Home About us Contact
|
Home About us Contact | ||
|
|
||
High Level Expression (high + level_expression)
Selected AbstractsRibosome bypassing elicited by tRNA depletionMOLECULAR MICROBIOLOGY, Issue 5 2003Dale Lindsley Summary Ribosome bypassing refers to the ability of the ribosome::peptidyl-tRNA complex to slide down the message without translation to a site several or dozens of nucleotides downstream and resume protein chain elongation there. The product is an isoform of a protein with a ,coding' gap corresponding to the region of the message which was bypassed. Previous work showed that ribosome bypassing was strongly stimulated at ,hungry' codons calling for a tRNA whose aminoacylation was limited. We have now used the ,minigene' phenomenon to ascertain whether depletion of the pool of specific isoacceptors has a similar effect. High level expression of plasmid-borne minigenes results in the sequestration as peptidyl-tRNA of tRNA cognate to the last triplet of the minigene, thereby limiting protein synthesis for lack of the tRNA in question. We find that induction of a minigene ending in AUA stimulates bypassing at an AUA codon, but not in a control sequence with AGA at the test position; induction of a minigene ending in AGA stimulates bypassing at the latter but not the former. Induction of the AUA minigene also stimulates both leftward and rightward frameshifting at ,shifty' sequences containing an AUA codon. The normal, background frequency of bypassing at an AUA codon is markedly reduced by increasing the cellular level of the tRNA which reads the codon. Thus, the frequency of bypassing can be increased or decreased by lowering or raising the concentration of a relevant tRNA isoacceptor. These observations suggest that the occurrence of ribosome bypassing reflects the length of the pause at a given codon. [source] Relationships between the ethanol utilization (alc) pathway and unrelated catabolic pathways in Aspergillus nidulansFEBS JOURNAL, Issue 17 2003Michel Flipphi The ethanol utilization pathway in Aspergillus nidulans is a model system, which has been thoroughly elucidated at the biochemical, genetic and molecular levels. Three main elements are involved: (a) high level expression of the positively autoregulated activator AlcR; (b) the strong promoters of the structural genes for alcohol dehydrogenase (alcA) and aldehyde dehydrogenase (aldA); and (c) powerful activation of AlcR by the physiological inducer, acetaldehyde, produced from growth substrates such as ethanol and l -threonine. We have previously characterized the chemical features of direct inducers of the alc regulon. These studies allowed us to predict which type of carbonyl compounds might induce the system. In this study we have determined that catabolism of different amino acids, such as l -valine, l -isoleucine, l -arginine and l -proline, produces aldehydes that are either not accumulated or fail to induce the alc system. On the other hand, catabolism of d -galacturonic acid and putrescine, during which aldehydes are transiently accumulated, gives rise to induction of the alc genes. We show that the formation of a direct inducer from carboxylic esters does not depend on alcA -encoded alcohol dehydrogenase I or on AlcR, and suggest that a cytochrome P450 might be responsible for the initial formation of a physiological aldehyde inducer. [source] Maternal embryonic leucine zipper kinase transcript abundance correlates with malignancy grade in human astrocytomasINTERNATIONAL JOURNAL OF CANCER, Issue 4 2008Suely K.N. Marie Abstract We have performed cDNA microarray analyses to identify gene expression differences between highly invasive glioblastoma multiforme (GBM) and typically benign pilocytic astrocytomas (PA). Despite the significant clinical and pathological differences between the 2 tumor types, only 63 genes were found to exhibit 2-fold or greater overexpression in GBM as compared to PA. Forty percent of these genes are related to the regulation of the cell cycle and mitosis. QT-PCR validation of 6 overexpressed genes: MELK, AUKB, ASPM, PRC1, IL13RA2 and KIAA0101 confirmed at least a 5-fold increase in the average expression levels in GBM. Maternal embryonic leucine zipper kinase (MELK) exhibited the most statistically significant difference. A more detailed investigation of MELK expression was undertaken to study its oncogenic relevance. In the examination of more than 100 tumors of the central nervous system, we found progressively higher expression of MELK with astrocytoma grade and a noteworthy uniformity of high level expression in GBM. Similar level of overexpression was also observed in medulloblastoma. We found neither gene promoter hypomethylation nor amplification to be a factor in MELK expression, but were able to demonstrate that MELK knockdown in malignant astrocytoma cell lines caused a reduction in proliferation and anchorage-independent growth in in vitro assays. Our results indicate that GBM and PA differ by the expression of surprisingly few genes. Among them, MELK correlated with malignancy grade in astrocytomas and represents a therapeutic target for the management of the most frequent brain tumors in adult and children. © 2007 Wiley-Liss, Inc. [source] Bridging the gap: A GFP-based strategy for overexpression and purification of membrane proteins with intra and extracellular C-terminiPROTEIN SCIENCE, Issue 4 2010Jennifer M. Hsieh Abstract Low expression and instability during isolation are major obstacles preventing adequate structure-function characterization of membrane proteins (MPs). To increase the likelihood of generating large quantities of protein, C-terminally fused green fluorescent protein (GFP) is commonly used as a reporter for monitoring expression and evaluating purification. This technique has mainly been restricted to MPs with intracellular C-termini (Cin) due to GFP's inability to fluoresce in the Escherichia coli periplasm. With the aid of Glycophorin A, a single transmembrane spanning protein, we developed a method to convert MPs with extracellular C-termini (Cout) to Cin ones providing a conduit for implementing GFP reporting. We tested this method on eleven MPs with predicted Cout topology resulting in high level expression. For nine of the eleven MPs, a stable, monodisperse protein-detergent complex was identified using an extended fluorescence-detection size exclusion chromatography procedure that monitors protein stability over time, a critical parameter affecting the success of structure-function studies. Five MPs were successfully cleaved from the GFP tag by site-specific proteolysis and purified to homogeneity. To address the challenge of inefficient proteolysis, we explored expression and purification conditions in the absence of the fusion tag. Contrary to previous studies, optimal expression conditions established with the fusion were not directly transferable for overexpression in the absence of the GFP tag. These studies establish a broadly applicable method for GFP screening of MPs with Cout topology, yielding sufficient protein suitable for structure-function studies and are superior to expression and purification in the absence GFP fusion tagging. [source] Anti-allergic effects of PG102, a water-soluble extract prepared from Actinidia arguta, in a murine ovalbumin-induced asthma modelCLINICAL & EXPERIMENTAL ALLERGY, Issue 2 2009D. Kim Summary Background Asthma is a chronic inflammatory disease of the lung and its incidence has been increasing around the world. We previously reported that oral administration of a water-soluble extract prepared from Actinidia arguta, code-named PG102, could modulate the level of Th1 and Th2 cytokines and suppress the production of immunoglobulin E (IgE) in the ovalbumin (OVA)-immunized murine model as well as in the in vitro cell culture system, and furthermore could significantly improve dermatitis conditions in the NC/Nga murine model. These data suggested that PG102 might have therapeutic effects in a broad range of allergic diseases. Objective To assess the possible anti-allergic effects of PG102 in the OVA-induced murine asthma model. Methods The quality of PG102 was standardized, using its effects on the production of IgE, IL-5, and IL-13, in in vitro cell culture systems. To test effects on asthma, BALB/c mice were orally administrated with PG102, followed by OVA sensitization and challenge to induce asthmatic symptoms. Airway hyperresponsiveness (AHR), bronchoalveolar lavage fluid, serum, and lung tissue were analysed by using various methods. Results PG102 could decrease the level of IgE, IL-5, and IL-13 in in vitro cell culture systems with IC50 being 1.12,1.43 mg/mL. PG102 could ameliorate asthmatic symptoms, including AHR and eosinophilia in the lungs. Such improvement of asthmatic symptoms by PG102 was accompanied by the down-regulation of IL-5 and IgE. In PG102-treated mice, high level expression of heme oxygenase-1, a potent anti-inflammatory enzyme, was observed in alveolar inflammatory cells, while the mRNA levels of foxp3, TGF-,1, and IL-10, important markers for regulatory T cells, were also up-regulated in the lung tissue. Conclusions PG102 may have potential as a safe and effective reagent for the prevention or treatment of asthma. [source] Differential regulation of nitric oxide synthase isoforms in experimental acute Chagasic cardiomyopathyCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 1 2000B. Chandrasekar We have previously demonstrated induction and high level expression of IL-1,, IL-6 and tumour necrosis factor-alpha in the myocardium during the acute stage of experimental Trypanosoma cruzi infection (Chagas' disease). The myocardial depressive effects of these cytokines are mediated in part by the induction of nitric oxide synthase (NOS), production of nitric oxide (NO) and formation of peroxynitrite. In this study we investigated the expression, activity and localization of NOS isoforms, and the levels of NO, malondialdehyde (a measure of oxidative stress), and peroxynitrite in rats at 1·5, 5, 10 and 15 days after infection with T. cruzi trypomastigotes. The myocardial inflammatory infiltrate and number of amastigote nests increased over the course of infection. A significant increase in tissue nitrate + nitrite levels, NOS2 mRNA, and NOS2 enzyme activity was observed at all time points in the infected compared with uninfected animals. The enzyme activity of constitutive NOS, tissue malondialdehyde levels, and NOS3 mRNA levels was only transiently increased after infection. The protein levels of the NOS isoforms paralleled their mRNA expression. While no positive nitrotyrosine immunoreactivity was detected in control myocardium, its levels increased in infected animals over time. Thus, by 1·5 days post-infection, when no parasite or immune cell infiltration could be detected, the myocardium expressed high levels of NOS and NO metabolites. Nevertheless, the early production of NO in the myocardium was not sufficient to clear the parasites. [source] | |||||||||||||