Biomedical subjects

Stefanie Müller

Publications and source records attributed to Stefanie Müller.

10 recordsLinked to original sources

PubMed2007

The orphan response regulator HP1021 of Helicobacter pylori regulates transcription of a gene cluster presumably involved in acetone metabolism.

Helicobacter pylori is a gastric pathogen for which no nonhuman reservoir is known. In accordance with the tight adaptation to its unique habitat, the human stomach, H. pylori is endowed with a very restricted repertoire of regulatory proteins. Nevertheless, the three complete two-component systems of H. pylori were shown to be involved in the regulation of important virulence traits like motility and acid resistance and in the control of metal homeostasis. HP1021 is an orphan response regulator with an atypical receiver domain whose inactivation has a considerable impact on the growth of H. pylori. Here we report the identification of HP1021-regulated genes by whole-genome transcriptional profiling. We show that the transcription of the essential housekeeping genes nifS and nifU, which are required for the assembly of Fe-S clusters, is activated by HP1021. Furthermore, we demonstrate that the expression of a gene cluster comprising open reading frames hp0690 to hp0693 and hp0695 to hp0697 which is probably involved in acetone metabolism is strongly upregulated by HP1021. Evidence is provided for a direct regulation of the hp0695-to-hp0697 operon by the binding of HP1021 to its promoter region.

Acetone↗

PubMed2006

Inhibition of filovirus replication by the zinc finger antiviral protein.

The zinc finger antiviral protein (ZAP) was recently shown to inhibit Moloney murine leukemia virus and Sindbis virus replication. We tested whether ZAP also acts against Ebola virus (EBOV) and Marburg virus (MARV). Antiviral effects were observed after infection of cells expressing the N-terminal part of ZAP fused to the product of the zeocin resistance gene (NZAP-Zeo) as well as after infection of cells inducibly expressing full-length ZAP. EBOV was inhibited by up to 4 log units, whereas MARV was inhibited between 1 to 2 log units. The activity of ZAP was dependent on the integrity of the second and fourth zinc finger motif, as tested with cell lines expressing NZAP-Zeo mutants. Heterologous expression of EBOV- and MARV-specific sequences fused to a reporter gene suggest that ZAP specifically targets L gene sequences. The activity of NZAP-Zeo in this assay was also dependent on the integrity of the second and fourth zinc finger motif. Time-course experiments with infectious EBOV showed that ZAP reduces the level of L mRNA before the level of genomic or antigenomic RNA is affected. Transient expression of ZAP decreased the activity of an EBOV replicon system by up to 95%. This inhibitory effect could be partially compensated for by overexpression of L protein. In conclusion, the data demonstrate that ZAP exhibits antiviral activity against filoviruses, presumably by decreasing the level of viral mRNA.

Animals↗

PubMed2006

Mutational analysis of the lassa virus promoter.

The promoter sequences directing viral gene expression and genome replication of arenaviruses reside within the 3' and 5' termini of each RNA segment. The terminal 19 nucleotides at both ends are highly conserved among all arenavirus species and are almost completely complementary to each other. This study aimed at characterizing the Lassa virus promoter in detail. The relevance of each position in the promoter was studied by site-directed mutagenesis using the Lassa virus minireplicon system. The data indicate that the Lassa virus promoter functions as a duplex, regulates transcription and replication in a coordinated manner, and is composed of two functional elements, a sequence-specific region from residue 1 to 12 and a variable complementary region from residue 13 to 19. The first region appears to interact with the replication complex mainly via base-specific interactions, while in the second region solely base pairing between 3' and 5' promoter ends is important for promoter function.

Base Pairing↗

PubMed2006

Mesenchymal stem cells display coordinated rolling and adhesion behavior on endothelial cells.

To explore the initial steps by which transplanted mesenchymal stem cells (MSCs) interact with the vessel wall in the course of extravasation, we studied binding of human MSCs to endothelial cells (ECs). In a parallel plate flow chamber, MSCs bound to human umbilical vein ECs (HUVECs) similar to peripheral-blood mononuclear cells (PBMCs) or CD34(+) hematopoietic progenitors at shear stresses of up to 2 dynes/cm(2). This involved rapid extension of podia, rolling, and subsequent firm adhesion that was increased when ECs were prestimulated with TNF-alpha. MSC binding was suppressed when ECs were pretreated with function-blocking anti-P-selectin antibody, and rolling of MSCs was induced on immobilized P-selectin, indicating that P-selectin was involved in this process. Preincubation of HUVECs with anti-VCAM-1 or of MSCs with anti-VLA-4 antibodies suppressed binding of MSCs to HUVECs but did not enhance inhibition by anti-P-selectin, indicating that both P-selectin and VCAM-1 are equally required for this process. Intravital microscopy demonstrated the capacity of MSCs to roll and adhere to postcapillary venules in vivo in a mouse model in a P-selectin-dependent manner. Thus, MSCs interact in a coordinated fashion with ECs under shear flow, engaging P-selectin and VCAM-1/VLA-4.

Animals↗

PubMed2006

Regulation of expression of atypical orphan response regulators of Helicobacter pylori.

The human gastric pathogen Helicobacter pylori exhibits a remarkably small repertoire of transcriptional regulators including three complete two-component systems as well as the orphan response regulators HP1021 and HP1043. Both HP1021 and HP1043 show atypical receiver sequences and are required for the normal cell growth of H. pylori. Recently, we demonstrated that phosphorylation of HP1021 and HP1043 according to the two-component paradigm is not a prerequisite for the cell growth-associated functions of these response regulators, raising the question of how the activity of this regulatory proteins is modulated. Here, we report that strict transcriptional control of its expression is not involved in the cell-growth associated function of HP1021. We show that expression of hp1043 is controlled both on the post-transcriptional or post-translational level and by transcriptional regulation. Furthermore, we provide evidence that hp1043 can be replaced by the orthologous gene cj0355 from Campylobacter jejuni.

Adaptation, Physiological↗

PubMed2006

Dinuclear alkylamine platinum(II) complexes of [1,2-bis(4-fluorophenyl)ethylenediamine]platinum(II): influence of endocytosis and copper and organic cation transport systems on cellular uptake.

Various possible pathways for the uptake of cationic alkylamine platinum(II) complexes into the MCF-7 breast cancer cells were studied with di[meso-1,2-bis(4-fluorophenyl)ethylenediamine]di[sulfinylbis(methane)-S][mu-1,6-diaminohexaneN:N']diplatinum(II) disulfate (m-4F-PtDMSO-DAH) as an example. It was demonstrated that m-4F-PtDMSO-DAH competed neither for the copper transporter nor the organic cation transporters (OCT and OATP). Instead, adsorptive endocytosis by macropinocytosis played an essential role. Inhibitors of this processes such as amiloride, N-ethyl-N-isopropylamiloride (EIPA), wortmannin, and cytochalasin D decreased the intracellular uptake of m-4F-PtDMSO-DAH dramatically. These results support the understanding of the pharmacological behavior of this promising drug family, which showed no cross resistance with cisplatin.

Amines↗

PubMed2006

Hyperhomocysteinemia--the biochemical link between a weak heart and brittle bones?

Osteoporosis, chronic heart failure (CHF) and mild to moderate hyperhomocysteinemia (HHCY) can frequently be found in elderly individuals and often occur in the same individual. Due to demographic changes in the number of elderly people the total number of individuals suffering from osteoporosis and/or CHF, and hence the cost to society, will increase dramatically over the next 50 years. Thus, prevention of these diseases by identifying and modifying risk factors is a major issue. Recent large population-based prospective studies suggested HHCY as an independent risk factor for CHF and osteoporosis. However, the mechanisms that link HHCY to CHF and osteoporosis are almost unknown. Moreover, until now both diseases have been considered as independent diseases. The finding that heart and bones share a common biochemical risk factor raises the question if there is a biochemical link between these two diseases? This manuscript reviews the existing literature about HHCY and osteoporosis, about HHCY and CHF, and about possible mechanisms that link HHCY to both diseases. Existing data suggest that HHCY could be a useful paradigm with most interesting therapeutic implications, because HCY lowering therapy could favourably influence the course in CHF and osteoporotic patients.

Animals↗

PubMed2005

Repeated sequence motifs in the Helicobacter pylori P1408 promoter do not affect its transcription.

The ArsRS two-component system controls the pH-dependent transcription of several target genes involved in the acid resistance of Helicobacter pylori. In its phosphorylated form the response regulator ArsR activates transcription of the urease genes and it has been reported that ArsR approximately P binds to a 26 bp consensus motif which is present in the promoter regions of the ORFs hp1408, hp119 and hp1432 encoding proteins of unknown function. Here we show that the upstream region of ORF hp1408 exhibits considerable sequence variation in different isolates of H. pylori. By the construction of fusions of the P(1408) promoter from different H. pylori strains to the reporter gene gfp in the genetic background of H. pylori G27 we demonstrate that these sequence variations do not significantly affect acid-induced transcription. Furthermore, we show that a P(1408) core promoter comprising only the -10 promoter element and the 26 bp ArsR binding site overlapping the -35 region is sufficient for eliciting the normal acid response of ORF hp1408.

Bacterial Proteins↗

PubMed2004

Replicon system for Lassa virus.

Lassa virus is endemic to West Africa and causes hemorrhagic fever in humans. To facilitate the functional analysis of this virus, a replicon system was developed based on Lassa virus strain AV. Genomic and antigenomic minigenomes (MG) were constructed consisting of the intergenic region of S RNA and a reporter gene (Renilla luciferase) in antisense orientation, flanked by the 5' and 3' untranslated regions of S RNA. MGs were expressed under the control of the T7 promoter. Nucleoprotein (NP), L protein, and Z protein were expressed from plasmids containing the T7 promoter and internal ribosomal entry site. Transfection of cells stably expressing T7 RNA polymerase (BSR T7/5) with MG in the form of DNA or RNA and plasmids for the expression of NP and L protein resulted in high levels of Renilla luciferase expression. The replicon system was optimized with respect to the ratio of the transfected constructs and by modifying the 5' end of the MG. Maximum activity was observed 24 to 36 h after transfection with a signal-to-noise ratio of 2 to 3 log units. Northern blot analysis provided evidence for replication and transcription of the MG. Z protein downregulated replicon activity close to background levels. Treatment with ribavirin and alpha interferon inhibited replicon activity, suggesting that both act on the level of RNA replication, transcription, or ribonucleoprotein assembly. In conclusion, this study describes the first replicon system for a highly pathogenic arenavirus. It is a tool for investigating the mechanisms of replication and transcription of Lassa virus and may facilitate the testing of antivirals outside a biosafety level 4 laboratory.

Animals↗

PubMed2003

Identification of a novel coronavirus in patients with severe acute respiratory syndrome.

BACKGROUND: The severe acute respiratory syndrome (SARS) has recently been identified as a new clinical entity. SARS is thought to be caused by an unknown infectious agent. METHODS: Clinical specimens from patients with SARS were searched for unknown viruses with the use of cell cultures and molecular techniques. RESULTS: A novel coronavirus was identified in patients with SARS. The virus was isolated in cell culture, and a sequence 300 nucleotides in length was obtained by a polymerase-chain-reaction (PCR)-based random-amplification procedure. Genetic characterization indicated that the virus is only distantly related to known coronaviruses (identical in 50 to 60 percent of the nucleotide sequence). On the basis of the obtained sequence, conventional and real-time PCR assays for specific and sensitive detection of the novel virus were established. Virus was detected in a variety of clinical specimens from patients with SARS but not in controls. High concentrations of viral RNA of up to 100 million molecules per milliliter were found in sputum. Viral RNA was also detected at extremely low concentrations in plasma during the acute phase and in feces during the late convalescent phase. Infected patients showed seroconversion on the Vero cells in which the virus was isolated. CONCLUSIONS: The novel coronavirus might have a role in causing SARS.

Adult↗