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Results for “RNA, Competitive Endogenous”

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PubMed2000

Activation of nuclear factor-kappaB in C6 rat glioma cells after transfection with glia maturation factor.

The 17-kDa endogenous brain protein glia maturation factor (GMF) was transfected into C6 rat glioma cells using a replication-defective human adenovirus vector. The cells overexpressed GMF but did not secrete the protein into the medium. Transfection with GMF led to the activation of the transcription factor nuclear factor-kappaB (NF-kappaB), as evidenced by electrophoretic mobility shift assay of the nuclear extract, using a double-stranded oligonucleotide probe containing the consensus binding sequence for NF-kappaB. The specificity of binding was demonstrated by competition with unlabeled probe and by the nonbinding of the mutant probe. Binding was detectable as early as 3 h after transfection, peaked at 6 and 12 h, and gradually declined thereafter. The observed NF-kappaB activation was reduced by cotransfection with catalase and by the presence of high concentrations of pyruvate in the medium, suggesting the involvement of H2O2. The p38 mitogen-activated protein kinase inhibitor SB-203580 also suppressed the GMF-activated NF-kappaB, suggesting the involvement of the p38 signal transduction cascade. On the other hand, the phorbol ester phorbol 12-myristate 13-acetate activated NF-kappaB whether or not GMF was overexpressed. Along with NF-kappaB activation was an enhanced expression of superoxide dismutase (SOD), which was suppressed if NF-kappaB nuclear translocation was blocked by its specific decoy DNA, implicating NF-kappaB as an upstream mediator of this antioxidant enzyme. The p38 inhibitor SB-203580 also blocked the GMF-activated SOD. As NF-kappaB and SOD are both pro-survival signals, the results suggest a cytoprotective role for endogenous GMF in glial cells.

Animals↗

PubMed2003

Regulatory effects of gonadotropin-releasing hormone (GnRH) I and GnRH II on the levels of matrix metalloproteinase (MMP)-2, MMP-9, and tissue inhibitor of metalloproteinases-1 in primary cultures of human extravillous cytotrophoblasts.

An intricate balance between the production of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs), modulates the overall proteolytic activity of trophoblasts during human implantation. In these studies we have examined the ability of classical GnRH I and the second form of this hormone (GnRH II) to regulate MMP-2, MMP-9, and TIMP-1 mRNA and protein levels in extravillous cytotrophoblasts propagated from explants of first trimester chorionic villi. GnRH I and GnRH II were found to increase MMP-2 and MMP-9 mRNA and protein levels in these primary cell cultures in a dose- and time-dependent manner using quantitative competitive-PCR and ELISA. In contrast, these two hormones decreased trophoblastic TIMP-1 mRNA and protein levels. Cetrorelix, a GnRH receptor antagonist, inhibited the regulatory effects of GnRH I, but not GnRH II, on MMP-2, MMP-9, and TIMP-1 expression in these cells. Collectively, these observations suggest that GnRH I and GnRH II differentially regulate MMP-2, MMP-9, and TIMP-1 expression in human trophoblasts, possibly via distinct receptor-mediated intracellular signaling pathways.

Cells, Cultured↗

PubMed1997

Functional importance of Shc tyrosine 317 on insulin signaling in Rat1 fibroblasts expressing insulin receptors.

Shc is phosphorylated on Tyr-317, which serves as a docking site for Grb2. To investigate the specific role of Shc phosphorylation and Shc.Grb2 coupling on insulin signaling, we generated expression vectors for wild-type (WT-Shc) and a mutant Shc with a Tyr-317 --> Phe substitution (317Y/F-Shc) and stably transfected them into Rat1 fibroblasts expressing insulin receptors (HIRc). From different clonal cell lines, cells expressing 10 times greater amounts of WT-Shc or 317Y/F-Shc compared with endogenous Shc were chosen for analysis of insulin signaling. Insulin-induced Shc phosphorylation and subsequent association with Grb2 was enhanced in WT-Shc cells. Because of competition between Shc and IRS-1 for interaction with the insulin receptor, insulin-stimulated tyrosine phosphorylation of IRS-1 was decreased in WT-Shc cells compared with that in HIRc cells. Likewise, reduction of endogenous Shc expression by antisense Shc mRNA resulted in increased insulin stimulation of IRS-1 phosphorylation. Although 317Y/F-Shc was also able to interact with insulin receptor, decreased amounts of Shc phosphorylation and Shc association with Grb2 were observed in 317Y/F-Shc cells, indicating that 317Y/F-Shc functions as a dominant-negative mutant. The kinetics of mitogen-activated protein (MAP) kinase activation closely paralleled the kinetics of Shc phosphorylation. Thus, insulin stimulation of MAP kinase activation occurred more rapidly and was prolonged in WT-Shc cells, while the activation was delayed and transient in 317Y/F-Shc cells compared with that in HIRc cells. Importantly, WT-Shc cells displayed enhanced sensitivity to insulin stimulation of thymidine and bromodeoxyuridine incorporation, whereas the sensitivity was decreased in 317Y/F-Shc cells. These results indicate that Shc Tyr-317 phosphorylation plays an important role, via coupling with Grb2 and competition with IRS-1, in signal transduction to MAP kinase by insulin, ultimately leading to mitogenesis in Rat1 fibroblasts.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Repression of 5-aminolevulinate synthase gene by the potent tumor promoter, TPA, involves multiple signal transduction pathways.

The potent tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) induces activator protein-1 (AP-1) transcription factors, early response genes involved in a diverse set of transcriptional regulatory processes, and protein kinase C (PKC) activity. This work was designed to explore the signal transduction pathways involved in TPA regulation of 5-aminolevulinate synthase (ALAS) gene expression, the mitochondrial matrix enzyme that catalyzes the first and rate-limiting step of heme biosynthesis. We have previously reported that TPA causes repression of ALAS gene, but the signaling pathways mediating this effect remain elusive. The present study investigates the role of different cascades often implicated in the propagation of phorbol ester signaling. To explore this, we combined the transient overexpression of regulatory proteins involved in these pathways and the use of small cell permeant inhibitors in human hepatoma HepG2 cells. In these experimental conditions, we analyzed TPA action upon endogenous ALAS mRNA levels, as well as the promoter activity of a fusion reporter construct, harboring the TPA-responsive region of ALAS gene driving chloramphenicol acetyl transferase gene expression. We demonstrated that the participation of alpha isoform of PKC, phosphatidylinositol 3-kinase (PI3K), extracellular-signal regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK) is crucial for the end point response. Remarkably, in this case, ERK activation is achieved in a Ras/Raf/MEK-independent manner. We also propose that p90RSK would be a convergent point between PI3K and ERK pathways. Furthermore, we elucidated the crosstalk among the components of the cascades taking part in TPA-mediated ALAS repression. Finally, by overexpression of a constitutively active p90RSK and the coactivator, cAMP-response element protein (CREB)-binding protein (CBP), we reinforced our previous model, that implies competition between AP-1 and CREB for CBP.

5-Aminolevulinate Synthetase↗

PubMed2002

Increased intestinal ABCA1 expression contributes to the decrease in cholesterol absorption after plant stanol consumption.

The hypocholesterolemic effect of plant stanols is explained by a decreased intestinal cholesterol absorption due to a competition between plant stanols and cholesterol for incorporation into mixed micelles. Earlier we had suggested that plant stanols have a so far unknown action inside the enterocytes. The recent discovery of the involvement of ATP binding cassette (ABC) transporters in cholesterol absorption was a lead to further explore the hypocholesterolemic mechanism of plant stanols. We found that mixed micelles enriched with sitostanol or with cholesterol plus sitostanol were potent inducers of ABCA1 expression in caco-2 cells, an accepted model to study human intestinal lipoprotein metabolism. Based on these findings, we now hypothesize that plant stanols--and possibly plant sterols--increase ABCA1-mediated cholesterol efflux back into the intestinal lumen. We further hypothesize that intracellular levels of plant stanols are monitored by the same sensors (SREBP-2 and LXR) as those that monitor cholesterol. Consequently, increased plant stanol levels within the enterocyte activate cholesterol efflux through ABCA1- but not SREBP-2-mediated endogenous cholesterol synthesis even if intracellular cholesterol concentrations are lowered through consumption of plant stanols. If our hypothesis is correct, then the LXR pathway may be a target for dietary regulation of intestinal lipid metabolism.

ATP Binding Cassette Transporter 1↗

PubMed2003

Differential effects of gonadotropin-releasing hormone I and II on the urokinase-type plasminogen activator/plasminogen activator inhibitor system in human decidual stromal cells in vitro.

To date, the factors capable of regulating the coordinate expression of the urokinase-type plasminogen activator (uPA) and its endogenous inhibitor, plasminogen activator inhibitor (PAI-1), at the maternal-fetal interface remain poorly characterized. In these studies we examined the ability of the classical form of gonadotropin-releasing hormone (GnRH) I and the second, mammalian form of this hormone, GnRH II, to regulate uPA and PAI-1 mRNA and protein expression levels in cultures of stromal cells isolated from first trimester decidual tissues using quantitative competitive-PCR and ELISA, respectively. GnRH I and GnRH II increased uPA mRNA and protein expression levels in these primary cell cultures in a dose- and time-dependent manner. In contrast, GnRH I increased, whereas GnRH II decreased PAI-1 mRNA and protein expression levels in these cells. Cetrorelix, a GnRH receptor antagonist, inhibited the regulatory effects of GnRH I, but not GnRH II, on uPA and PAI-1 expression levels in these decidual stromal cell cultures. Taken together, these observations suggest that GnRH I and GnRH II differentially regulate the balance between uPA and PAI-1 expression levels in the human decidua, possibly via distinct receptor-mediated signaling pathways.

Adult↗

PubMed2005

Cytokine mRNA levels in brain and peripheral tissues of the rat: relationships with plus-maze behavior.

There is evidence that interleukin (IL)-2 may be related to anxiety as measured in the elevated plus-maze. Recently, we showed that normal adult male Wistar rats can differ systematically in this test of avoidance behavior, that is, time spent on the open arms of the elevated plus-maze. Rats with low open arm time had higher striatal levels of IL-2 mRNA than those with high open arm time, but did not differ significantly in expression of other striatal cytokine mRNA. Here, we investigated whether these expression effects are anatomically specific to the striatum. Therefore, we asked in this double-blind study whether elevated plus-maze behavior may also be related to endogenous levels of cytokine mRNA in other brain regions, which play a role for anxiety, namely the amygdala, hippocampus, and the prefrontal cortex. Additionally, and as peripheral controls, immuno-neuro-endocrine relevant tissues (adrenal glands, spleen) were analyzed. Based on open arm time in the elevated plus-maze, male Wistar rats were divided into sub-groups with either low or high open arm time behavior. Then, IL-1beta, IL-2, IL-6, and tumor necrosis factor (TNF)-alpha cDNA levels were measured post-mortem using semi-quantitative, competitive, reverse transcription polymerase chain reaction. First, we found that cytokine expressions differed considerably between and within these central and peripheral tissues. Secondly, rats with high compared to low open arm time behavior showed higher IL-2 mRNA levels in the prefrontal cortex, which is an inverse pattern to what we recently found in the striatum. These results provide new evidence indicating that cytokine mRNA in the brain can be related to elevated plus-maze behavior and that this relationship is site (prefrontal cortex, striatum)- and cytokine mRNA-specific (IL-2).

Adrenal Glands↗

PubMed1994

Distinct signaling pathways mediate phorbol-ester-induced and cytokine-induced inhibition of erythropoietin gene expression.

Hypoxia-induced erythropoietin (Epo) production in vitro is suppressed by interleukin 1 beta (IL-1 beta), tumor necrosis factor alpha (TNF) and phorbol esters. Herein, the Epo-synthesizing human hepatoma cell line HepG2 was used to investigate whether protein kinase C (PKC) is involved in the inhibitory action of the cytokines. Within 1 h after the onset of hypoxia, Epo mRNA levels were markedly increased in untreated HepG2 cells as quantitated by competitive reverse transcription PCR. The cytokines IL-1 beta and TNF prevented this hypoxia-induced increase in Epo mRNA levels. In phorbol-ester-treated cells first inhibitory effects on Epo mRNA levels were observed only after 3 h. Western blot analyses revealed the presence of four isoenzymes of PKC in HepG2 cells. None of these isoenzymes was translocated in response to TNF or IL-1 beta, suggesting that the cytokines do not activate PKC in HepG2 cells. In contrast, phorbol esters translocated and, upon prolonged exposure, down-regulated PKC isoenzymes alpha and epsilon. Activation of protein kinase A by dibutyryl-cAMP partially antagonized the cytokine-dependent inhibition of Epo production but did not influence the inhibitory effect of phorbol esters. Endogenous cAMP levels in HepG2 cells were unchanged by cytokine treatment. Obviously, at least two signaling pathways exist that can confer inhibition of Epo production in HepG2 cells. One of these may be mediated by down-regulation of the PKC alpha or epsilon isoenzyme. The other pathway, however, which is triggered by IL-1 beta and TNF, is independent of PKC.

1-Methyl-3-isobutylxanthine↗

PubMed2002

Endogenous DNA lesions can inhibit the binding of the AP-1 (c-Jun) transcription factor.

The repair of DNA damage, caused by both endogenous and exogenous sources, is necessary to remove lesions that either miscode or block DNA or RNA polymerases. We propose that damage also must be repaired to maintain sequence-specific DNA-protein interactions. In this paper, we have systematically studied two lesions that interfere with one important DNA landmark, the thymine methyl group. Oxidation of the thymine methyl group in DNA generates 5-hydroxymethyluracil (HmU) whereas the misincorporation of dUMP into DNA generates uracil (U), replacing the methyl group with a hydrogen. Both substitutions are shown to inhibit binding of the AP-1 (c-Jun) transcription factor. The energy cost of the perturbation, approximately 0.4 kcal/mol, is similar in magnitude for both U and HmU substitutions and is additive when multiple substitutions are present. A third lesion, substitution of the central C:G base pair of the AP-1 DNA binding domain with the pro-mutagenic U:G mispair, unexpectedly increases AP-1 binding, allowing the transcription factor to interfere with uracil DNA glycosylase activity. Our results support the hypothesis that an additional role for DNA repair systems is to maintain the integrity of sequence-specific DNA-protein interactions, a role of particular importance in long-lived organisms.

Adenine↗

PubMed1997

[Tissue-specific expression of human endogenous retrovirus mRNA and its regulation by cytokines in vitro].

To investigate biological roles of human endogenous retroviruses (ERVs), the author examined the viral mRNA expression in the normal systemic organs in vivo and its regulation by cytokines in cultured cells. The following evidence suggesting biological activities of a human ERV, ERV3, was obtained. First, the ERV3 mRNA was demonstrated at different levels in organs, and at consistently high levels in adrenal glands from all individuals and in all adrenocortical adenomas examined, by Northern hybridization. In situ hybridization revealed that the ERV3 expression was localized in all three layers of the adrenal cortex, but not in the medulla. These results suggest that the ERV3 expression may relate to the cellular differentiation and/or steroid production of adrenocortical cells. Second, the amount of ERV3 mRNA in cultured endothelial cells from human umbilical vein was significantly increased with any of TNF-alpha, IL-1 beta or IL-1 alpha stimulation but decreased with IFN-gamma treatment, by a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) with competitive PCR. The collective evidence suggests that the ERV3 expression may be upregulated at the inflammatory sites of vessels in vivo, and that the ERV3 expression may, therefore, play certain pathogenic roles in diseases, including collagen and vascular diseases in man.

Adrenal Cortex↗

PubMedDate not supplied

A preliminary investigation of the effects of maternal ethanol intake during gestation and lactation on brain adenosine A(1) receptor expression in rat offspring.

Ethanol exposure during fetal development can result in behavioral and neurological deficits, including reduced cognitive functions, retarded growth, and craniofacial abnormalities. Adenosine is an endogenous neuromodulator that fine-tunes the release and/or synaptic activities of several neurotransmitters, including glutamate, dopamine, and serotonin. Our aim was to determine whether ethanol exposure during early development affects adenosine receptors, particularly the A1 receptor subtype, in adult rats. Female rats were given water or 15% (vol/vol) ethanol in water prior to mating and throughout gestation and lactation. Sixty-day-old male rat offspring from these dams were randomly selected and assayed for adenosine A1 receptor expression in four brain areas: cortex, cerebellum, hippocampus, and striatum. Our results indicate that ethanol intake by dams decreased body and brain weights of offspring and reduced both A1 receptor mRNA and protein density in cortex and cerebellum. These preliminary findings indicate that ethanol intake by dams during pregnancy and lactation can affect adenosine A1 receptor signalling in the offspring. A pair-fed controlled study is warranted to explore these findings further.

Animals↗

PubMed2004

Prostaglandin E2 protects the heart from ischemia-reperfusion injury via its receptor subtype EP4.

BACKGROUND: In the heart with acute myocardial infarction, production of prostaglandin (PG) E2 increases significantly. In addition, several subtypes of PGE2 receptors (EPs) have been reported to be expressed in the heart. The role of PGE2 in cardiac ischemia-reperfusion (I/R) injury, however, remains unknown. We intended to clarify the role of PGE2 via EP4, an EP subtype, in I/R injury using mice lacking EP4 (EP4-/- mice). METHODS AND RESULTS: In murine cardiac ventricle, competitive reverse transcription-polymerase chain reaction revealed the highest expression level of EP4 mRNA among EP mRNAs. EP4-/- mice had larger infarct size than wild-type mice in a model of I/R; the left anterior descending coronary artery was occluded for 1 hour, followed by 24 hours of reperfusion. In addition, isolated EP4-/- hearts perfused according to the Langendorff technique had greater functional and biochemical derangements in response to I/R than wild-type hearts. In vitro, AE1-329, an EP4 agonist, raised cAMP concentration remarkably in noncardiomyocytes, whereas the action was weak in cardiomyocytes. When 4819-CD, another EP4 agonist, was administered 1 hour before coronary occlusion, it reduced infarct size significantly in wild-type mice. Notably, a similar cardioprotective effect was observed even when it was administered 50 minutes after coronary occlusion. CONCLUSIONS: Both endogenous PGE2 and an exogenous EP4 agonist protect the heart from I/R injury via EP4. The potent cardioprotective effects of 4819-CD suggest that the compound would be useful for treatment of acute myocardial infarction.

Animals↗

PubMed2006

Expression and characterization of vitamin C transporter in the human trophoblast cell line HTR-8/SVneo: effect of steroids, flavonoids and NSAIDs.

Vitamin C plays an important role in embryogenesis and fetal growth as well as in the progression of pregnancy and delivery. Therefore, it is important to understand the mechanism that mediates its transport to the fetus as well as the possible influences by endogenous and exogenous substances on its placental uptake. The aim of this study was to investigate placental sodium-dependent vitamin C transporters (SVCT) 1 and 2. By means of RT-PCR, we found that SVCT2, but not SVCT1, mRNA is expressed in human trophoblast cell line HTR-8/SVneo. Our method was able to confirm SVCT2 mRNA expression in human first-trimester chorionic villi but not in term placental tissue. Cell line kinetic studies of [(14)C] ascorbic acid (AA) uptake indicated a one-site model and a saturable process. Fetal bovine serum (FBS) and epidermal growth factor (EGF) do not influence the transport properties, although they significantly increase the expression of SVCT2. Steroid hormones (17beta-estradiol, progesterone and cortisol), flavonoids (genistein and quercetin) and non-steroidal anti-inflammatory drugs (NSAIDs) (indomethacin and diclofenac) inhibit [(14)C]AA uptake in a dose-dependent and non-competitive manner. On the contrary, the process is not influenced by aspirin. Our study suggests the use of HTR-8/SVneo cells as a suitable model for trophoblast vitamin C transport investigation.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1995

Identification of inducible mixed function oxidase system in rat glioma C6 cell line.

The mixed function oxidase system consists of NADPH-cytochrome P450 reductase (P450 reductase) and various isoforms of cytochrome P450 (P450), which can catalyze the oxidation of a broad range of endogenous and exogenous compounds. In this study, we examined the rat glioma C6 cell line for the presence of P450 reductase and three isozymes of cytochrome P450, 1A1, 2B1, and 2B2, by reverse transcription followed by PCR (RT-PCR). Rat glioma C6 cells were treated with hepatic P450 inducers phenobarbital (PB) or benzo(a)anthracene (BA). Cytochromes P450 1A1, P450 2B1, and P450 2B2, and P450 reductase, were detected in all the different treatment groups. Restriction digestion was used to confirm the PCR fragments and the expected digestion products were obtained. The induction of P450 1A1 and 2B was quantified using competitive PCR. Ten- and five-fold inductions of P450 1A and 2B mRNA after BA or PB treatments, respectively, were detected by competitive PCR. Microsomes prepared from rat glioma C6 cells showed cytochrome P450 spectra with absorption at 450 nm. Ethoxyresorufin O-deethylase activity (11.5 +/- 1.7 pmol/min/mg of microsomal protein) and pentoxyresorufin O-dealkylation activity (8.9 +/- 1.4 pmol/min/mg of microsomal protein) confirmed the induction of P450 1A and 2B at the protein level in response to BA or PB treatments, respectively. These experiments provide further evidence that the rat glioma C6 cell line contains an active mixed function oxidase system that can be induced by hepatic P450 inducers.

Animals↗

PubMed2001

Sarcoplasmic reticulum Ca(2+) atpase (SERCA) 1a structurally substitutes for SERCA2a in the cardiac sarcoplasmic reticulum and increases cardiac Ca(2+) handling capacity.

Ectopic expression of the sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA) 1a pump in the mouse heart results in a 2.5-fold increase in total SERCA pump level. SERCA1a hearts show increased rates of contraction/relaxation and enhanced Ca(2+) transients; however, the cellular mechanisms underlying altered Ca(2+) handling in SERCA1a transgenic (TG) hearts are unknown. In this study, using confocal microscopy, we demonstrate that SERCA1a protein traffics to the cardiac SR and structurally substitutes for the endogenous SERCA2a isoform. SR Ca(2+) load measurements revealed that TG myocytes have significantly enhanced SR Ca(2+) load. Confocal line-scan images of field-stimulated SR Ca(2+) release showed an increased rate of Ca(2+) removal in TG myocytes. On the other hand, ryanodine receptor binding activity was decreased by approximately 30%. However, TG myocytes had a greater rate of spontaneous ryanodine receptor opening as measured by spark frequency. Whole-cell L-type Ca(2+) current density was reduced by approximately 50%, whereas the time course of inactivation was unchanged in TG myocytes. These studies provide important evidence that SERCA1a can substitute both structurally and functionally for SERCA2a in the heart and that SERCA1a overexpression can be used to enhance SR Ca(2+) transport and cardiac contractility.

Animals↗

PubMed1999

Lipoxin A4: a new class of ligand for the Ah receptor.

The Ah receptor is a ligand-activated transcription factor that mediates many of the biological actions of a large class of environmental compounds. Support for a role of the Ah receptor in normal physiology also has been reported, but an endogenous regulating ligand has not been identified. We have examined candidate endogenous lipophilic substances and report here the ability of the arachidonic acid metabolite, lipoxin A4, to bind to and activate the Ah receptor in Hepa-1 cells. Lipoxin A4 produced a concentration-dependent response in a DRE-driven CAT reporter construct, with a greater than 10-fold increase in CAT activity at 0. 3 microM. Lipoxin A4 transformed the Ah receptor to an active DRE-binding form in a concentration-dependent manner as indicated by gel mobility shift analysis. Results of Ah receptor competitive binding experiments indicated that at a concentration of 100 nM, lipoxin A4 produced a half-maximum displacement (EC50) of [3H]TCDD binding. Results of Northern blot analyses indicated a transient increase in mRNA levels of the Ah receptor-responsive gene CYP1A1, which peaked at 4 h, consistent with the kinetics observed for lipoxin A4-induced CYP1A1 enzyme activity. Further, lipoxin A4 was found to be a competitive inhibitor for the CYP1A1 enzyme, with a calculated Ki = 1.1 microM. These results establish lipoxin A4 as a new class of Ah receptor ligand, one that differs dramatically from classical Ah receptor ligands.

Animals↗

PubMed2002

BDNF-triggered events in the rat hippocampus are required for both short- and long-term memory formation.

Information storage in the brain is a temporally graded process involving different memory types or phases. It has been assumed for over a century that one or more short-term memory (STM) processes are involved in processing new information while long-term memory (LTM) is being formed. Because brain-derived neutrophic factor (BDNF) modulates both short-term synaptic function and activity-dependent synaptic plasticity in the adult hippocampus, we examined the role of BDNF in STM and LTM formation of a hippocampal-dependent one-trial fear-motivated learning task in rats. Using a competitive RT-PCR quantitation method, we found that inhibitory avoidance training is associated with a rapid and transient increase in BDNF mRNA expression in the hippocampus. Bilateral infusions of function-blocking anti-BDNF antibody into the CA, region of the dorsal hippocampus decreased extracellular signal-regulated kinase 2 (ERK2) activation and impaired STM retention scores. Inhibition of ERK1/2 activation by PD098059 produced similar effects. In contrast, intrahippocampal administration of recombinant human BDNF increased ERK1/2 activation and facilitated STM. The infusion of anti-BDNF antibody impaired LTM when given 15 min before or 1 and 4 hr after training, but not at 0 or 6 hr posttraining, indicating that two hippocampal BDNF-sensitive time windows are critical for LTM formation. At the same time points, PD098059 produced no LTM deficits. Thus, our results indicate that endogenous BDNF is required for both STM and LTM formation of an inhibitory avoidance learning. Additionally, they suggest that this requirement involves ERK1/2-dependent and -independent mechanisms.

Animals↗

PubMed2004

Sensitivity and reproducibility of standardized-competitive RT-PCR for transcript quantification and its comparison with real time RT-PCR.

BACKGROUND: Probe based detection assays form the mainstay of transcript quantification. Problems with these assays include varying hybridization efficiencies of the probes used for transcript quantification and the expense involved. We examined the ability of a standardized competitive RT-PCR (StaRT PCR) assay to quantify transcripts of 4 cell cycle associated genes (RB, E2F1, CDKN2A and PCNA) in two cell lines (T24 & LD419) and compared its efficacy with the established Taqman real time quantitative RT-PCR assay. We also assessed the sensitivity, reproducibility and consistency of StaRT PCR. StaRT PCR assay is based on the incorporation of competitive templates (CT) in precisely standardized quantities along with the native template (NT) in a PCR reaction. This enables transcript quantification by comparing the NT and CT band intensities at the end of the PCR amplification. The CT serves as an ideal internal control. The transcript numbers are expressed as copies per million transcripts of a control gene such as beta-actin (ACTB). RESULTS: The NT and CT were amplified at remarkably similar rates throughout the StaRT PCR amplification cycles, and the coefficient of variation was least (<3.8%) when the NT/CT ratio was kept as close to 1:1 as possible. The variability between the rates of amplification in different tubes subjected to the same StaRT PCR reaction was very low and within the range of experimental noise. Further, StaRT PCR was sensitive enough to detect variations as low as 10% in endogenous actin transcript quantity (p < 0.01 by the paired student's t-test). StaRT PCR correlated well with Taqman real time RT-PCR assay in terms of transcript quantification efficacy (p < 0.01 for all 4 genes by the Spearman Rank correlation method) and the ability to discriminate between cell types and confluence patterns. CONCLUSION: StaRT PCR is thus a reliable and sensitive technique that can be applied to medium-high throughput quantitative transcript measurement. Further, it correlates well with Taqman real time PCR in terms of quantitative and discriminatory ability. This label-free, inexpensive technique may provide the ability to generate prognostically important molecular signatures unique to individual tumors and may enable identification of novel therapeutic targets.

Binding, Competitive↗