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1
Content available New face of the “RNA world”
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PL
For a very long time, RNA was considered just the medium by which information flows from DNA into the cell. The model proposed in the 1960s assumed that proteins are the main products and regulators of the gene expression process. In this context, the results of the Human Genome Project and the discoveries of RNA interference and small regulatory RNAs (srRNAs) came as a true surprise. The first ones demonstrated that less than 5% of the human genome encodes proteins. The second showed that RNA, especially 20-30 nt-long molecules should be placed among the most important factors controlling gene expression. srRNAs are capable of affecting the release and flow of genetic information in many different ways. They can induce changes in the genome structure, inhibit transcription, mediate mRNA degradation and repress translation. Interestingly, in different organisms, different pathways are used to regulate gene expression. It has recently been estimated that, in humans, the expression of 35-40% of genes is controlled by srRNA. As a result, RNA is currently believed to be a central molecule in many biological processes.
EN
Colon anastomosis is therapeutically challenging because multiple, usually undetectable factors influence a spectrum of repair mechanisms. We hypothesized that low molecular weight heparins, routinely administered perioperatively, may differentially affect gene expression related to colon healing. Twenty pairs of untreated and enoxaparin-treated rats underwent left-side hemicolectomy with a primary end-to-end anastomosis. Normal colon and anastomotic bowel segments were resected on day 0 and on days 1, 3, 5, and 7 after surgery, respectively. Serial anastomosis transverse cross-sections were evaluated microscopically and by microarray (Rat Genome 230 2.0, Affymetrix). Differentially expressed probe sets were annotated with Gene Ontology. We also examined the influence of enoxaparin on fibroblast proliferation and viability in vitro. Among the 5476 probe sets, we identified differential expression at each healing time point, yielding 79 subcategories. Most indicated genes were involved in wound healing, including multicellular organismal development, locomotory behavior, immune response, cell adhesion, inflammatory response, cell-cell signaling, blood vessel development, and tissue remodeling. Although we found no intensity differences in histological features of healing between enoxaparin-treated and control rats, treatment did induce significant expression changes during early healing. Of these changes, 83 probe sets exhibited at least twofold changes and represented different functional annotations, including inflammatory response, regulation of transcription, regulation of apoptosis, and angiogenesis. Fibroblast culture confirmed an anti-viability effect of enoxaparin. Enoxaparin affects colon wound-related gene expression profiles, but further studies will resolve whether heparin treatment is a risk factor after intestinal surgery, at least in some patients.
EN
Recently we observed that the transcription factors Sp1 and Sp3 bind to the CTCCTCCTC sequence located between positions -194 and -172 of the αV promoter region and are directly involved in the regulation of transcriptional activity of the αV gene in human umbilical vascular endothelial cells (HUVECs) (Czyz & Cierniewski, 1999, Eur. J. Biochem. 265, 638). In this report we provide evidence that the GATA-1 factor regulates αV expression during differentiation of pluripotent K562 cells induced either by phorbol 12-myristate 13-acetate (PMA) or butyric acid (BA) through interaction with the GATA element in the αV gene promoter. DNase I footprinting analysis revealed that region -413 to -408, covering the GATA binding site, was protected by nuclear extract from K562 cells. There was no protection of this region by HUVEC nuclear extract. Electrophoretic mobility shift assay (EMSA) analysis of nuclear extract of K562 cells treated with BA revealed an increase in GATA binding activity, which was associated with reduced αV mRNA and αV protein on the cell surface. Stimulation of K562 cells with PMA resulted in opposite effects: lower expression of GATA-1 was associated with increased levels of αV. We conclude that the GATA-1 transcription factor specifically binds to the GATA element in the αV gene promoter and negatively regulates αV gene expression.
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Filamentous fungi belonging to the Fusarium genus are responsible for large economic losses due to their high pathogenicity and toxigenicity. Fusarium sp. may produce variety of mycotoxins, one of them is zearalenone (ZEA). The presence of the PKS4 gene shows the possibility of zearalenone biosynthesis by Fusarium sp. In this study, in four Fusarium graminearum and one Fusarium poae strains the presence of PKS4 genes and ZEA concentrations were determined. The presence of the PKS4 gene was confirmed by classical polymerase chain reaction (PCR) in three of four strains of F. graminearum. One strain with no PKS4 gene detected was found while still producing ZEA. In the present study, a real-time PCR assay has been successfully performed for the relative expression of Fusarium strains based on new designed primers targeting the PKS4 gene involved in ZEA biosynthesis. Result shows that P56/4 strain of F. graminearum has the highest mRNA level, in the range of 12, what correlates to the high production of this mycotoxin. In this study, a real-time PCR assay has been successfully developed for the prediction of the production of ZEA by F. graminearum strains by PCR real-time techniques based on primers targeting the gene, PKS4, involved in ZEA biosynthesis. The special significance was pointed to occurring genes polymorphism.
EN
Background: The aim of this study was to establish whether the gene expression of estrogen receptor alpha (encoded by ESR1) correlates with the expression of glutathione peroxidase 1 (encoded by GPX1) in the tumor and adjacent tumor-free breast tissue, and whether this correlation is affected by breast cancer. Such relationships may give further insights into breast cancer pathology with respect to the status of estrogen receptor. Methods: We used the quantitative real-time PCR technique to analyze differences in the expression levels of the ESR1 and GPX1 genes in paired malignant and non-malignant tissues from breast cancer patients. Results: ESR1 and GPX1 expression levels were found to be significantly down-regulated by 14.7% and 7.4% (respectively) in the tumorous breast tissue when compared to the non-malignant one. Down-regulation of these genes was independent of the tumor histopathology classification and clinicopathological factors, while the ESR1 mRNA level was reduced with increasing tumor grade (G1: 103% vs. G2: 85.8% vs. G3: 84.5%; p<0.05). In the non-malignant and malignant breast tissues, the expression levels of ESR1 and GPX1 were significantly correlated with each other (Rs=0.450 and Rs=0.360; respectively). Conclusion: Our data suggest that down-regulation of ESR1 and GPX1 was independent of clinicopathological factors. Down-regulation of ESR1 gene expression was enhanced by the development of the disease. Moreover, GPX1 and ESR1 gene expression was interdependent in the malignant breast tissue and further work is needed to determine the mechanism underlying this relationship.
7
Content available remote Type 1 fimbriae in commensal Escherichia coli derived from healthy humans
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EN
Type 1 fimbriae are one of the most important factors of Escherichia coli adaptation to different niches in the host. Our study indicated that the genetic marker - fimH gene occurred commonly in commensal E. coli derived from healthy humans but expression of the type 1 fimbriae was not observed. Identification of fim structural subunit genes (fimA-fimH) and recombinase fimE and fimB genes showed that many of the strains were carrying an incomplete set of genes and the genes expression study revealed that in strains with complete set of fim genes, the fimC gene, encoding the chaperone protein, was not expressed.
EN
Hereditary predisposition to breast cancer determined in large part by loss of function mutations in one of two genes BRCA1 and BRCA2. Besides BRCA1 and BRCA2 other genes are also likely to be involved in hereditary predisposition to breast cancer. TopBP1 protein is involved in DNA replication, DNA damage checkpoint response and transcriptional regulation. Expression of TopBP1 gene at the mRNA level was analyzed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) in 94 samples of hereditary breast cancer. Analysis of TopBP1 mRNA level showed that expression of TopBP1 is significantly downregulated in poorly differentiated breast cancer (grade III according Bloom-Richardson system (P<0.05).
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Statins are inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key enzyme of the sterol biosynthesis pathway. Statin therapy is commonly regarded as well tolerated. However, serious adverse effects have also been reported, especially during high-dose statin therapy. The aim of our study was to investigate the effect of statins on gene expression profiles in human hepatoma HepG2 cells using Affymetrix Human Genome U133 Plus 2.0 arrays. Expression of 102, 857 and 1091 genes was changed substantially in HepG2 cells treated with simvastatin, fluvastatin and atorvastatin, respectively. Pathway and gene ontology analysis showed that many of the genes with changed expression levels were involved in a broad range of metabolic processes. The presented data clearly indicate substantial differences between the tested statins.
EN
Titanium dental implants often induce the foreign body immune response. The duration of the inflammatory process determines the initial stability and biocompatibility of the implant. The challenge for bone tissue engineering is to develop implant biocompatible and bioactive surface coatings that regulate the inflammatory response and enhance osseointegration. Pectins, plant-derived polysaccharides, have been shown to be potential candidates for surface coating due to their possible roles in improving osseointegration and bone healing. The aim of this study was to evaluate in vitro the effect of plant-derived pectin rhamnogalacturonan-I (RG-I) nanocoating on pro- and anti-inflammatory human polymorphonuclear leucocytes (PMN) responses to E. coli LPS or P. gingivalis bacteria. In this study unmodified RG-I and structurally modified RG-I from potato were examined. All in vitro studies were performed on tissue culture polystyrene surfaces (TCPS) or titanium (Ti) discs coated with unmodified and modified RG-Is. Changes in PMN gene expression occurred on both surfaces. The presence of RG-Is down-regulated proinflammatory genes, IL1B, IL8, TNFA. Our results clearly showed that pectin RG-I nanocoating decreased the level of proinflammatory genes expression in stimulated PMN and may therefore be considered as a potential candidate for modulation of the inflammatory response elicited by insertion of implants into living tissue.
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In patients without substantial alcohol use, triglyceride accumulation in the liver can lead to nonalcoholic fatty liver disease (NAFLD) that may progress to nonalcoholic steatohepatitis (NASH). The differential diagnosis between NAFLD and NASH can be accomplished only by morphological examination. Although the relationship between mitochondrial dysfunction and the progression of liver pathologic changes has been described, the exact mechanisms initiating primary liver steatosis and its progression to NASH are unknown. We selected 16 genes encoding mitochondrial proteins which expression was compared by quantitative RT-PCR in liver tissue samples taken from patients with NAFLD and NASH. We found that 6 of the 16 examined genes were differentially expressed in NAFLD versus NASH patients. The expression of hepatic HK1, UCP2, ME2, and ME3 appeared to be higher in NASH than in NAFLD patients, whereas HMGCS2 and hnRNPK expression was lower in NASH patients. Although the severity of liver morphological injury in the spectrum of NAFLD-NASH may be defined at the molecular level, expression of these selected 6 genes cannot be used as a molecular marker aiding histological examination. Moreover, it is still unclear whether these differences in hepatic gene expression profiles truly reflect the progression of morphological abnormalities or rather indicate various metabolic and hormonal states in patients with different degrees of fatty liver disease.
EN
Introduction: Tissue inhibitors of metalloproteinases (TIMP) and the matrix metalloproteinases (MMP) are involved in the spread of cancer. Methods: We have evaluated the matrix metalloproteinases' (MMP-10, MMP-7) and their inhibitors' (tissue inhibitors of metalloproteinases - TIMP-1, TIMP-2) mRNA expression in 61 esophageal cancer samples from patients who had undergone surgery, by using real-time quantitative RT-PCR, and correlated the results with the patient clinicopathologic features. Results: MMP-10, MMP-7, TIMP-1, TIMP-2 were overexpressed in 73%, 85%, 55% and 42% of esophageal cancer samples, respectively. The expression of MMP-10, TIMP-1, and TIMP-2 correlated with the tumor size. The MMP-7 overexpression was associated with the tumour stage (I, II vs III, p=0.05) and lymph node metastasis (N0 vs N1, p=0.037). Conclusions: We conclude that in the resected esophageal cancer an increased mRNA expression of MMP-7, MMP-10 and TIMP-1 correlated with clinicopathologic features. We suggest that these genes may play a role during progression of the disease.
EN
Background: The aim of the study was to designate changes in the expression of HSPA1A, HSPB1 and LDHb in elite rowers after completing a test “till exhaustion” on a rowing ergometer. Finally, we searched for the answer whether there are significant correlations between the expression of the genes and anaerobic threshold (AnT) or the maximal oxygen uptake (VO2max). Material/Methods: The research was conducted on the sample of 9 Polish lightweight male rowers (23.7 ±3.77 yrs, 72.7 ±1.76 kg, 183.6 ±4.58 cm). To determine AnT and VO2max, the subjects performed the test “till exhaustion” with an increasing load on a rowing ergometer. Directly before and after the test, blood samples were collected from the ulnar vein in order to isolate genetic material. RNA was extracted from white cells of venous blood by the chemical method. 2 μg RNA for the reverse transcription was used and the expression of HSPA1A, HSPB1 and LDHb was determined by Real time PCR reaction. To assess the intensity of expression, the ΔΔCt method was used. Results: The study showed an increased expression of HSPA1A and HSPB1 and a decreased one of LDHb. Moreover, post-training changes of the genes activity in white blood cells occurred immediately and could be determined directly after the termination of exertion. Conclusions: No significant correlations between the expression of the genes and anaerobic threshold (AnT), maximal oxygen uptake (VO2max) were stated
15
Content available remote An introduction to DNA chips: principles, technology, applications and analysis.
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EN
This review describes the recently developed GeneChip technology that provides efficient access to genetic information using miniaturised, high-density arrays of DNA or oligonucleotide probes. Such microarrays are powerful tools to study the molecular basis of interactions on a scale that would be impossible using conventional analysis. The recent development of the microarray technology has greatly accelerated the investigation of gene regulation. Arrays are mostly used to identify which genes are turned on or off in a cell or tissue, and also to evaluate the extent of a gene's expression under various conditions. Indeed, this technology has been successfully applied to investigate simultaneous expression of many thousands of genes and to the detection of mutations or polymorphisms, as well as for their mapping and sequencing.
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Content available remote Learning Rough Set Classifiers from Gene Expressions and Clinical Data
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EN
Biological research is currently undergoing a revolution. With the advent of microarray technology the behavior of thousands of genes can be measured simultaneously. This capability opens a wide range of research opportunities in biology, but the technology generates a vast amount of data that cannot be handled manually. Computational analysis is thus a prerequisite for the success of this technology, and research and development of computational tools for microarray analysis are of great importance. One application of microarray technology is cancer studies where supervised learning may be used for predicting tumor subtypes and clinical parameters. We present a general Rough Set approach for classification of tumor samples analyzed with microarrays. This approach is tested on a data set of gastric tumors, and we develop classifiers for six clinical parameters. One major obstacle in training classifiers from microarray data is that the number of objects is much smaller that the number of attributes. We therefore introduce a feature selection method based on bootstrapping for selecting genes that discriminate significantly between the classes, and study the performance of this method. Moreover, the efficacy of several learning and discretization methods implemented in the ROSETTA system [18] is examined. Their performance is compared to that of linear and quadratic discrimination analysis. The classifiers are also biologically validated. One of the best classifiers is selected for each clinical parameter, and the connection between the genes used in these classifiers and the parameters are compared to the establish knowledge in the biomedical literature.
EN
Arabidopsis microRNA162 (miRNA162) level regulation was studied under abiotic stresses, such as drought and salinity. The TaqMan® microRNA assay proved that A. thaliana miRNA162 level was elevated under these stresses, confirming its salt and drought responsiveness. The promoter region analyses of A. thaliana miRNA162a and b genes (MIR162a and MIR162b) identified numerous salinity and drought responsive elements. However, our results indicated that Arabidopsis MIR162a was presumably the main locus responsible for the mature ath-miRNA162 accumulation under the stresses tested, and the MIR162b was generally rather weakly expressed, both in control and under the stress conditions. The MIR162a structure was confirmed to be complex and the pri-miRNA162a hairpin structure was shown to span an alternative exon and an intron. The MIR162a transcription generated a few pri-miRNA162a splicing isoforms that could be functional and non-functional. Upon drought and salinity stresses, the regulation of the pri-miRNA162a alternative splicing pattern revealed an increase of a functional pri-miR162a isoform and a preferential distal polyA site selection under the stress conditions. Apart from the potential transcriptional regulation of the miRNA genes (MIRs) expression, the data obtained point to an essential role of posttranscriptional regulation of Arabidopsis microRNA162 level.
18
Content available remote Metabolic enzymes that bind RNA: yet another level of cellular regulatory network?
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EN
Several enzymes that were originally characterized to have one defined function in intermediatory metabolism are now shown to participate in a number of other cellular processes. Multifunctional proteins may be crucial for building of the highly complex networks that maintain the function and structure in the eukaryotic cell possessing a relatively low number of protein-encoding genes. One facet of this phenomenon, on which I will focus in this review, is the interaction of metabolic enzymes with RNA. The list of such enzymes known to be associated with RNA is constantly expanding, but the most intriguing question remains unanswered: are the metabolic enzyme-RNA interactions relevant in the regulation of cell metabolism? It has been proposed that metabolic RNA-binding enzymes participate in general regulatory circuits linking a metabolic function to a regulatory mechanism, similar to the situation of the metabolic enzyme aconitase, which also functions as iron-responsive RNA-binding regulatory element. However, some authors have cautioned that some of such enzymes may merely represent "molecular fossils" of the transition from an RNA to a protein world and that the RNA-binding properties may not have a functional significance. Here I will describe enzymes that have been shown to interact with RNA (in several cases a newly discovered RNA-binding protein has been identified as a well-known metabolic enzyme) and particularly point out those whose ability to interact with RNA seems to have a proven physiological significance. I will also try to depict the molecular switch between an enzyme's metabolic and regulatory functions in cases where such a mechanism has been elucidated. For most of these enzymes relations between their enzymatic functions and RNA metabolism are unclear or seem not to exist. All these enzymes are ancient, as judged by their wide distribution, and participate in fundamental biochemical pathways.
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Content available Regulatory RNAs in the brain
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EN
Nervous system is characterized by its uniqueness in cells origin, their variability, electrical properties of the nervous cell membrane, response to external signals, neuronal network and changes in synapses activity that are the basis of higher brain functions, such as learning and memory. Brain is a superior organ of human body with an extremely efficient regulation system. Apart from protein and small-molecule regulators, ribonucleic acids (RNAs), especially noncoding proteins (ncRNAs), play a crucial controlling role in the brain. They are present in every cell, from bacteria to primates and have regulatory, catalytic as well as structural function. Many specific ncRNAs have been identified in human brain, responsible for development and functioning. Disturbances in ncRNA synthesis and mechanism of action are connected to diseases such as autism, schizophrenia, Alzheimer disease, Prader-Willi syndrome and others.
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During the last few years a growing amount of data has accumulated showing phospholipid par tic i pa tion in nu clear sig nal transduction. Very re cent data strongly sup port the hy poth e sis that sig nal transduction in the nu cleus is au to nomic. Lo cal pro duc tion of inositol polyphosphates, be gin ning with the ac ti va tion of phospholipase C is required for their specific function in the nucleus. Enzymes which modify poly- phosphoinositols may control gene expression. Much less information is available about the role of other lipids in nuclear signal transduction. The aim of this minireview is to stress what is cur rently known about nu clear lipids with re spect to nu clear sig nal transduction.
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