Dezoxinukleozid kinázok szerepe a DNS repair és az apoptózis folyamatában = The role of deoxynucleoside kinases in DNA repair and apoptosis

A genotoxikus anyagok és a besugárzás DNS-károsodást váltanak ki, amelynek szenzora a p53 tumor-szuppresszor fehérje. A kezelések hatására a limfocitákon és a vad típusú p53-at expresszáló DG-75 sejtvonalon többszörös dezoxicitidin-kináz (dCK) aktiválódást észleltünk, ellentétben a p53-at nem expresszáló BL-41 és Ramos sejtekkel. A pifithrin-alfával és a Ca2+-kelátor BAPTA-AM-mel a dCK-aktiválódás felfüggeszthető. Az eredmények azt valószínűsítik, hogy a p53 és a Ca2+ lényeges szerepet játszik a dCK aktiválódásban. Az aktivált enzim stabilabb, immunreaktivitása fokozott a kontroll enziméhez képest. Limitált tripszinolízis segítségével bebizonyítottunk, hogy a dCK aktiválása közben konformáció-változás történik. Az aktivált dCK foszfoprotein-foszfatáz emésztésével, a tisztított dCK kétdimenziós elektroforézisével, valamint affinitás-kromatográfiával elsőként mutattuk ki, hogy a dezoxicitidin-kináz fehérjének legalább 30%-a foszforilált. Azonban a foszforilációs helyek azonosítása tömeg-spektroszkópiai analízissel nem sikerült, valószínűleg az endogén dCK kis mennyisége és az általunk használt tömegspektroszkópiás módszer nem eléggé érzékeny volta miatt. A dAdo is hatékonyan stimulálta a dCK-t, párhuzamosan a sejtek kaszpáz-3-aktivitása és a dATP-pool is szignifikánsan (150-170%) emelkedett, miközben a pirimidin-poolok 30-50%-kal csökkentek. Az eredmények azt bizonyítják, hogy a dCK aktiválódásának fontos szerepe lehet a dAdo-mediálta citotoxicitásban. | Both genotoxic agents and irradiation cause DNA damage that activates the p53 tumour suppressor protein. Deoxycytidine kinase (dCK) activity was stimulated several fold in irradiated lymphocytes and in DG-75 cells expressing wild-type p53, but not in p53-/- BL-41 and Ramos cells. Enhancement of dCK activity can be totally released with p53 inhibitors or with the calcium chelator BAPTA-AM. These results suggest that both p53 and Ca2+ play an important role in the activation of dCK.The activated enzyme is more stable and its native immunoreactivity is increased as compared to the control species. Using limited tripsinolysis, we have proven that dCK undergoes a conformational change during activation. We also demonstrated by several assays (phosphoproteine phosphatase digestion, two-dimensional electrophoresis, affinity chromatography) that 30% of the cellular dCK pool is phosphorylated. However, we failed to identify the phosphorylated amino acid residues in the dCK sequence, probably due to the too low amounts of endogenous dCK in cells, and the mass spectrometry method we had used might not have been sensitive enough. dAdo effectively stimulates dCK, and we could observe parallel activation of caspase 3 and a simultaneous 150-170% increase of the dATP pool, paralleled by 30-50% decrease of pyrimidine pools. These results underline the importance of dCK activation in the mechanism of dAdo-mediated cytotoxicity

Hypoxia-induced transcription of dopamine D3 and D4 receptors in human neuroblastoma and astrocytoma cells

<p>Abstract</p> <p>Background</p> <p>Dopaminergic pathways that influence mood and behaviour are severely affected in cerebral hypoxia. In contrast, hypoxia promotes the differentiation of dopaminergic neurons. In order to clarify the hypoxic sensitivity of key dopaminergic genes, we aimed to study their transcriptional regulation in the context of neuroblastoma and astrocytoma cell lines exposed to 1% hypoxia.</p> <p>Results</p> <p>Quantitative RT-PCR assays revealed that the transcription of both type D3 and D4 postsynaptic dopamine receptors (DRD3 and DRD4) was induced several fold upon 2-day hypoxia in a cell-specific manner, while the vascular endothelial growth factor gene was activated after 3-hr incubation in hypoxia. On the other hand, mRNA levels of type 2 dopamine receptor, dopamine transporter, monoamino oxidase and catechol-O-methyltransferase were unaltered, while those of the dopamine receptor regulating factor (DRRF) were decreased by hypoxia. Notably, 2-day hypoxia did not result in elevation of protein levels of DRD3 and DRD4.</p> <p>Conclusion</p> <p>In light of the relatively delayed transcriptional activation of the DRD3 and DRD4 genes, we propose that slow-reacting hypoxia sensitive transcription factors might be involved in the transactivation of DRD3 and DRD4 promoters in hypoxia.</p

Altered gene expression profiles in the hippocampus and prefrontal cortex of type 2 diabetic rats

<p>Abstract</p> <p>Background</p> <p>There has been an increasing body of epidemiologic and biochemical evidence implying the role of cerebral insulin resistance in Alzheimer-type dementia. For a better understanding of the insulin effect on the central nervous system, we performed microarray-based global gene expression profiling in the hippocampus, striatum and prefrontal cortex of streptozotocin-induced and spontaneously diabetic Goto-Kakizaki rats as model animals for type 1 and type 2 diabetes, respectively.</p> <p>Results</p> <p>Following pathway analysis and validation of gene lists by real-time polymerase chain reaction, 30 genes from the hippocampus, such as the inhibitory neuropeptide galanin, synuclein gamma and uncoupling protein 2, and 22 genes from the prefrontal cortex, e.g. galanin receptor 2, protein kinase C gamma and epsilon, <it>ABCA1 </it>(ATP-Binding Cassette A1), <it>CD47 </it>(Cluster of Differentiation 47) and the <it>RET </it>(Rearranged During Transfection) protooncogene, were found to exhibit altered expression levels in type 2 diabetic model animals in comparison to non-diabetic control animals. These gene lists proved to be partly overlapping and encompassed genes related to neurotransmission, lipid metabolism, neuronal development, insulin secretion, oxidative damage and DNA repair. On the other hand, no significant alterations were found in the transcriptomes of the corpus striatum in the same animals. Changes in the cerebral gene expression profiles seemed to be specific for the type 2 diabetic model, as no such alterations were found in streptozotocin-treated animals.</p> <p>Conclusions</p> <p>According to our knowledge this is the first characterization of the whole-genome expression changes of specific brain regions in a diabetic model. Our findings shed light on the complex role of insulin signaling in fine-tuning brain functions, and provide further experimental evidence in support of the recently elaborated theory of type 3 diabetes.</p

High Throughput Multiplex SNP-analysis In Chronic Obstructive Pulmonary Disease and Lung Cancer

Background A number of human inflammatory diseases and tumors have been shown to cause alterations in the glycosylation pattern of plasma proteins in a specific manner. These highly variable and versatile post-translational modifications fine-tune protein functions by influencing sorting, folding, enzyme activity and subcellular localization. However, relatively little is known about regulatory factors of this procedure and about the accurate causative connection between glycosylation and disease. Objective The aim of the present study was to investigate whether certain single nucleotide polymorphisms (SNPs) in genes encoding glycosyltransferases and glycosidases could be associated with elevated risk for chronic obstructive pulmonary disease (COPD) and lung adenocarcinoma. Methods A total of 32 SNPs localized in genes related to N-glycosylation were selected for the association analysis. Polymorphisms with putative biological functions (missense or regulatory variants) were recruited. SNPs were genotyped by a TaqMan OpenArray platform. A single base extension based method in combination with capillary gel electrophoresis was used for verification. Results The TaqMan OpenArray approach provided accurate and reliable genotype data (global call rate: 94.9%, accuracy: 99.6%). No significant discrepancy was detected between the obtained and expected genotype frequency values (Hardy–Weinberg equilibrium) in the healthy control sample group in case of any SNP confirming reliable sampling and genotyping. Allele frequencies of the rs3944508 polymorphism localized in the 3’ UTR of the MGAT5 gene significantly differed in the sample groups compared. Conclusion Our results suggest that the rs34944508 SNP might modulate the risk for lung cancer by influencing the expression of MGAT5. This enzyme catalyzes the addition of N-acetylglucosamine (GlcNAc) in beta 1-6 linkage to the alpha-linked mannose of biantennary N-linked oligosaccharides, thus, increases branching that characteristic for invasive malignancies

A common polymorphism of the human cardiac sodium channel alpha subunit (SCN5A) gene is associated with sudden cardiac death in chronic ischemic heart disease

Cardiac death remains one of the leading causes of mortality worldwide. Recent research has shed light on pathophysiological mechanisms underlying cardiac death, and several genetic variants in novel candidate genes have been identified as risk factors. However, the vast majority of studies performed so far investigated genetic associations with specific forms of cardiac death only (sudden, arrhythmogenic, ischemic etc.). The aim of the present investigation was to find a genetic marker that can be used as a general, powerful predictor of cardiac death risk. To this end, a case-control association study was performed on a heterogeneous cohort of cardiac death victims (n=360) and age-matched controls (n=300). Five single nucleotide polymorphisms (SNPs) from five candidate genes (beta2 adrenergic receptor, nitric oxide synthase 1 adaptor protein, ryanodine receptor 2, sodium channel type V alpha subunit and transforming growth factor-beta receptor 2) that had previously been shown to associate with certain forms of cardiac death were genotyped using sequence-specific real-time PCR probes. Logistic regression analysis revealed that the CC genotype of the rs11720524 polymorphism in the SCN5A gene encoding a subunit of the cardiac voltage-gated sodium channel occurred more frequently in the highly heterogeneous cardiac death cohort compared to the control population (p=0.019, odds ratio: 1.351). A detailed subgroup analysis uncovered that this effect was due to an association of this variant with cardiac death in chronic ischemic heart disease (p=0.012, odds ratio =1.455). None of the other investigated polymorphisms showed association with cardiac death in this context. In conclusion, our results shed light on the role of this non-coding polymorphism in cardiac death in ischemic cardiomyopathy. Functional studies are needed to explore the pathophysiological background of this association. © 2015 Marcsa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Association between anxiety and non-coding genetic variants of the galanin neuropeptide

Galanin, an inhibitory neuropeptide and cotransmitter has long been known to co-localize with noradrenaline and serotonin in the central nervous system. Several human studies demonstrated altered galanin expression levels in major depressive disorder and anxiety. Pharmacological modulation of galanin signaling and transgenic strategies provide further proof for the involvement of the galanin system in the pathophysiology of mood disorders. Little is known, however, on the dynamic regulation of galanin expression at the transcriptional level. The aim of the present study was to seek genetic association of non-coding single nucleotide variations in the galanin gene with anxiety and depression.Six single nucleotide polymorphisms (SNP) occurring either in the regulatory 5' or 3' flanking regions or within intronic sequences of the galanin gene have been genotyped with a high-throughput TaqMan OpenArray qPCR system in 526 healthy students (40% males). Depression and anxiety scores were obtained by filling in the Hospital Anxiety and Depression Scale (HADS) questionnaire. Data were analyzed by ANCOVA and Bonferroni correction was applied for multiple testing. Linkage disequilibrium (LD) analysis was used to map two haploblocks in the analyzed region.A single-locus and a haplotype genetic association proved to be statistically significant. In single-marker analysis, the T allele of the rs1042577 SNP within the 3' untranslated region of the galanin gene associated with greater levels of anxiety (HADS scores were 7.05±4.0 vs 6.15±.15; p = 0.000407). Haplotype analysis revealed an association of the rs948854 C_rs4432027_C allele combination with anxiety [F(1,1046) = 4.140, p = 0.042141, η2 = 0.004, power = 0.529]. Neither of these associations turned out to be gender-specific. These promoter polymorphisms are supposed to participate in epigenetic regulation of galanin expression by creating potentially methylatable CpG dinucleotides. The functional importance of the rs1042577_T allele remains to be elucidated

Glial Cell Line-Derived Neurotrophic Factor (GDNF) as a Novel Candidate Gene of Anxiety.

Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor for dopaminergic neurons with promising therapeutic potential in Parkinson's disease. A few association analyses between GDNF gene polymorphisms and psychiatric disorders such as schizophrenia, attention deficit hyperactivity disorder and drug abuse have also been published but little is known about any effects of these polymorphisms on mood characteristics such as anxiety and depression. Here we present an association study between eight (rs1981844, rs3812047, rs3096140, rs2973041, rs2910702, rs1549250, rs2973050 and rs11111) GDNF single nucleotide polymorphisms (SNPs) and anxiety and depression scores measured by the Hospital Anxiety and Depression Scale (HADS) on 708 Caucasian young adults with no psychiatric history. Results of the allele-wise single marker association analyses provided significant effects of two single nucleotide polymorphisms on anxiety scores following the Bonferroni correction for multiple testing (p = 0.00070 and p = 0.00138 for rs3812047 and rs3096140, respectively), while no such result was obtained on depression scores. Haplotype analysis confirmed the role of these SNPs; mean anxiety scores raised according to the number of risk alleles present in the haplotypes (p = 0.00029). A significant sex-gene interaction was also observed since the effect of the rs3812047 A allele as a risk factor of anxiety was more pronounced in males. In conclusion, this is the first demonstration of a significant association between the GDNF gene and mood characteristics demonstrated by the association of two SNPs of the GDNF gene (rs3812047 and rs3096140) and individual variability of anxiety using self-report data from a non-clinical sample

Diabetes-specific Modulation of Peripheral Blood Gene Expression Signatures in Colorectal Cancer

Background: Type 2 diabetes (T2DM) and colorectal cancer (CRC) are both known to modulate gene expression patterns in peripheral blood leukocytes (PBLs). Objective: As T2DM has been shown to increase the incidence of CRC, we were prompted to check whether diabetes affects mRNA signatures in PBLs isolated from CRC patients. Methods: Twenty-two patients were recruited to the study and classified into four cohorts (healthy controls; T2DM; CRC; CRC and T2DM). Relative expression levels of 573 cell signaling gene transcripts were determined by reverse transcription real-time PCR assays run on low-density OpenArray platforms. Enrichment analysis was performed with the g:GOSt profiling tool to order differentially expressed genes into functional pathways. Results: 49 genes were found to be significantly up- or downregulated in tumorous diabetic individuals as compared to tumor-free diabetic controls, while 11 transcripts were differentially regulated in patients with CRC versus healthy, tumor-free and nondiabetic controls. Importantly, these gene sets were completely distinct, implying that diabetes exerts a profound influence on the transcription of signaling genes in CRC. The top 5 genes showing the most significant expression differences in both contexts were PCK2, MAPK9, CCND1, HMBS, TLR3 (p≤0.0040) and CREBBP, PPIA, NFKBIL1, MDM2 and SELPLG (p≤0.0121), respectively. Functional analysis revealed that most significantly affected pathways were cytokine, interleukin and PI3K/Akt/mTOR signaling cascades as well as mitotic regulation. Conclusion: We propose that differentially expressed genes listed above might be potential biomarkers of CRC and should be studied further on larger patient groups. Diabetes might promote colorectal carcinogenesis by impairing signaling pathways in PBLs