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Find video protocols related to scientific articles indexed in Pubmed.
Repair of single-point mutations by polypurine reverse Hoogsteen hairpins.
Hum Gene Ther Methods
PUBLISHED: 10-14-2014
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Abstract Polypurine reverse Hoogsteen hairpins (PPRHs) are formed by two intramolecularly bound antiparallel homopurine domains linked by a five-thymidine loop. One of the homopurine strands binds with antiparallel orientation by Watson-Crick bonds to the polypyrimidine target sequence, forming a triplex. We had previously reported the ability of PPRHs to effectively bind dsDNA displacing the fourth strand away from the newly formed triplex. The main goal of this work was to explore the possibility of repairing a point mutation in mammalian cells using PPRHs as tools. These repair-PPRHs contain different combinations of extended sequences of DNA with the corrected nucleotide to repair the point mutation. As a model we used the dihydrofolate reductase gene. On the one hand, we demonstrate in vitro that PPRHs bind specifically to their polypyrimidine target sequence, opening the two strands of the dsDNA, and allowing the binding of a given repair oligonucleotide to the displaced strand of the DNA. Subsequently, we show at a cellular level (Chinese ovary hamster cells) that repair-PPRHs are able to correct a single-point mutation in a dihydrofolate reductase minigene bearing a nonsense mutation, both in an extrachromosomal location and when the mutated plasmid was stably transfected into the cells. Finally, this methodology was successfully applied to repair a single-point mutation at the endogenous locus, using the DA5 cell line with a deleted nucleotide in exon six of the dhfr gene.
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Walnut polyphenol metabolites, urolithins A and B, inhibit the expression of the prostate-specific antigen and the androgen receptor in prostate cancer cells.
Food Funct
PUBLISHED: 09-13-2014
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Walnuts have been gathering attention for their health-promoting properties. They are rich in polyphenols, mainly ellagitannins (ETs) that after consumption are hydrolyzed to release ellagic acid (EA). EA is further metabolized by microbiota to form urolithins, such as A and B, which are absorbed. ETs, EA and urolithins have shown to slow the proliferation and growth of different types of cancer cells but the mechanisms remain unclear. We investigate the role of urolithins in the regulatory mechanisms in prostate cancer, specifically those related to the androgen receptor (AR), which have been linked to the development of this type of cancer. In our study, urolithins down-regulated the mRNA and protein levels of both prostate specific antigen (PSA) and AR in LNCaP cells. The luciferase assay performed with a construct containing three androgen response elements (AREs) showed that urolithins inhibit AR-mediated PSA expression at the transcriptional level. Electrophoretic mobility shift assays revealed that urolithins decreased AR binding to its consensus response element. Additionally, urolithins induced apoptosis in LNCaP cells, and this effect correlated with a decrease in Bcl-2 protein levels. In summary, urolithins attenuate the function of the AR by repressing its expression, causing a down-regulation of PSA levels and inducing apoptosis. Our results suggest that a diet rich in ET-containing foods, such as walnuts, could contribute to the prevention of prostate cancer.
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Stability and Immunogenicity Properties of the Gene-Silencing Polypurine Reverse Hoogsteen Hairpins.
Mol. Pharm.
PUBLISHED: 11-27-2013
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Gene silencing by either small-interference RNAs (siRNA) or antisense oligodeoxynucleotides (aODN) is widely used in biomedical research. However, their use as therapeutic agents is hindered by two important limitations: their low stability and the activation of the innate immune response. Recently, we developed a new type of molecule to decrease gene expression named polypurine reverse Hoogsteen hairpins (PPRHs) that bind to polypyrimidine targets in the DNA. Herein, stability experiments performed in mouse, human, and fetal calf serum and in PC3 cells revealed that the half-life of PPRHs is much longer than that of siRNAs in all cases. Usage of PPRHs with a nicked-circular structure increased the binding affinity to their target sequence and their half-life in FCS when bound to the target. Regarding the innate immune response, we determined that the levels of the transcription factors IRF3 and its phosphorylated form, as well as NF-?B were increased by siRNAs and not by PPRHs; that the expression levels of several proinflammatory cytokines including IL-6, TNF-?, IFN-?, IFN-ß, IL-1ß, and IL-18 were not significantly increased by PPRHs; and that the cleavage and activation of the proteolytic enzyme caspase-1 was not triggered by PPRHs. These determinations indicated that PPRHs, unlike siRNAs, do not activate the innate inflammatory response.
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Polypurine reverse Hoogsteen hairpins as a gene therapy tool against survivin in human prostate cancer PC3 cells in vitro and in vivo.
Biochem. Pharmacol.
PUBLISHED: 07-24-2013
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As a new approach for gene therapy, we recently developed a new type of molecule called polypurine reverse Hoogsteen hairpins (PPRHs). We decided to explore the in vitro and in vivo effect of PPRHs in cancer choosing survivin as a target since it is involved in apoptosis, mitosis and angiogenesis, and overexpressed in different tumors. We designed four PPRHs against the survivin gene, one of them directed against the template strand and three against different regions of the coding strand. These PPRHs were tested in PC3 prostate cancer cells in an in vitro screening of cell viability and apoptosis. PPRHs against the promoter sequence were the most effective and caused a decrease in survivin mRNA and protein levels. We confirmed the binding between the selected PPRHs and their target sequences in the survivin gene. In addition we determined that both the template- and the coding-PPRH targeting the survivin promoter were interfering with the binding of transcription factors Sp1 and GATA-3, respectively. Finally, we conducted two in vivo efficacy assays using the Coding-PPRH against the survivin promoter and performing two routes of administration, namely intratumoral and intravenous, in a subcutaneous xenograft tumor model of PC3 prostate cancer cells. The results showed that the chosen Coding-PPRH proved to be effective in decreasing tumor volume, and reduced the levels of survivin protein and the formation of blood vessels. These findings represent the preclinical proof of principle of PPRHs as a new silencing tool for cancer gene therapy.
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Validation of miRNA-mRNA interactions by electrophoretic mobility shift assays.
BMC Res Notes
PUBLISHED: 06-20-2013
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MicroRNAs are small non-coding RNAs involved in gene expression regulation by targeting specific regions in the 3[prime]-UTR of the mRNA of their target genes. This binding leads to a decrease in the protein levels of such genes either by mRNA degradation or mRNA destabilization and translation inhibition. The interaction between a miRNA and its target mRNAs is usually studied by co-transfection of a reporter expression vector containing the 3[prime]-UTR region of the mRNA and an inhibitory or precursor molecule for the miRNA. This approach, however, does not measure the direct and physical interaction between a miRNA and a specific mRNA.
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Cocoa flavanol metabolites activate HNF-3?, Sp1, and NFY-mediated transcription of apolipoprotein AI in human cells.
Mol Nutr Food Res
PUBLISHED: 01-07-2013
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To identify the mechanisms by which cocoa induces HDL levels and since apolipoprotein AI (ApoAI) is the major protein in HDLs, we analyzed, upon incubation with cocoa metabolites, ApoAI mRNA levels, its transcriptional regulation, and the levels of the transcription factors involved in this process.
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Therapeutic targeting of tumor growth and angiogenesis with a novel anti-S100A4 monoclonal antibody.
PLoS ONE
PUBLISHED: 01-01-2013
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S100A4, a member of the S100 calcium-binding protein family secreted by tumor and stromal cells, supports tumorigenesis by stimulating angiogenesis. We demonstrated that S100A4 synergizes with vascular endothelial growth factor (VEGF), via the RAGE receptor, in promoting endothelial cell migration by increasing KDR expression and MMP-9 activity. In vivo overexpression of S100A4 led to a significant increase in tumor growth and vascularization in a human melanoma xenograft M21 model. Conversely, when silencing S100A4 by shRNA technology, a dramatic decrease in tumor development of the pancreatic MiaPACA-2 cell line was observed. Based on these results we developed 5C3, a neutralizing monoclonal antibody against S100A4. This antibody abolished endothelial cell migration, tumor growth and angiogenesis in immunodeficient mouse xenograft models of MiaPACA-2 and M21-S100A4 cells. It is concluded that extracellular S100A4 inhibition is an attractive approach for the treatment of human cancer.
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The redox state of cytochrome c modulates resistance to methotrexate in human MCF7 breast cancer cells.
PLoS ONE
PUBLISHED: 01-01-2013
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Methotrexate is a chemotherapeutic agent used to treat a variety of cancers. However, the occurrence of resistance limits its effectiveness. Cytochrome c in its reduced state is less capable of triggering the apoptotic cascade. Thus, we set up to study the relationship among redox state of cytochrome c, apoptosis and the development of resistance to methotrexate in MCF7 human breast cancer cells.
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A lyophilized red grape pomace containing proanthocyanidin-rich dietary fiber induces genetic and metabolic alterations in colon mucosa of female C57BL/6J mice.
J. Nutr.
PUBLISHED: 07-20-2011
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Diet plays a decisive role in promoting or preventing colon cancer. However, the specific effects of some nutrients remain unclear. The capacity of fruit and vegetables to prevent cancer has been associated with their fiber and antioxidant composition. We investigated whether consumption of a lyophilized red grape pomace containing proanthocyanidin-rich dietary fiber (grape antioxidant dietary fiber, GADF) by female C57BL/6J mice would affect the serum metabolic profile or colon mucosa gene expression using NMR techniques and DNA microarray, respectively. The mice were randomly assigned to 2 groups that for 2 wk consumed a standard rodent diet and were gavaged with 100 mg/kg body weight GADF suspended in water or an equivalent volume of plain tap water (10 mL/kg body weight). The amount of fiber supplemented was calculated to equal the current recommended daily levels of fiber consumption for humans. The inclusion of dietary GADF induced alterations in the expression of tumor suppressor genes and proto-oncogenes as well as the modulation of genes from pathways, including lipid biosynthesis, energy metabolism, cell cycle, and apoptosis. Overexpression of enzymes pertaining to the xenobiotic detoxifying system and endogenous antioxidant cell defenses was also observed. In summary, the genetic and metabolic profiles induced by GADF were consistent with the preventive effects of fiber and polyphenols. On the basis of these observations, we propose that GADF may contribute to reducing the risk of colon cancer.
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Underexpression of miR-224 in methotrexate resistant human colon cancer cells.
Biochem. Pharmacol.
PUBLISHED: 07-01-2011
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MicroRNAs (miRNAs) are small non-coding RNAs involved in RNA silencing that play a role in many biological processes. They are involved in the development of many diseases, including cancer. Extensive experimental data show that they play a role in the pathogenesis of cancer as well as the development of drug resistance during treatments. The aim of this work was to detect differentially expressed miRNAs in MTX-resistant cells. Thus, miRNA microarrays of sensitive and MTX-resistant HT29 colon cancer cells were performed. The results were analyzed using the GeneSpring GX11.5 software. Differentially expressed miRNAs in resistant cells were identified and miR-224, which was one of the most differentially expressed miRNAs and with high raw signal values, was selected for further studies. The underexpression of miR-224 was also observed in CaCo-2 and K562 cells resistant to MTX. Putative targets were predicted using TargetScan 5.1 software and integrated with the data from expression microarrays previously performed. This approach allowed us to identify miR-224 targets that were differentially expressed more than 2-fold in resistant cells. Among them CDS2, DCP2, HSPC159, MYST3 and SLC4A4 were validated at the mRNA level by qRT-PCR. Functional assays using an anti-miR against miR-224 desensitized the cells towards MTX, mimicking the resistant phenotype. On the other hand, siRNA treatment against SLC4A4 or incubation of Poly Purine Reverse Hoogsteen (PPRH) hairpins against CDS2 or HSPC159 increased sensitivity to MTX. These results revealed a role for miR-224 and its targets in MTX resistance in HT29 colon cancer cells.
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Gene expression profiles in rat mesenteric lymph nodes upon supplementation with conjugated linoleic acid during gestation and suckling.
BMC Genomics
PUBLISHED: 04-11-2011
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Diet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA), a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life.
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Coding polypurine hairpins cause target-induced cell death in breast cancer cells.
Hum. Gene Ther.
PUBLISHED: 02-16-2011
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Polypurine reverse-Hoogsteen hairpins (PPRHs) are double-stranded DNA molecules formed by two polypurine stretches linked by a pentathymidine loop, with intramolecular reverse-Hoogsteen bonds that allow a hairpin structure. PPRHs bind to polypyrimidine targets by Watson-Crick bonds maintaining simultaneously a hairpin structure due to intramolecular Hoogsteen bonds. Previously, we described the ability of Template-PPRHs to decrease mRNA levels because these PPRHs target the template DNA strand interfering with the transcription process. Now, we designed Coding-PPRHs, a new type of PPRHs that directly target the pre-mRNA. The dihydrofolate reductase (dhfr) gene was selected as a target in breast cancer therapy. These PPRHs caused a high degree of cytotoxicity and a decrease in DHFR mRNA and protein levels, but by a different mechanism of action than Template-PPRHs. Coding-PPRHs interfere with the splicing process by competing with U2 auxiliary factor 65 for binding to the polypyrimidine target sequence, leading to a lower amount of mature mRNA. These new PPRHs showed high specificity as no off-target effects were found. The application of these molecules as therapeutic tools was tested in breast cancer cells resistant to methotrexate, obtaining a noticeable cytotoxicity even though the dhfr locus was amplified. Coding-PPRHs can be considered as new molecules to decrease gene expression at the mRNA level and an alternative to other antisense molecules.
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CYP1A1 is overexpressed upon incubation of breast cancer cells with a polyphenolic cocoa extract.
Eur J Nutr
PUBLISHED: 02-08-2011
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To evaluate the effect of cocoa flavonoids in breast cancer cells at the molecular level, a functional genomic analysis was performed using a polyphenolic cocoa extract (PCE) in MCF-7 and SKBR3 cell lines.
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UDP-glucuronosyltransferase 1A6 overexpression in breast cancer cells resistant to methotrexate.
Biochem. Pharmacol.
PUBLISHED: 07-16-2010
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Methotrexate is a chemotherapeutic agent used in breast cancer treatment, but the occurrence of resistance limits its therapeutic use. A microarrays analysis between sensitive and methotrexate resistant MCF7 and MDA-MB-468 breast cancer cells pointed out the UDP-glucuronosyltransferase 1A (UGT1A) family as a common deregulated node in both cell lines. This family of genes is involved in Phase II metabolism. UGT1A6 was the main isoform responsible for UGT1A family overexpression in these cells. Its overexpression was not due to gene amplification. Transfection of a vector encoding for UGT1A6 in sensitive cells counteracted the cytotoxicity caused by methotrexate. Methotrexate increased the transcriptional activity from a luciferase reporter driven by the UGT1A6 promoter and induced UGT1A6 mRNA and enzymatic activity. Promoter analysis suggested that UGT1A6 induction by methotrexate could be driven by the transcription factors ARNT (HIF-1) and AhR/ARNT. Cells incubated with anticancer drugs susceptible to glucuronidation, such as tamoxifen or irinotecan, together with methotrexate, showed a lesser degree of cytotoxicity, due to UGT1A6 induction. The pharmacological effect of this induction should be taken into account when combining methotrexate with other drugs that are glucuronidated.
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Overexpression of S100A4 in human cancer cell lines resistant to methotrexate.
BMC Cancer
PUBLISHED: 06-01-2010
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Methotrexate is a chemotherapeutic drug that is used in therapy of a wide variety of cancers. The efficiency of treatment with this drug is compromised by the appearance of resistance. Combination treatments of MTX with other drugs that could modulate the expression of genes involved in MTX resistance would be an adequate strategy to prevent the development of this resistance.
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Networking of differentially expressed genes in human cancer cells resistant to methotrexate.
Genome Med
PUBLISHED: 05-22-2009
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The need for an integrated view of data obtained from high-throughput technologies gave rise to network analyses. These are especially useful to rationalize how external perturbations propagate through the expression of genes. To address this issue in the case of drug resistance, we constructed biological association networks of genes differentially expressed in cell lines resistant to methotrexate (MTX).
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Polypurine hairpins directed against the template strand of DNA knock down the expression of mammalian genes.
J. Biol. Chem.
PUBLISHED: 03-03-2009
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We analyzed whether polypurine hairpins (PPRHs) had the ability to knock down gene expression. These hairpins are formed by two antiparallel purine domains linked by a loop that allows the formation of Hoogsteen bonds between both domains and Watson-Crick bonds with the target polypyrimidine sequence, forming triplex structures. To set up the experimental conditions, the human dhfr gene was used as a model. The PPRHs were designed toward the template strand of DNA. The transfection of the human breast cancer cell line SKBR3 with these template hairpins against the dhfr gene produced higher than 90% of cell mortality. Template PPRHs produced a decrease in DHFR mRNA, protein, and its corresponding enzymatic activity. In addition, the activity of DHFR PPRHs was tested against breast cancer cells resistant to methotrexate, observing high cell mortality. Given the difficulty in finding long polypyrimidine stretches, we studied how to compensate for the presence of purine interruptions in the polypyrimidine target sequence. The stability of PPRH was measured, resulting in a surprisingly long half-life of about 5 days. Finally, to test the generality of usage, template PPRHs were employed against two important genes involved in cell proliferation, telomerase and survivin, producing 80 and 95% of cell death, respectively. Taken together our results show the ability of antiparallel purine hairpins to bind the template strand of double strand DNA and to decrease gene transcription. Thus, PPRHs can be considered as a new type of molecules to modulate gene expression.
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Inhibition of cancer cell growth by ruthenium(II) cyclopentadienyl derivative complexes with heteroaromatic ligands.
J. Inorg. Biochem.
PUBLISHED: 01-06-2009
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Inhibition of the growth of LoVo human colon adenocarcinoma and MiaPaCa pancreatic cancer cell lines by two new organometallic ruthenium(II) complexes of general formula [Ru(eta(5)-C(5)H(5))(PP) L][CF(3)SO(3)], where PP is 1,2-bis(diphenylphosphino)ethane and L is 1,3,5-triazine (Tzn) 1 or PP is 2x triphenylphosphine and L is pyridazine (Pyd) 2 has been investigated. Crystal structures of compounds 1 and 2 were determined by X-ray diffraction studies. Atomic force microscopy (AFM) images suggest different mechanisms of interaction with the plasmid pBR322 DNA; while the mode of binding of compound 1 could be intercalation between base pairs of DNA, compound 2 might be involved in a covalent bond formation with N from the purine base.
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Identification of novel Sp1 targets involved in proliferation and cancer by functional genomics.
Biochem. Pharmacol.
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Sp1 is a transcription factor regulating many genes through its DNA binding domain, containing three zinc fingers. We were interested in identifying target genes regulated by Sp1, particularly those involved in proliferation and cancer. Our approach was to treat HeLa cells with a siRNA directed against Sp1 mRNA to decrease the expression of Sp1 and, in turn, the genes activated by this transcription factor. Sp1-siRNA treatment led to a great number of differentially expressed genes as determined by whole genome cDNA microarray analysis. Underexpressed genes were selected since they represent putative genes activated by Sp1 and classified in six Gene Onthology categories, namely proliferation and cancer, mRNA processing, lipid metabolism, glucidic metabolism, transcription and translation. Putative Sp1 binding sites were found in the promoters of the selected genes using the Match™ software. After literature mining, 11 genes were selected for further validation. Underexpression by qRT-PCR was confirmed for the 11 genes plus Sp1 in HeLa cells after Sp1-siRNA treatment. EMSA and ChIP assays were performed to test for binding of Sp1 to the promoters of these genes. We observed binding of Sp1 to the promoters of RAB20, FGF21, IHPK2, ARHGAP18, NPM3, SRSF7, CALM3, PGD and Sp1 itself. Furthermore, the mRNA levels of RAB20, FGF21 and IHPK2 and luciferase activity for these three genes related to proliferation and cancer, were determined after overexpression of Sp1 in HeLa cells, to confirm their regulation by Sp1. Involvement of these three genes in proliferation was validated by gene silencing using polypurine reverse hoogsteen hairpins.
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Coffee polyphenols change the expression of STAT5B and ATF-2 modifying cyclin D1 levels in cancer cells.
Oxid Med Cell Longev
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Epidemiological studies suggest that coffee consumption reduces the risk of cancer, but the molecular mechanisms of its chemopreventive effects remain unknown.
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Transcriptional profiling of striatal neurons in response to single or concurrent activation of dopamine D2, adenosine A(2A) and metabotropic glutamate type 5 receptors: focus on beta-synuclein expression.
Gene
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G protein-coupled receptor oligomerization is a concept which is changing the understanding of classical pharmacology. Both, oligomerization and functional interaction between adenosine A(2A,) dopamine D(2) and metabotropic glutamate type 5 receptors have been demonstrated in the striatum. However, the transcriptional consequences of receptors co-activation are still unexplored. We aim here to determine the changes in gene expression of striatal primary cultured neurons upon isolated or simultaneous receptor activation. Interestingly, we found that 95 genes of the total analyzed (15,866 transcripts and variants) changed their expression in response to simultaneous stimulation of all three receptors. Among these genes, we focused on the ?-synuclein (?-Syn) gene (SCNB). Quantitative PCR verified the magnitude and direction of change in expression of SCNB. Since ?-Syn belongs to the homologous synuclein family and may be considered a natural regulator of ?-synuclein (?-Syn), it has been proposed that ?-Syn might act protectively against ?-Syn neuropathology.
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Influence of habitat quality, population size, patch size, and connectivity on patch-occupancy dynamics of the middle spotted woodpecker.
Conserv. Biol.
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Despite extensive research on the effects of habitat fragmentation, the ecological mechanisms underlying colonization and extinction processes are poorly known, but knowledge of these mechanisms is essential to understanding the distribution and persistence of populations in fragmented habitats. We examined these mechanisms through multiseason occupancy models that elucidated patch-occupancy dynamics of Middle Spotted Woodpeckers (Dendrocopos medius) in northwestern Spain. The number of occupied patches was relatively stable from 2000 to 2010 (15-24% of 101 patches occupied every year) because extinction was balanced by recolonization. Larger and higher quality patches (i.e., higher density of oaks >37 cm dbh [diameter at breast height]) were more likely to be occupied. Habitat quality (i.e., density of large oaks) explained more variation in patch colonization and extinction than did patch size and connectivity, which were both weakly associated with probabilities of turnover. Patches of higher quality were more likely to be colonized than patches of lower quality. Populations in high-quality patches were less likely to become extinct. In addition, extinction in a patch was strongly associated with local population size but not with patch size, which means the latter may not be a good surrogate of population size in assessments of extinction probability. Our results suggest that habitat quality may be a primary driver of patch-occupancy dynamics and may increase the accuracy of models of population survival. We encourage comparisons of competing models that assess occupancy, colonization, and extinction probabilities in a single analytical framework (e.g., dynamic occupancy models) so as to shed light on the association of habitat quality and patch geometry with colonization and extinction processes in different settings and species.
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