrate. MRP1, together with GSH, is hypothesized to have a role in the defence against oxidative stress.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Effects of PTK787/ZK 222584, a specific inhibitor of vascular endothelial growth factor receptor tyrosine kinases, on primary tumor, metastasis, vessel density, and blood flow in a murine renal cell carcinoma model.

Antiangiogenic therapy is a promising new strategy to inhibit tumor growth and formation of metastases. Vascular endothelial growth factor (VEGF) and its receptors, VEGF-receptor 1 (VEGF-R1; FLT-1) and VEGF-R2 (KDR), have been shown to play a major role in tumor angiogenesis. PTK787/ZK 222584, a specific inhibitor of both VEGF-receptor tyrosine kinases, was investigated for its antitumoral and antiangiogenic activity in a murine renal cell carcinoma model. After intrarenal application of the renal carcinoma cells, mice develop a primary tumor and metastases to the lung and to the abdominal lymph nodes. Daily oral therapy with PTK787/ZK 222584 at a dose of 50 mg/kg resulted in a significant decrease of 61 and 67% in primary tumors after 14 and 21 days, respectively. The occurrence of lung metastases was significantly inhibited at both time points (98% reduction and 78% reduction, respectively). After 14 days, no lymph node metastases developed in the PTK787/ZK 222584-treated group, whereas after 21 days of treatment, the lymph node metastases were reduced by 87%. Vessel density in tumor tissues, detected by immunohistochemistry with an anti-CD31 antibody, was significantly decreased by PTK787/ZK 222584. Using color Doppler imaging ultrasound, significant changes in blood flow in the tumor feeding renal artery were found under treatment with PTK787/ZK 222584. Blood flow changes correlated with changes in vessel density but not with tumor volume. The compound was well tolerated in all in vivo experiments and had no significant effects on body weight or general well-being of the animals. This was in contrast to the animals treated with the antiangiogenic agent TNP-470. s.c. therapy with 30 mg/kg TNP-470 every other day had to be discontinued after 13 days because of animal weight loss (>20%) and ataxia. These results demonstrate that PTK787/ZK 222584 is a potent inhibitor of tumor growth, metastases formation, and tumor vascularization in murine renal cell carcinoma. Furthermore, we have been able to demonstrate that color Doppler imaging ultrasound can be used to measure blood flow to a tumor and that flow correlates with vessel density. Thus, this may be a valuable noninvasive method for monitoring the effects of antiangiogenic agents such as PTK787/ZK 222584 on tumor vasculature.

Angiogenesis Inhibitors↗

PubMed2000

Dissecting the translocase and integrase functions of the Escherichia coli SecYEG translocon.

Recent evidence suggests that in Escherichia coli, SecA/SecB and signal recognition particle (SRP) are constituents of two different pathways targeting secretory and inner membrane proteins to the SecYEG translocon of the plasma membrane. We now show that a secY mutation, which compromises a functional SecY-SecA interaction, does not impair the SRP-mediated integration of polytopic inner membrane proteins. Furthermore, under conditions in which the translocation of secretory proteins is strictly dependent on SecG for assisting SecA, the absence of SecG still allows polytopic membrane proteins to integrate at the wild-type level. These results indicate that SRP-dependent integration and SecA/SecB-mediated translocation do not only represent two independent protein delivery systems, but also remain mechanistically distinct processes even at the level of the membrane where they engage different domains of SecY and different components of the translocon. In addition, the experimental setup used here enabled us to demonstrate that SRP-dependent integration of a multispanning protein into membrane vesicles leads to a biologically active enzyme.

Adenosine Triphosphatases↗

PubMed2000

Mediators of neuronal activation in the rat brainstem following intestinal anaphylaxis.

Brainstem neurones become activated following intestinal antigen challenge but the signalling mechanisms have not been resolved. Our aim was to determine the extent of brainstem activation after intestinal anaphylaxis induced by chicken egg albumin (EA). An increase in Fos-positive neurones in the nucleus tractus solitarius (nTS) was observed following EA (P<0.05). Fos-expression was decreased following pretreatment with pyrilamine and ondansetron i.p. and to a similar extent when both antagonists were administered together (all P<0.05 vs. control). Indomethacin had no effect on Fos-expression after antigen challenge. 5-HT and histamine but not prostanoids, released following intestinal anaphylaxis, induce nTS activation via histamine H(1)- and 5-HT(3) receptors. Information on the intestinal inflammatory status is relayed centrally and may play a role in reflexes and behavioural responses to activation of the immune system.

Anaphylaxis↗

PubMed2000

Differential expression of sphingolipids in MRP1 overexpressing HT29 cells.

We have obtained a novel multidrug resistant cell line, derived from HT29 G(+) human colon carcinoma cells, by selection with gradually increasing concentrations of the anti-mitotic, microtubule-disrupting agent colchicine. This HT29(col) cell line displayed a 25-fold increase in colchicine resistance and exhibited cross-resistance to doxorubicin, VP16, vincristine and taxol. Immunoblotting, combined with RT-PCR showed that the multidrug resistance phenotype was conferred by specific overexpression of the multidrug resistance protein 1. Confocal scanning laser microscopy revealed that multidrug resistance protein 1 specifically localized in the plasma membrane of HT29(col) cells. In a functional assay, using the fluorescent multidrug resistance protein 1 substrate 5-carboxyfluorescein, an increased efflux activity of HT29(col) cells was measured, as compared to the wild-type HT29 G(+) cells. MK571, a specific inhibitor of multidrug resistance protein 1, blocked the 5-carboxyfluorescein efflux, but only partially reversed resistance to colchicine, indicating that additional multidrug resistance mechanisms operate in HT29(col) cells. In conclusion, these results show for the first time overexpression of a functional multidrug resistance protein 1 under colchicine pressure, indicating that colchicine is not a P-glycoprotein-specific substrate. Colchicine-induced overexpression of multidrug resistance protein 1 is accompanied by a changed sphingolipid composition, i.e., enhanced levels of glucosylceramide and galactosylceramide. In addition, ceramide, a lipid messenger molecule involved in apoptosis-related signal transduction processes, was much more abundant in HT29(col) cells, which is indicative of a stress response.

ATP-Binding Cassette Transporters↗

PubMed2000

Validation of urea as an endogenous reference compound for the in vivo calibration of microdialysis probes.

Microdialysis is a widely used experimental technique, which offers the opportunity to measure drug and metabolite concentrations in the interstitial space fluid in animals and humans. However, microdialysis probes need to be calibrated in vivo to obtain a recovery factor, which describes the relative drug transfer across the membrane. Recently, novel time-saving calibration techniques, based on the use of reference compounds, have been developed. In particular, the use of endogenous urea levels has been advocated. In the present study we set out to validate the use of the urea reference technique for microdialysis probe calibration in healthy volunteers, employing glucose and paracetamol as model analytes. Urea calibration was compared with the results of two standard calibration techniques, i.e. the no net flux technique and the retrodialysis technique. For glucose, recovery values, calculated by the urea reference technique differed significantly from those values, which were assessed by the no net flux technique (p < 0.05), whereas for paracetamol recovery values did not differ significantly, albeit a high intramethod variability was observed (CV=66%). As a conclusion, we could not confirm the hypothesis that recovery values calculated by the urea reference technique provide equivalent results compared with standard calibration techniques.

Acetaminophen↗

PubMed2000

Early-branching or fast-evolving eukaryotes? An answer based on slowly evolving positions.

The current paradigm of eukaryotic evolution is based primarily on comparative analysis of ribosomal RNA sequences. It shows several early-emerging lineages, mostly amitochondriate, which might be living relics of a progressive assembly of the eukaryotic cell. However, the analysis of slow-evolving positions, carried out with the newly developed slow-fast method, reveals that these lineages are, in terms of nucleotide substitution, fast-evolving ones, misplaced at the base of the tree by a long branch attraction artefact. Since the fast-evolving groups are not always the same, depending on which macromolecule is used as a marker, this explains most of the observed incongruent phylogenies. The current paradigm of eukaryotic evolution thus has to be seriously re-examined as the eukaryotic phylogeny is presently best summarized by a multifurcation. This is consistent with the Big Bang hypothesis that all extant eukaryotic lineages are the result of multiple cladogeneses within a relatively brief period, although insufficiency of data is also a possible explanation for the lack of resolution. For further resolution, rare evolutionary events such as shared insertions and/or deletions or gene fusions might be helpful.

Actins↗

PubMed2000

Uranium(VI) sulfilimine complexes: a new class of nitrogen analogues of the uranyl ion.

The compound tetraphenylphosphonium tetrachlorooxo-S,S-diphenylsulfiliminatouranium, [Ph4P][UOCl4(NSPh2)], has been prepared in high yield from [Ph4P][UOCl5] and [Ph2S=NSiMe3]. An X-ray structure of this compound shows that the uranium atom has a pseudooctahedral geometry with oxygen and nitrogen atoms in trans positions. The structure of the analogous phosphoriminato complex [Ph4P][UOCl4(NPPh3)] has been determined for comparison. Derivatization of the sulfide group shows that only a limited range of functionalization confers stability toward reduction. The emission spectrum of the first electronic excited state reveals a greatly reduced energy compared with that of the uranyl ion. This red shift in the transition is consistent with the weakening of the U-N bond relative to the U-O bond.

Journal Article↗

PubMed2000

The homodimeric ATP-binding cassette transporter LmrA mediates multidrug transport by an alternating two-site (two-cylinder engine) mechanism.

The bacterial LmrA protein and the mammalian multidrug resistance P-glycoprotein are closely related ATP-binding cassette (ABC) transporters that confer multidrug resistance on cells by mediating the extrusion of drugs at the expense of ATP hydrolysis. The mechanisms by which transport is mediated, and by which ATP hydrolysis is coupled to drug transport, are not known. Based on equilibrium binding experiments, photoaffinity labeling and drug transport assays, we conclude that homodimeric LmrA mediates drug transport by an alternating two-site transport (two-cylinder engine) mechanism. The transporter possesses two drug-binding sites: a transport-competent site on the inner membrane surface and a drug-release site on the outer membrane surface. The interconversion of these two sites, driven by the hydrolysis of ATP, occurs via a catalytic transition state intermediate in which the drug transport site is occluded. The mechanism proposed for LmrA may also be relevant for P-glycoprotein and other ABC transporters.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Deletion of the multidrug resistance protein MRP1 gene in acute myeloid leukemia: the impact on MRP activity.

Deletion of the multidrug resistance gene MRP1 has been demonstrated in acute myeloid leukemia (AML) patients with inversion of chromosome 16 (inv[16]). These AML patients are known to have a relatively favorable prognosis, which suggests that MRP1 might play an important role in determining clinical outcome. This study analyzed MRP1 deletion by fluorescent in situ hybridization (FISH), with a focus on inv(16) AML patients. Functional activity of multidrug resistance protein (MRP) was studied in a flow cytometric assay with the use of the MRP substrate carboxyfluorescein (CF) and the inhibitor MK-571. MRP1, MRP2, and MRP6 messenger RNA (mRNA) expression was determined with reverse transcriptase-polymerase chain reaction (RT-PCR). The results were compared with normal bone marrow cells. MRP1 deletion was detected in 7 AML patients; 2 cases showed no MRP1 FISH signals, and 5 cases had 1 MRP1 signal, whereas in 4 AML patients with inv(16) no MRP1 deletions were observed. A variability in MRP activity, expressed as CF efflux-blocking by MK-571, was observed (efflux-blocking factors varied between 1.2 and 3.6); this correlated with the number of MRP1 genes (r = 0.91, P <. 01). MRP activity in the AML cases was not different from normal hematopoietic cells. MRP1 mRNA was detected in patients with 1 or 2 MRP1 FISH signals, but not in patients with no MRP1 signals. MRP2 and MRP6 mRNA were expressed predominantly in AML samples with 1 MRP1 signal, whereas in normal bone marrow cells no MRP2 and MRP6 mRNA was observed. In conclusion, this study shows that MRP activity varies among inv(16) AML cases and does not differ from that in normal hematopoietic cells; this might be in part due to the up-regulation of other MRP genes.

Acute Disease↗

PubMed2000

Association of nonsense mutation of dystrophin gene with disruption of sarcoglycan complex in X-linked dilated cardiomyopathy.

BACKGROUND: In a systematic analysis of inherited forms of cardiomyopathy, we previously identified a family with X-linked dilated cardiomyopathy characterised by a mutation in the rod region of dystrophin. We have now attempted to eludicate the genetic mechanism involved in this disease, as well as the role of dystrophin-associated glycoproteins. METHODS: The affected dystrophin epitope, which lacks binding to the dys-1 antibody, was analysed by single-strand conformation polymorphism analysis, reverse-transcription PCR, and DNA sequencing. Effects on dystrophin-associated glycoproteins were studied by immunohistochemistry and western blotting. FINDINGS: A translation-termination mutation (C4148T) in exon 29 of the dystrophin gene was found in all affected family members. Alternative splicing rescued the reading frame and led to the expression of a dystrophin molecule lacking 50 aminoacids both in cardiac and skeletal muscle. Immunohistochemical analysis of the dystrophin-associated proteins revealed a reduction of beta-sarcoglycan and delta-sarcoglycan in the sarcolemma of cardiac muscle but not skeletal muscle tissue. However, western blotting revealed similar amounts of sarcoglycan subunits in both tissues. INTERPRETATION: The molecular mechanism of this subtype of X-linked cardiomyopathy may be explained by a conformational change in exon-29-deleted dystrophin, resulting in disruption of the sarcoglycan assembly in heart muscle but not skeletal muscle.

Cardiomyopathy, Dilated↗

PubMed2000

Assembly of human papillomavirus type 16 pseudovirions in Saccharomyces cerevisiae.

Studies of the encapsidation of papillomavirus (PV) DNA, and production of preparative amounts of PVs in vitro, have met with only limited success. To circumvent this problem we established a system in yeast to generate infectious HPV-16 pseudovirions. Saccharomyces cerevisiae strain 1699 was transformed with a construct to allow production of HPV-16 virus-like particles (VLPs). This strain was then transformed with a second construct (target plasmid), the same size as the HPV-16 genome and containing the HPV-16 upstream regulatory region (URR) and the HPV-16 E2 open reading frame. In addition, the target plasmid contained the green fluorescent protein gene to monitor delivery of the target plasmid into mammalian cells after infection. We conclude that this system allows HPV DNA encapsidation because (1) HPV-16 VLPs of two different types (heavy and light) were detected by CsCl gradient centrifugation, (2) DNase I-resistant DNA was detected by PCR/Southern blot analysis in fractions of CsCl gradients at a density corresponding to heavy VLPs, (3) in vitro infection of mammalian cells, including primary mouse splenocytes, with pseudovirions resulted in delivery of the reporter gene as demonstrated by FACS analysis for GFP expression, and (4) after injection of pseudovirions into mice, in vivo reporter gene expression was detected by confocal microscopy in sections of muscle tissue. We conclude that HPV-16 pseudovirions produced in yeast may be useful both for in vitro transduction and for gene delivery in vivo.

3T3 Cells↗