La catabolisme anaérobique de l'arginine chez Bacillus licheniformis et sa régulation par les protéines ArgR et ArcR

Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

The arcABDC Gene Cluster, Encoding the Arginine Deiminase Pathway of Bacillus licheniformis, and Its Activation by the Arginine Repressor ArgR

The arginine deiminase pathway enables Bacillus licheniformis to grow anaerobically on arginine. Both the presence of arginine and anaerobiosis are needed to trigger induction of the pathway. In this study we have cloned and sequenced the arc genes encoding the pathway. They appear clustered in an operon-like structure in the order arcA (arginine deiminase), arcB (ornithine carbamoyltransferase), arcD (putative arginine-ornithine antiporter), arcC (carbamate kinase). It was found that B. licheniformis has an arginine repressor, ArgR, homologous to the B. subtilis arginine repressor AhrC. Mutants affected in argR were isolated. These mutants have lost both repression by arginine of the anabolic ornithine carbamoyltransferase and induction of the arginine deiminase pathway. Electrophoretic band shift experiments and DNase I footprinting revealed that in the presence of arginine, ArgR binds to a site upstream from the arc promoter. The binding site is centered 108 nucleotides upstream from the transcription start point and contains a single Arg box

Regulation of anaerobic arginine catabolism in Bacillus licheniformis by a protein of the Crp/Fnr family

Arginine anaerobic catabolism occurs in Bacillus licheniformis through the arginine deiminase pathway, encoded by the gene cluster arcABDC. We report here the involvement of a new protein, ArcR, in the regulation of the pathway. ArcR is a protein of the Crp/Fnr family encoded by a gene located 109 bp downstream from arcC. It binds to a palindromic sequence, very similar to an Escherichia coli Crp binding site, located upstream from arcA. Residues in the C-terminal domain of Crp that form the DNA binding motif, in particular residues Arg-180 and Glu-181 that make specific bonds with DNA, are conserved in ArcR, suggesting that the complexes formed with DNA by Crp and ArcR are similar. Moreover, the pattern of DNase I hypersensitivity sites induced by the binding of ArcR suggests that ArcR bends the DNA in the same way as Crp. From the absence of anaerobic induction following inactivation of arcR and from the existence of a binding site upstream of the arcA transcription start point, it can be inferred that ArcR is an activator of the arginine deiminase pathway. Copyright (C) 2000 Federation of European Microbiological Societies.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Disabling VEGF-Response of Purkinje Cells by Downregulation of <em>KDR</em> via miRNA-204-5p

The vascular endothelial growth factor (VEGF) is well known for its wide-ranging functions, not only in the vascular system, but also in the central (CNS) and peripheral nervous system (PNS). To study the role of VEGF in neuronal protection, growth and maturation processes have recently attracted much interest. These effects are mainly mediated by VEGF receptor 2 (VEGFR-2). Current studies have shown the age-dependent expression of VEGFR-2 in Purkinje cells (PC), promoting dendritogenesis in neonatal, but not in mature stages. We hypothesize that microRNAs (miRNA/miR) might be involved in the regulation of VEGFR-2 expression during the development of PC. In preliminary studies, we performed a miRNA profiling and identified miR204-5p as a potential regulator of VEGFR-2 expression. In the recent study, organotypic slice cultures of rat cerebella (postnatal day (p) 1 and 9) were cultivated and VEGFR-2 expression in PC was verified via immunohistochemistry. Additionally, PC at age p9 and p30 were isolated from cryosections by laser microdissection (LMD) to analyse VEGFR-2 expression by quantitative RT-PCR. To investigate the influence of miR204-5p on VEGFR-2 levels in PC, synthetic constructs including short hairpin (sh)-miR204-5p cassettes (miRNA-mimics), were microinjected into PC. The effects were analysed by confocal laser scanning microscopy (CLSM) and morphometric analysis. For the first time, we could show that miR204-5p has a negative effect on VEGF sensitivity in juvenile PC, resulting in a significant decrease of dendritic growth compared to untreated juvenile PC. In mature PC, the overexpression of miR204-5p leads to a shrinkage of dendrites despite VEGF treatment. The results of this study illustrate, for the first time, which miR204-5p expression has the potential to play a key role in cerebellar development by inhibiting VEGFR-2 expression in PC

Keratin23 (KRT23) Knockdown Decreases Proliferation and Affects the DNA Damage Response of Colon Cancer Cells

<div><p>Keratin 23 (KRT23) is strongly expressed in colon adenocarcinomas but absent in normal colon mucosa. Array based methylation profiling of 40 colon samples showed that the promoter of KRT23 was methylated in normal colon mucosa, while hypomethylated in most adenocarcinomas. Promoter methylation correlated with absent expression, while increased KRT23 expression in tumor samples correlated with promoter hypomethylation, as confirmed by bisulfite sequencing. Demethylation induced KRT23 expression <i>in vitro.</i> Expression profiling of shRNA mediated stable KRT23 knockdown in colon cancer cell lines showed that KRT23 depletion affected molecules of the cell cycle and DNA replication, recombination and repair. <i>In vitro</i> analyses confirmed that KRT23 depletion significantly decreased the cellular proliferation of SW948 and LS1034 cells and markedly decreased the expression of genes involved in DNA damage response, mainly molecules of the double strand break repair homologous recombination pathway. KRT23 knockdown decreased the transcript and protein expression of key molecules as e.g. MRE11A, E2F1, RAD51 and BRCA1. Knockdown of KRT23 rendered colon cancer cells more sensitive to irradiation and reduced proliferation of the KRT23 depleted cells compared to irradiated control cells.</p></div

Irradiation of colon cancer cells.

<p>A) SW948-ctrl or SW948-sh1506 with stable KRT23 knockdown were irradiated with 0 GY or 5 GY of γ-rays, respectively and seeded on RTCA16-well plates with 16.000 cells/well (n = 4). Non-irradiated SW948-sh1506 cells showed a reduced proliferation rate compared to non-irradiated SW948-ctrl cells. Irradiated SW948-ctrl cells continued proliferation after a short lag period, while the proliferation of the irradiated KRT23-depleted SW948-sh1506 cells decreased after 72 h post-irradiation. The Cell Index CI of the irradiated SW948-sh1506 cells markedly dropped down at about 96 h post-irradiation suggesting a detaching of the cells, possibly induced by cell death upon irradiation of the KRT23 depleted cells. B) A MTT viability assay co-performed at 120 h post-irradiation together with RTCA showed that the viability of KRT23 depleted SW948-sh1506 cells was reduced by 60% upon irradiation with 5GY (p = 8.1E-08) compared to 30% in the SW948-ctrl cells (p = 6.4E-05). C) Visual inspection at 7 days post-irradiation showed a markedly reduced number of cells in KRT23 depleted SW948-sh1506 cells irradiated with 5GY compared to non-irradiated cells.</p

Label-Free Raman Spectroscopic Imaging Monitors the Integral Physiologically Relevant Drug Responses in Cancer Cells

Predictions about the cellular efficacy of drugs tested <i>in vitro</i> are usually based on the measured responses of a few proteins or signal transduction pathways. However, cellular proteins are highly coupled in networks, and observations of single proteins may not adequately reflect the <i>in vivo</i> cellular response to drugs. This might explain some large discrepancies between <i>in vitro</i> drug studies and drug responses observed in patients. We present a novel <i>in vitro</i> marker-free approach that enables detection of cellular responses to a drug. We use Raman spectral imaging to measure the effect of the epidermal growth factor receptor (EGFR) inhibitor panitumumab on cell lines expressing wild-type Kirsten-Ras (K-Ras) and oncogenic K-Ras mutations. Oncogenic K-Ras mutation blocks the response to anti-EGFR therapy in patients, but this effect is not readily observed <i>in vitro</i>. The Raman studies detect large panitumumab-induced differences <i>in vitro</i> in cells harboring wild-type K-Ras as seen in A in red but not in cells with K-Ras mutations as seen in B; these studies reflect the observed patient outcomes. However, the effect is not observed when extracellular-signal-regulated kinase phosphorylation is monitored. The Raman spectra show for cells with wild-type K-Ras alterations based on the responses to panitumumab. The subcellular component with the largest spectral response to panitumumab was lipid droplets, but this effect was not observed when cells harbored K-Ras mutations. This study develops a noninvasive, label-free, <i>in vitro</i> vibrational spectroscopic test to determine the integral physiologically relevant drug response in cell lines. This approach opens a new field of patient-centered drug testing that could deliver superior patient therapies