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Agnes Zvara

Publications and source records attributed to Agnes Zvara.

18 recordsLinked to original sources
PubMed2007

Melatonin-induced gene expression changes and its preventive effects on adriamycin-induced lipid peroxidation in rat liver.

Adriamycin (ADR) provokes lipid peroxidation process, while melatonin (MEL) is a free radical scavenger that has been found to protect against lipid peroxidation in vitro and in many experimental models. In the present study, the effects of ADR and the combination of ADR and MEL were analyzed on the modulation of fatty acid composition, lipid peroxidation and gene expression in rat liver. Sixty genes were selected for the study of relative gene expression changes in the liver. ADR treatment decreased the polyunsaturated fatty acids C22:6 n-3 and C20:4 n-6 in rat liver mitochondria. When the treatment of ADR was followed by MEL, decrease in these fatty acids could not be detected. A significant increase in lipid peroxidation was observed after administration of ADR, which was restored to control values by post-treatment with MEL. Gene expression profiles of ADR- versus ADR+MEL-treated rat livers indicated that both treatments induced significant changes. Quantitative real-time polymerase chain reaction analysis of 60 genes involved in oxidative stress revealed that cyp1b1, which is involved in electron transport, cyclin-dependent kinase inhibitor 1a that possesses cyclin-dependent protein kinase inhibitor activity, was induced at a more pronounced level in the ADR+MEL-treated samples than in the ADR-treated ones. Several genes having roles in heat-shock response were downregulated in MEL-treated animals, such as hsp40, hsp70 and hsp90 proteins reflecting the reduced oxidative stress in these animals. Global gene expression analysis will highlight the gene expression changes accompanying oxidative damage and its prevention in more details.

Animals↗
PubMed2006

Improved DOP-PCR-based representational whole-genome amplification using quantitative real-time PCR.

In many cases, only a minute amount of partially degraded genomic DNA can be extracted from archived clinical samples. Diverse whole-genome amplification methods are applied to provide sufficient amount of DNA for comparative genome hybridization, single-nucleotide polymorphism, and microsatellite analyses. In these applications, the reliability of the amplification techniques is particularly important. In PCR-based approaches, the plateau effect can seriously alter the original relative copy number of certain chromosomal regions. To eliminate this distorting effect, we improved the standard degenerate oligonucleotide-primed PCR (DOP-PCR) technique by following the amplification status with quantitative real-time PCR (QRT-PCR). With real-time detection of the products, we could eliminate DNA overamplification. Probes were prepared from 10 different tumor samples: primary and metastatic melanoma tissues, epidermoid and bronchioloalveolar lung carcinomas, 2 renal cell carcinomas, 2 colorectal carcinomas, and a Conn and Cushing adenoma. Probes were generated by using nonamplified and amplified genomic DNA with DOP-PCR and DOP-PCR combined with QRT-PCR. To demonstrate the reliability of the QRT-PCR based amplification protocol, altogether 152 relative copy number changes of 44 regions were determined. There was 85.6% concordance in copy number alterations between the QRT-PCR protocol and the nonamplified samples, whereas this value was only 63.8% for the traditional DOP-PCR. Our results demonstrate that our protocol preserves the original copy number of different chromosomal regions in amplified genomic DNA than standard DOP-PCR techniques more accurately.

DNA Primers↗
PubMed2005

Capsaicin-sensitive sensory neurons regulate myocardial function and gene expression pattern of rat hearts: a DNA microarray study.

We have previously shown that capsaicin-sensitive sensory nerves contribute to the regulation of normal cardiac function and to the development of cardiac adaptation to ischemic stress; however, the underlying molecular mechanisms remain unknown. Therefore, here we assessed cardiac functional alterations and relative gene expression changes by DNA microarray analysis of 6400 genes in rat hearts 7 days after the end of systemic capsaicin treatment protocol leading to selective sensory chemodenervation. Capsaicin pretreatment resulted in a cardiac dysfunction characterized by elevation of left ventricular end-diastolic pressure and led to altered expression of 80 genes of known function or homology to known sequences. Forty-seven genes exhibited significant up-regulation and 33 genes were down-regulated (changes ranged from -3.9 to +4.8-fold). The expression changes of 10 selected genes were verified, and an additional 11 genes were examined by real-time quantitative PCR. This is the first demonstration that gene expression changes in the heart due to capsaicin pretreatment included vanilloid receptor-1 (capsaicin receptor), transient receptor potential protein, GABA receptor rho-3 subunit, 5-hydroxytryptamine 3 receptor B, neurokinin receptor 2, endothelial nitric oxide synthase, matrix metalloproteinase-13, cytochrome P450, farnesyl-transferase, ApoB, and leptin. None of the genes have been previously shown to be involved in the mechanism of the cardiac functional effects of sensory chemodenervation by capsaicin. We conclude that capsaicin-sensitive sensory nerves play a significant role in the regulation of a variety of neuronal and non-neuronal genes in the heart and possibly in other tissues as well.

Animals↗
PubMed2005

Identification of germ plasm-enriched mRNAs in Drosophila melanogaster by the cDNA microarray technique.

The development of embryonic germ cells in Drosophila depends on the germ plasm, the most posterior part of the ooplasm. The germ plasm is devoted to the formation of future germ cells and is known to contain all the factors that are necessary to induce germ cell fate. Besides having a characteristic organelle and protein distribution, the germ plasm also contains a large number of localized RNA species that have been shown to play crucial roles in germ cell determination. To identify germ plasm-enriched, localized transcripts, we used a two-step method composed of cDNA microarray (containing 3200 annotated Drosophila cDNAs) and in situ RNA hybridization techniques. We compared germ plasm deficient, normal and ectopic germ plasm conditions in the cDNA microarray experiments. RNA species whose concentration increased when ectopic germ plasm was present and decreased when the germ plasm was missing were selected. These candidates were then subjected to a second screen which compared the distribution of the given RNA in wild type embryos and in eggs with ectopic germ plasm. Finally, 17 RNA species were found to be enriched in the germ plasm. Based on these data, we estimate that around 1% of the Drosophila genes encode for germ plasm-enriched, localized transcripts. We conclude that this combination of microarray and in situ hybridization techniques is a simple but powerful experimental design for the genome-wide identification of genes coding for germ plasm localized transcripts.

Animals↗
PubMed2005

Real-time polymerase chain reaction-based exponential sample amplification for microarray gene expression profiling.

Conventional approaches to target labeling for gene expression analysis using microarray technology typically require relatively large amounts of RNA, a serious limitation when the available sample is limited. Here we describe an alternative exponential sample amplification method by using quantitative real-time polymerase chain reaction (QRT-PCR) to follow the amplification and eliminate the overamplified cDNA which could distort the quantitative ratio of the starting mRNA population. Probes generated from nonamplified, PCR-amplified, and real-time-PCR-amplified cDNA samples were generated from lipopolysaccharide-treated and nontreated mouse macrophages and hybridized to mouse cDNA microarrays. Signals obtained from the three protocols were compared. Reproducibility and reliability of the methods were determined. The Pearson correlation coefficients for replica experiments were r=0.927 and r=0.687 for QRT-PCR-amplification and PCR-overamplification protocols, respectively. Chi2 test showed that overamplification resulted in major biases in expression ratios, while these alterations could be eliminated by following the cycling status with QRT-PCR. Our exponential sample amplification protocol preserves the original expression ratios and allows unbiased gene expression analysis from minute amounts of starting material.

Animals↗
PubMed2005

Impact of haloperidol and risperidone on gene expression profile in the rat cortex.

Despite the clinical efficacy of the most thoroughly studied conventional neuroleptic agent haloperidol, and the atypical antipsychotic risperidone is well established, little information is available on their molecular effects. Recent advances in high-density DNA microarray techniques allow the possibility to analyze thousands of genes simultaneously for their differential gene expression patterns in various biological processes, and to determine mechanisms of drug action. The aim of this series of experiments was to gain experience in antipsychotic gene-expression profiling and characterize (in the parlance of genomics) the "antipsychotic transcriptome." In this prospective animal study, broad-scale gene expression profiles were characterized for brains of rats treated with antipsychotics and compared with those of sham controls. We used DNA microarrays containing 8000 sequences to measure the expression patterns of multiple genes in rat fronto-temporo-parietal cortex after intraperitoneal treatment with haloperidol or risperidone. A number of transcripts were differentially expressed between control and treated samples, of which only 36 and 89 were found to significantly differ in expression as a result of exposure to haloperidol or risperidone, respectively (P<0.05). Acutely, 13 genes were more highly expressed and 15 transcripts were found to be significantly less abundant, whereas chronically nine genes were up-regulated and none of them was repressed in haloperidol-treated cortices. Risperidone acutely induced 43 and repressed 46 genes, and chronically over-expressed 6 and down-regulated 11 transcripts. Selected genes were assayed by real-time PCR, then normalized to beta-actin. These assays confirmed the significance of the array results for all transcripts tested. Despite their differing receptor affinity and selectivity, our findings indicate that haloperidol and risperidone interfere with cell survival, neural plasticity, signal transduction, ionic homeostasis and metabolism in a similar manner.

Animals↗
PubMed2005

Over-expression of dopamine D2 receptor and inwardly rectifying potassium channel genes in drug-naive schizophrenic peripheral blood lymphocytes as potential diagnostic markers.

Schizophrenia is one of the most common neuropsychiatric disorders affecting nearly 1% of the human population. Current diagnosis of schizophrenia is based on complex clinical symptoms. The use of easily detectable peripheral molecular markers could substantially help the diagnosis of psychiatric disorders. Recent studies showed that peripheral blood lymphocytes (PBL) express subtypes of D1 and D2 subclasses of dopamine receptors. Recently, dopamine receptor D3 (DRD3) was found to be over-expressed in schizophrenic PBL and proposed to be a diagnostic and follow-up marker for schizophrenia. In this study we screened PBL of 13 drug-naive/drug-free schizophrenic patients to identify additional markers of schizophrenia. One of the benefits of our study is the use of blood samples of non-medicated, drug-naive patients. This excludes the possibility that changes detected in gene expression levels might be attributed to the medication rather than to the disorder itself. Among others, genes for dopamine receptor D2 (DRD2) and the inwardly rectifying potassium channel (Kir2.3) were found to be over-expressed in microarray analysis. Increased mRNA levels were confirmed by quantitative real-time PCR (QRT-PCR) using the SybrGreen method and dual labeled TaqMan probes. The use of both molecular markers allows a more rapid and precise prediction of schizophrenia and might help find the optimal medication for schizophrenic patients.

Adult↗
PubMed2004

Cholesterol diet-induced hyperlipidemia influences gene expression pattern of rat hearts: a DNA microarray study.

To profile gene expression patterns involved in the direct myocardial effect of cholesterol-enriched diet-induced hyperlipidemia, we monitored global gene expression changes by DNA microarray analysis of 3200 genes in rat hearts. Twenty-six genes exhibited significant up-regulation and 25 showed down-regulation in hearts of rats fed a 2% cholesterol-enriched diet for 8 weeks as compared to age-matched controls. The expression changes of 12 selected genes were also assessed by real-time quantitative polymerase chain reaction. Genes with altered expression in the heart due to hyperlipidemia included procollagen type III, cofilin/destrin, tensin, transcription repressor p66, synaptic vesicle protein 2B, Hsp86, chaperonin subunit 5epsilon, metallothionein, glutathione S-transferase, protein kinase C inhibitor, ATP synthase subunit c, creatine kinase, chloride intracellular channel 4, NADH oxidoreductase and dehydrogenase, fibronectin receptor beta chain, CD81 antigen, farnesyltransferase, calreticulin, disintegrin, p120 catenin, Smad7, etc. Although some of these genes have been suspected to be related to cardiovascular diseases, none of the genes has been previously shown to be involved in the mechanism of the cardiac effect of hyperlipidemia.

Animals↗
PubMed2004

Survival motor neuron SMN1 and SMN2 gene promoters: identical sequences and differential expression in neurons and non-neuronal cells.

Spinal muscular atrophy (SMA) is a recessive disorder involving the loss of motor neurons from the spinal cord. Homozygous absence of the survival of motor neuron 1 gene (SMN1) is the main cause of SMA, but disease severity depends primarily on the number of SMN2 gene copies. SMN protein levels are high in normal spinal cord and much lower in the spinal cord of SMA patients, suggesting neuron-specific regulation for this ubiquitously expressed gene. We isolated genomic DNA from individuals with SMN1 or SMN2 deletions and sequenced 4.6 kb of the 5' upstream regions of the these. We found that these upstream regions, one of which is telomeric and the other centromeric, were identical. We investigated the early regulation of SMN expression by transiently transfecting mouse embryonic spinal cord and fibroblast primary cultures with three transgenes containing 1.8, 3.2 and 4.6, respectively, of the SMN promoter driving beta-galactosidase gene expression. The 4.6 kb construct gave reporter gene expression levels five times higher in neurons than in fibroblasts, due to the combined effects of a general enhancer and a non-neuronal cell silencer. The differential expression observed in neurons and fibroblasts suggests that the SMN genes play a neuron-specific role during development. An understanding of the mechanisms regulating SMN promoter activity may provide new avenues for the treatment of SMA.

Analysis of Variance↗
PubMed2004

Transcript identification and profiling during salt stress and recovery of Populus euphratica.

Populus euphratica Oli. is a salt-tolerant species that can cope with up to 450 mM NaCl under hydroponic conditions and can tolerate high accumulations of Na+ and Cl- in roots and leaves when grown in 300 mM NaCl. Transcript responses to salt stress and recovery were monitored by microarray hybridization of 315 cDNAs preselected by suppression subtractive hybridization. Transcripts of a heat-shock protein and a hydroxyproline-rich glycoprotein accumulated 1.5 and 3 h, respectively, after adding 300 mM NaCl to the culture medium. Transcripts significantly up-regulated by salt stress included ionic and osmotic homeostasis elements such as magnesium transporter-like protein, syntaxin-like protein, seed imbibition protein and plasma membrane intrinsic protein; metabolism regulators like cytochrome P450, zinc finger protein, cleavage factor and aminotransferase; and the photosynthesis-activating enzyme Rubisco activase and photorespiration-related glycolate oxidase. Several photosynthesis-related transcripts were down-regulated in response to 72 h of salt stress but were up-regulated after long-term recovery (48 h). Sucrose synthase, ABC transporter, calmodulin, Pop3 peptide and aquaporin appeared to be actively involved in the process of plant recovery from salt stress. Several transcripts encoding proteins of unknown function were regulated by salt stress. Selected transcripts exhibiting altered transcript profiles in response to salt stress were also analyzed by real-time quantitative PCR. Transcript analysis during salt stress and recovery of this woody species revealed several genes and corresponding proteins deserving special attention in future studies of salt tolerance in woody species.

Electron Probe Microanalysis↗
PubMed2003

Modification by docosahexaenoic acid of age-induced alterations in gene expression and molecular composition of rat brain phospholipids.

Advanced age is associated with reduced brain levels of long-chain polyunsaturated fatty acids, arachidonic acid (AA) and docosahexaenoic acid (DHA). Memory impairment is also a common phenomenon in this age. Two-year-old, essential fatty acid-sufficient rats were fed with fish oil (11% DHA) for 1 month, and fatty acid as well as molecular composition of the major phospholipids, phosphatidylcholine and phosphatidylethanolamine (PE), was compared with that of 2-month-old rats on the same diet. DHA but not AA was significantly reduced in brains of old rats but was restored to the level of young rats when they received rat chow fortified with fish oil. This effect was pronounced with diacyl 18:0/22:6 PE species, whereas levels of 18:1/22:6 and 16:0/22:6 remained unchanged in all of the three PE subclasses. Fish oil reduced the AA in the old rat brains, diacyl and alkenylacyl 18:0/20:4 PE being most affected. Phosphatidylcholines gave less pronounced response. Six genes were up-regulated, whereas no significant changes were observed in brains of old rats receiving fish oil for 1 month. None of them except synuclein in young rat brains could be related to mental functions. Old rats on the fish-oil diet did not perform better in Morris water maze test than the control ones. A 10% increase in levels of diacyl 18:0/22:6 PE in young rat brains resulted in a significant improvement of learning capacity. The results are interpreted in terms of the roles of different phospholipid molecular species in cognitive functions coupled with differential responsiveness of the genetic machinery of neurons to n-3 polyunsaturated fatty acids.

Aging↗
PubMed2003

Gene expression and molecular composition of phospholipids in rat brain in relation to dietary n-6 to n-3 fatty acid ratio.

Rats were fed from conception till adulthood either with normal rat chow with a linoleic (LA) to linolenic acid (LNA) ratio of 8.2:1 or a rat chow supplemented with a mixture of perilla and soy bean oil giving a ratio of LA to LNA of 4.7:1. Fat content of the feed was 5%. Fatty acid and molecular species composition of ethanolamine phosphoglyceride was determined. Effect of this diet on gene expression was also studied. There was an accumulation of docosahexaenoic (DHA) and arachidonic acids (AA) in brains of the experimental animals. Changes in the ratio sn-1 saturated, sn-2 docosahexaenoic to sn-1 monounsaturated, sn-2 docosahexaenoic were observed. Twenty genes were found overexpressed in response to the 4.7:1 mixture diet and four were found down-regulated compared to normal rat chow. Among them were the genes related to energy household, lipid metabolism and respiration. The degree of up-regulation exceeded that observed with perilla with a ratio of LA to LNA 8.2:1 [Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 2619]. It was concluded that brain sensitively reacts to the fatty acid composition of the diet. It was suggested that alteration in membrane architecture and function coupled with alterations in gene expression profiles may contribute to the observed beneficial impact of n-3 type polyunsaturated fatty acids on cognitive functions.

Animals↗
PubMed2003

New molecular methods for classification, diagnosis and therapy prediction of hematological malignancies.

Normal functions of the cell are based on the precise regulation of various genes. If this strict regulation and the hierarchy of genes becomes upset due to flaws in this system, the result will be cellular dysfunction which eventually may lead to carcinogenic transformation. Two basic challenges of the classification of cancers are the discovery of new molecular markers characteristic to defined disease groups and the classification of already diagnosed or new cases into existing groups. This precise classification may open the door to tailored treatment or project the expected outcome of the disease. Today there is unlimited access available to the databases containing sequences and localization of the genes within the confines of Human Genome project. It provides significant help for the discovery of chromosome abnormalities and systematic analysis of gene expression patterns. This is important not only to understand normal functions of the cells, but it also contributes to the identification of new genes that are characteristic to given disease groups as markers and that are potential drug targets. Until the second half of the twentieth century the study of the function and regulation of genes was based on step-by-step investigation of individual genes. Regarding the fact, that the genomes of an increasing number of organisms have become known in whole or in part, numerous new techniques have been developed that facilitated the systematic analysis of gene functions. The aim of this study is to summarize the new, molecular based possibilities for classification, diagnosis and prognosis of hematological malignancies, as well as to summarize the main results of these areas.

Chromosome Aberrations↗
PubMed2003

Effect of classic preconditioning on the gene expression pattern of rat hearts: a DNA microarray study.

To profile gene expression patterns involved in ischemic preconditioning, we monitored global gene expression changes by DNA microarray analysis of 3200 rat-specific genes and by real-time quantitative polymerase chain reaction in rat hearts. Forty-nine genes with altered expression were found after ischemia/reperfusion as compared to control non-ischemic hearts and 31 genes were characteristic for classic preconditioning followed by ischemia/reperfusion as compared to ischemia/reperfusion without preconditioning. Genes with altered expression due to ischemia and/or preconditioning included those controlling protein degradation, stress responses, apoptosis, metabolic enzymes, regulatory proteins, and several unknown cellular functions. Metallothionein, natriuretic peptides, coagulation factor VII, cysteine proteinase inhibitor, peroxisome proliferator activator receptor gamma and myosin light chain kinase genes were previously suspected to be related to several cardiovascular diseases, however, most of these genes have not previously been shown to be related to myocardial ischemia/reperfusion. Some genes were observed to change specifically in response to preconditioning: oligoadenylate synthase, chaperonin subunit epsilon, a cGMP phosphodiesterase (PDE9A1), a secretory carrier membrane protein, an amino acid transporter, and protease 28 subunit. None of these genes has previously been shown to be involved in the mechanism of preconditioning.

Animals↗
PubMed2002

Murine peripherin gene sequences direct Cre recombinase expression to peripheral neurons in transgenic mice.

Spatially and temporally regulated somatic mutations can be achieved by using the Cre/loxP recombination system of bacteriophage P1. To develop a cell type-specific system of gene targeting in the peripheral nervous system, we generated the transgenic mouse lines expressing Cre recombinase under the control of the mouse peripherin gene promoter. The activity of the Cre recombinase during embryonic development was examined by mating the peripherin-Cre transgenic mice to the knock-in Cre-mediated recombination reporter strain, R26R. Analysis of F1 embryos from this cross showed specific excision of loxP-flanked sequences in the dorsal root ganglia, trigeminal ganglia, and olfactory epithelium, in a pattern very similar to the expression of the endogenous mouse peripherin gene, and the previously reported peripherin-lacZ transgenic mice. Thus, the peripherin-Cre mouse described here will provide a valuable tool for Cre-loxP-mediated conditional expression in the peripheral nervous system.

Animals↗
PubMed2002

The role of n-3 polyunsaturated fatty acids in brain: modulation of rat brain gene expression by dietary n-3 fatty acids.

Rats were fed either a high linolenic acid (perilla oil) or high eicosapentaenoic + docosahexaenoic acid (fish oil) diet (8%), and the fatty acid and molecular species composition of ethanolamine phosphoglycerides was determined. Gene expression pattern resulting from the feeding of n-3 fatty acids also was studied. Perilla oil feeding, in contrast to fish oil feeding, was not reflected in total fatty acid composition of ethanolamine phosphoglycerides. Levels of the alkenylacyl subclass of ethanolamine phosphoglycerides increased in response to feeding. Similarly, levels of diacyl phosphatidylethanolamine molecular species containing docosahexaenoic acid (18:0/22:6) were higher in perilla-fed or fish oil-fed rat brains whereas those in ethanolamine plasmalogens remained unchanged. Because plasmalogen levels in the brains of rats fed a n-3 fatty acid-enriched diet increased, it is plausible, however, that docosahexaenoic acid taken up from the food or formed from linolenic acid was deposited in this phospholipid subclass. Using cDNA microarrays, 55 genes were found to be overexpressed and 47 were suppressed relative to controls by both dietary regimens. The altered genes included those controlling synaptic plasticity, cytosceleton and membrane association, signal transduction, ion channel formation, energy metabolism, and regulatory proteins. This effect seems to be independent of the chain length of fatty acids, but the n-3 structure appears to be important. Because n-3 polyunsaturated fatty acids have been shown to play an important role in maintaining normal mental functions and docosahexaenoic acid-containing ethanolamine phosphoglyceride (18:0/22:6) molecular species accumulated in response to n-3 fatty acid feeding, a casual relationship between the two events can be surmised.

Animals↗
PubMed2002

RNA amplification results in reproducible microarray data with slight ratio bias.

Microarray expression analysis demands large amounts of RNA that are often not available. RNA amplification techniques have been developed to overcome this prcblem, but limited data are available regarding the reproducibility and maintenance of original transcript ratios. We optimized and validated two amplification techniques: a modified in vitro transcription for the linear amplification of 3 microg total RNA and a SMART PCR-based technique for the exponential amplification of 50 ng total RNA. To determine bias between transcript ratios, we compared the expression profiles in mouse testis versus spleen between the two amplification methods and a standard labeling protocol, using microarrays containing 4596 cDNAs spotted in duplicate. With each method, replicate hybridizations were highly reproducible. However, when comparing the amplification methods to standard labeling, correlation coefficients were lower. Twelve genes that exhibited inconsistent or contradictory expression ratios among the three methods were verified by quantitative RT-PCR. The amplification methods showed slightly more discrepancies in the expression ratios when compared to quantitative RT-PCR results but were more sensitive in terms of detecting expressed genes. In conclusion, although amplification methods introduce slight changes in the transcript ratios compared to standard labeling, they are highly reproducible. For small sample size, in vitro transcription is the preferred method, but one should never combine different labeling strategies within a single study.

Blotting, Southern↗