Direct isolation of specific RNA-interacting proteins using a novel affinity medium

Isolation of proteins that specifically interact with a given RNA or RNA regulation element is essential for studies on the molecular mechanisms of gene expression. Here, a novel method for direct isolation of such interacting proteins is described. It uses an affinity medium that consists of an interacting RNA with an artificially added ‘tail’, which is annealed to one end of a DNA ‘arm’, the other end of which is fixed covalently on the surface of aminosilanized glass powder. Thus the RNA itself is fully suspending, facilitating its interactions with proteins in its natural conformation. The proteins bound on the interacting RNA are eluted and subjected to SDS–PAGE, and the Coomassie-stained protein bands are cut and subjected to mass spectrometry (MS) analysis. Using this method, three proteins specifically interacting with the C/EBPβ 3′-untranslated region (3′-UTR) RNA were isolated and identified. This method is simple and convenient, and the DNA-glass powder medium can be used repeatedly

p21Waf1 expression is regulated by nuclear intermediate filament vimentin in neuroblastoma

<p>Abstract</p> <p>Background</p> <p>Human neuroblastoma (NB) cell lines may present with either one of the so-called S-and N-subtypes. We have previously reported a strong correlation between protein expression levels of vimentin, an S-subtype marker, and the p21^Waf1cyclin-dependent kinase inhibitor. We here investigated whether this correlation extend to the mRNA level in NB cell lines as well as in patients' tumors. We also further explored the relationship between expression of vimentin and p21, by asking whether vimentin could regulate p21 expression.</p> <p>Methods</p> <p>Vimentin and p21 mRNA levels in NB cell lines as well as in patients' tumors (<it>n </it>= 77) were quantified using Q-PCR. Q-PCR data obtained from tumors of high risk NB patients (<it>n </it>= 40) were analyzed in relation with the overall survival using the Log-rank Kaplan-Meier estimation. siRNA-mediated depletion or overexpression of vimentin in highly or low expressing vimentin cell lines, respectively, followed by protein expression and promoter activation assays were used to assess the role of vimentin in modulating p21 expression.</p> <p>Results</p> <p>We extend the significant correlation between vimentin and p21 expression to the mRNA level in NB cell lines as well as in patients' tumors. Overall survival analysis from Q-PCR data obtained from tumors of high risk patients suggests that lower levels of p21 expression could be associated with a poorer outcome. Our data additionally indicate that the correlation observed between p21 and vimentin expression levels results from p21 transcriptional activity being regulated by vimentin. Indeed, downregulating vimentin resulted in a significant decrease in p21 mRNA and protein expression as well as in p21 promoter activity. Conversely, overexpressing vimentin triggered an increase in p21 promoter activity in cells with a nuclear expression of vimentin.</p> <p>Conclusion</p> <p>Our results suggest that p21 mRNA tumor expression level could represent a refined prognostic factor for high risk NB patients. Our data also show that vimentin regulates p21 transcription; this is the first demonstration of a gene regulating function for this type III-intermediate filament.</p

Keratinocytes as Depository of Ammonium-Inducible Glutamine Synthetase: Age- and Anatomy-Dependent Distribution in Human and Rat Skin

In inner organs, glutamine contributes to proliferation, detoxification and establishment of a mechanical barrier, i.e., functions essential for skin, as well. However, the age-dependent and regional peculiarities of distribution of glutamine synthetase (GS), an enzyme responsible for generation of glutamine, and factors regulating its enzymatic activity in mammalian skin remain undisclosed. To explore this, GS localization was investigated using immunohistochemistry and double-labeling of young and adult human and rat skin sections as well as skin cells in culture. In human and rat skin GS was almost completely co-localized with astrocyte-specific proteins (e.g. GFAP). While GS staining was pronounced in all layers of the epidermis of young human skin, staining was reduced and more differentiated among different layers with age. In stratum basale and in stratum spinosum GS was co-localized with the adherens junction component ß-catenin. Inhibition of, glycogen synthase kinase 3β in cultured keratinocytes and HaCaT cells, however, did not support a direct role of ß-catenin in regulation of GS. Enzymatic and reverse transcriptase polymerase chain reaction studies revealed an unusual mode of regulation of this enzyme in keratinocytes, i.e., GS activity, but not expression, was enhanced about 8–10 fold when the cells were exposed to ammonium ions. Prominent posttranscriptional up-regulation of GS activity in keratinocytes by ammonium ions in conjunction with widespread distribution of GS immunoreactivity throughout the epidermis allows considering the skin as a large reservoir of latent GS. Such a depository of glutamine-generating enzyme seems essential for continuous renewal of epidermal permeability barrier and during pathological processes accompanied by hyperammonemia

Trends in Surgical Research in Head and Neck Cancer.

The task of surgical research is to improve the efficacy of available surgical therapeutic modalities, develop new ones, and balance this well with favorable functional outcome. Therefore, surgical research is composed of a translational and a clinical component. In translational surgical research, animal models are used to better understand the biology of head and neck cancers, but even more importantly, the biology of changes to the disease and the microenvironment created by surgical interventions. Animal models additionally allow for the development of image-guided surgery systems, novel strategies of intraoperative adjuvant treatment, and patient "avatars" to test innovative anticancer drug combinations. In clinical surgical research, surgical techniques are validated in clinical trials for effectiveness of tumor control and improvement of functional recovery of the patient. In conclusion, surgical research for head and neck cancer is an active field spanning across the entire breadth of basic and clinical science devoted to a better understanding of what surgery does to the disease and to the patient

Interaction in vitro of type III intermediate filament proteins with higher order structures of single-stranded DNA, particularly with G-quadruplex DNA

Cytoplasmic intermediate filament (cIF) proteins interact strongly with single-stranded (ss) DNAs and RNAs, particularly with G-rich sequences. To test the hypothesis that this interaction depends on special nucleotide sequences and, possibly, higher order structures of ssDNA, a random mixture of mouse genomic ssDNA fragments generated by a novel "whole ssDNA genome PCR" technique via RNA intermediates was subjected to three rounds of affinity binding to in vitro reconstituted vimentin IFs at physiological ionic strength with intermediate PCR amplification of the bound ssDNA segments. Nucleotide sequence and computer folding analysis of the vimentin-selected fragments revealed an enrichment in microsatellites, predominantly of the (GT)n type, telomere DNA, and C/T-rich sequences, most of which, however, were incapable of folding into stable stem-loop structures. Because G-rich sequences were underrepresented in the vimentin-bound fraction, it had to be assumed that such sequences require intramolecular folding or lateral assembly into multistrand structures to be able to stably interact with vimentin, but that this requirement was inadequately fulfilled under the conditions of the selection experiment. For that reason, the few vimentin-selected G-rich ssDNA fragments and a number of telomere models were analyzed for their capacity to form inter- and intramolecular Gquadruplexes (G4 DNAs) under optimized conditions and to interact as such with vimentin and its type III relatives, glial fibrillary acidic protein, and desmin. Band shift assays indeed demonstrated differential binding of the cIF proteins to parallel four-stranded G4 DNAs and, with lower affinity, to bimolecular G'2 and unimolecular G'4 DNA configurations, whereby the transition regions from four- to single-strandedness played an additional role in the binding reaction. In this respect, the binding activity of cIF proteins was comparable with that toward other noncanonical DNA structures, like ds/ss DNA forks, triplex DNA, four-way junction DNA and Z-DNA, which also involve configurational transitions in their interaction with the filament proteins. Association of the cIF proteins with the corresponding nonfolded G-rich ssDNAs was negligible. Considering the almost universal involvement of ssDNA regions and G-quadruplexes in nuclear processes, including DNA transcription and recombination as well as telomere maintenance and dynamics, it is plausible to presume that cIF proteins as complementary constituents of the nuclear matrix participate in the cell- and tissue-specific regulation of these processes

Isolation of SDS-stable complexes of the intermediate filament protein vimentin with repetitive, mobile, nuclear matrix attachment region, and mitochondrial DNA sequence elements from cultured mouse and human fibroblasts

Crosslinkage of vimentin to DNA in mouse L929 cells by formaldehyde and isolation of SDS-stable DNA-vimentin complexes from normal L929 cells and mouse and human embryo fibroblasts indicated close spatial relations between these components in the intact cell. The adducts, obtained by immunoprecipitation with anti-vimentin antibody, contained substantial quantities, not only of repetitive and mobile sequence elements such as centromeric satellite DNA, telomere DNA, microsatellites and minisatellites, long and short interspersed nucleotide elements, and retroposons, but also of mitochondrial (mt) DNA. Because the SDS-stable complexes could be isolated with distinctly higher yields from oxidatively stressed, senescent fibroblasts and were dissociated by boiling, they possibly arose from accidental condensation reactions mediated by unsaturated and dialdehydes, products of free radical-induced lipid peroxidation. They can therefore be considered vestiges of a general interaction of vimentin with cellular DNA. The sequence patterns of their DNA fragments were similar to those of extrachromosomal circular and linear DNA, including retroviral elements, markers and enhancers of genomic instability that also occur in the cytoplasm and are able to transport vimentin into the nucleus. Many of the fragments were also remarkably similar to AT-rich nuclear matrix attachment regions (MARs) in that they contained, in addition to various mobile elements, a palette of typical MAR motifs. With its tendency to multimerize and to interact with single-stranded and supercoiled DNA, vimentin thus behaves like a nuclear matrix protein and may as such participate in a variety of nuclear matrix-associated processes such as replication, recombination, repair, and transcription of DNA. These activities seem to be extendible to the mitochondrial compartment, as vimentin was also crosslinked to mtDNA, preferentially to its D-loop and hypervariable main control region. These sites are prone to point and deletion mutations and, like nuclear MARs, are associated with the cyto-karyomatrix. Moreover, as a developmentally regulated and tissue-specific cyto-karyomatrix protein, vimentin may contribute to the organization of chromatin, including centromeric and telomeric heterochromatin at the nuclear periphery, with all its consequences for genomic activities during embryogenesis and in adulthood of vertebrates. However, because of its high affinity for hypervariable, recombinogenic DNA sequences, vimentin is proposed to play a major role in both the preservation and the evolution of the nuclear and mitochondrial genome