960 resultados para GAD mRNA
Resumo:
Low to moderate doses of alcohol consumption induce heightened aggressive behavior in some, but not all individuals. Individual vulnerability for this nonadaptive behavior may be determined by an interaction of genetic and environmental factors with the sensitivity of alcohol`s effects on brain and behavior. We used a previously established protocol for alcohol oral self-administration and characterized alcohol-heightened aggressive (AHA) mice as compared with alcohol non-heightened (ANA) counterparts. A week later, we quantified mRNA steady state levels of several candidate genes in the serotonin [5-hydroxytryptamine (5-HT)] system in different brain areas. We report a regionally selective and significant reduction of all 5-HT receptor subtype transcripts, except for 5-HT(3), in the prefrontal cortex of AHA mice. Comparable gene expression profile was previously observed in aggressive mice induced by social isolation or by an anabolic androgenic steroid. Additional change in the 5-HT(1B) receptor transcripts was seen in the amygdala and hypothalamus of AHA mice. In both these areas, 5-HT(1B) mRNA was elevated when compared with ANA mice. In the hypothalamus, AHA mice also showed increased transcripts for 5-HT(2A) receptor. In the midbrain, 5-HT synthetic enzyme, 5-HT transporter and 5-HT receptors mRNA levels were similar between groups. Our results emphasize a role for postsynaptic over presynaptic 5-HT receptors in mice which showed escalated aggression after the consumption of a moderate dose of alcohol. This gene expression profile of 5-HT neurotransmission components in the brain of mice may suggest a vulnerability trait for alcohol-heightened aggression.
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Mechanisms regulating NADPH oxidase remain open and include the redox chaperone protein disulfide isomerase (PDI). Here, we further investigated PDI effects on vascular NADPH oxidase. VSMC transfected with wild-type PDI (wt-PDI) OF PDI mutated in all four redox cysteines (mut-PDI) enhanced (2.5-fold) basal cellular ROS production and membrane NADPH oxidase activity, with 3-fold increase in Nox1, but not Nox4 mRNA. However, further ROS production, NADPH oxidase activity and Nox1 mRNA increase triggered by angiotensin-II (AngII) were totally lost with PDI overexpression, suggesting preemptive Nox1 activation in such cells. PDI overexpression increased Nox4 mRNA after AngII stimulus, although without parallel ROS increase. We also show that Nox inhibition by the nitric oxide donor GSNO is independent of PDI. PDI silencing decreased specifically Nox1 mRNA and protein, confirming that PDI may regulate Nox1 at transcriptional level in VSMC. Such data further strengthen the role of PDI as novel NADPH oxidase regulator. (C) 2009 Elsevier Inc. All rights reserved.
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The cytoplasmic and nuclear protein Ki- 1 / 57 was first identified in malignant cells from Hodgkin`s lymphoma. Despite studies showing its phosphorylation, arginine methylation, and interaction with several regulatory proteins, the functional role of Ki- 1 / 57 in human cells remains to be determined. Here, we investigated the relationship of Ki- 1 / 57 with RNA functions. Through immunoprecipitation assays, we verified the association of Ki- 1 / 57 with the endogenous splicing proteins hnRNPQ and SFRS9 in HeLa cell extracts. We also found that recombinant Ki- 1 / 57 was able to bind to a poly- U RNA probe in electrophoretic mobility shift assays. In a classic splicing test, we showed that Ki- 1 / 57 can modify the splicing site selection of the adenoviral E1A minigene in a dose- dependent manner. Further confocal and. uorescence microscopy analysis revealed the localization of enhanced green. uorescent protein - Ki- 1 / 57 to nuclear bodies involved in RNA processing and or small nuclear ribonucleoprotein assembly, depending on the cellular methylation status and its N- terminal region. In summary, our findings suggest that Ki- 1 / 57 is probably involved in cellular events related to RNA functions, such as pre- mRNA splicing.
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The endocannabinoid system includes important signaling molecules that are involved in several homeostatic and neuroendocrine functions. In the present study, we evaluated the effects of the type 1 cannabinoid (CB(1)) receptor antagonist, rimonabant (10 mg/kg, p.o.), on hormone secretion, neuronal activation and mRNA expression in the hypothalamus following isotonic (I-) or hypertonic (H-) extracellular volume expansion (EVE). The total nitrate content in the PVN and SON was also assessed under the same experimental conditions. Our results showed that OT and AVP plasma concentrations were increased in response to H-EVE, while decreased AVP levels were found following I-EVE. Accordingly, both I- and H-EVE stimulated oxytocinergic neuronal activation, as evidenced by the increased number of c-Fos/OT double labeled neurons in the hypothalamus. The vasopressinergic cells of the PVN and SON, however, were only activated in response to H-EVE. Furthermore, increased amounts of both AVP and OT mRNAs were found in the hypothalamus following EVE. Pretreatment with rimonabant significantly potentiated hormone secretion and also vasopressinergic and oxytocinergic neuronal activation induced by EVE, although decreased AVP and OT mRNA expression was found in the hypothalami of rimonabant pretreated groups. In addition, the nitrate content in the PVN and SON was not altered in response to EVE or rimonabant pretreatment. Taken together, these results suggest that the CB(1) receptor may modulate several events that contribute to the development of appropriate responses to increased fluid volume and osmolality. (C) 2010 Elsevier Inc. All rights reserved.
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The objective of this article was to estimate quantitative differences for GAPDH transcripts and poly(A) mRNA: (i) between oocytes collected from cumulus-oocyte complexes (COCs) qualified morphologically as grades A and B; (ii) between grade A oocytes before and after in vitro maturation (IVM); and (iii) among in vitro-produced embryos at different developmental stages. To achieve this objective a new approach was developed to estimate differences between poly(A) mRNA when using small samples. The approach consisted of full-length cDNA amplification (acDNA) monitored by real-time PCR, in which the cDNA from half of an oocyte or embryo was used as a template. The GAPDH gene was amplified as a reverse transcription control and samples that were not positive for GAPDH transcripts were discarded. The fold differences between two samples were estimated using delta Ct and statistical analysis and were obtained using the pairwise fixed reallocation randomization test. It was found that the oocytes recovered from grade B COCs had quantitatively less poly(A) mRNA (p < 0.01) transcripts compared with grade A COCs (1 arbitrary unit expression rate). In the comparison with immature oocytes (I arbitrary unit expression rate), the quantity of poly(A) mRNA did not change during IVM, but declined following IVF and varied with embryo culture (p < 0.05). Amplification of cDNA by real-time PCR was an efficient method to estimate differences in the amount of poly(A) mRNA between oocytes and embryos. The results obtained from individual oocytes suggested an association between poly(A) mRNA abundance and different morphological qualities of oocytes from COCs. In addition, a poly(A) mRNA profile was characterized from oocytes undergoing IVM, fertilization and blastocyst heating.
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Background. Increased activity of multidrug resistance (MDR) genes has been associated with treatment failure in acute leukemias, although with controversial reports. The objective of the present study was to assess the expression profile of the genes related to MDR: ABCB1, ABCC1, ABCC3, ABCC2, and LRP/MVP in terms of the clinical and biological variable and the survival of children with acute lymphoblastic leukemia (ALL). Procedure. The levels of mRNA expression of the drug resistance genes ABCB1, ABCC1, ABCC3, ABCG2, and LRP/MVP were analyzed by quantitative real-time PCR using the median Values as cut-off points, in consecutive samples from 140 children with ALL at diagnosis. Results. Expression levels of the ABCG2 gene in the patient group as a whole (P=0.05) and of the ABCG2 and ABCC1 genes in patients classified as being at high risk were associated with higher rates of 5-year event-free survival (EFS) (P=0.04 and P=0.01). Expression levels of the ABCG2 gene below the median were associated with a greater chance of death related to treatment toxicity for the patient group as a whole (P=0.009) and expression levels below the median of the ABCG2 and ABCC1 genes were associated with a greater chance of death due to treatment toxicity for the high-risk group (P=0.02 and P=0.03, respectively). Conclusion. The present data suggest a low participation of the drug efflux genes in treatment failure in patients with childhood ALL. However, the low expression of some of these genes may be associated with a higher death risk related to treatment toxicity. Pediatr Blood Cancer 2009;53:996-1004. (C) 2009 Wiley-Liss, Inc.
Resumo:
This study evaluates the mRNA expression profile of genes TIMP1, TIMP2, MMP2 and MMP9 in diagnostic bone marrow samples from 134 consecutive ALL children by real-time quantitative PCR. A significant association was observed between higher expression levels of MMP9 and low risk group and absence of extramedullary infiltration and higher expression levels of TIMP2 and MMP2 with T-ALL. TIMP1 gene expression values higher than the median were associated with a significantly lower 5-year event free-survival in univariable (P = 0.04) and multivariable analysis (P = 0.01). Our data address new information in the complex interaction of the migration/adhesion genes and childhood ALL. (c) 2009 Elsevier Ltd. All rights reserved.
Resumo:
Humans and mice with loss-of-function mutations of the genes encoding kisspeptins (Kiss1) or kisspeptin receptor (Kiss1r) are infertile due to hypogonadotropic hypogonadism. Within the hypothalamus, Kiss1 mRNA is expressed in the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (Arc). In order to better study the different populations of kisspeptin cells we generated Kiss1-Cre transgenic mice. We obtained one line with Cre activity specifically within Kiss1 neurons (line J2-4), as assessed by generating mice with Cre-dependent expression of green fluorescent protein or beta-galactosidase. Also, we demonstrated Kiss1 expression in the cerebral cortex and confirmed previous data showing Kiss1 mRNA in the medial nucleus of amygdala and anterodorsal preoptic nucleus. Kiss1 neurons were more concentrated towards the caudal levels of the Arc and higher leptin-responsivity was observed in the most caudal population of Arc Kiss1 neurons. No evidence for direct action of leptin in AVPV Kiss1 neurons was observed. Me lanocortin fibers innervated subsets of Kiss1 neurons of the preoptic area and Arc, and both populations expressed melanocortin receptors type 4 (MC4R). Specifically in the preoptic area, 18-28% of Kiss1 neurons expressed MC4R. In the Arc, 90% of Kiss1 neurons were glutamatergic, 50% of which also were GABAergic. In the AVPV, 20% of Kiss1 neurons were glutamatergic whereas 75% were GABAergic. The differences observed between the Kiss1 neurons in the preoptic area and the Arc likely represent neuronal evidence for their differential roles in metabolism and reproduction. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.
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Background. Defects in apoptosis signaling have been considered to be responsible for treatment failure in many types of cancer, although with controversial results. The objective of the present study was to assess the expression profile of key apoptosis-related genes in terms of clinical and biological variables and of the survival of children with acute lymphoblastic leukemia (ALL). Procedure. The levels of mRNA expression of the apoptosis-related genes CASP3, CASP8, CASP9, FAS, and BCL2 were analyzed by quantitative real-time PCR in consecutive samples from 139 consecutive children with ALL at diagnosis treated by the Brazilian protocol (GBTLI-ALL 99). Gene expression levels and clinical and biological features were compared by the Mann-Whitney test. Event-free survival (EFS) was calculated by Kaplan-Meier plots and log-rank test. Results. A significant correlation was detected between CASP3, CASP8, CASP9, and FAS expression levels (P<0.01) in ALL samples. Higher levels of BCL2 were significantly associated with white blood cell (WBC) count <50,000/mm(3) at diagnosis (P=0.01) and low risk group classification (P=0.008). Lower expression levels of CASP3, CASP8 and FAS gene were associated with a poor response at day 7 according the GBTLI-ALL 99 protocol (P=0.03, P=0.02 and P=0.008, respectively). There was a relationship between FAS gene expression lower than the 75th percentile and lower 5-year EFS (P=0.02). Conclusion. These findings suggest an association between lower expression levels of the pro-apoptotic genes and a poor response to induction therapy at day 7 and prognosis in childhood ALL. Pediatr Blood Cancer 2010;55:100-107. (C) 2010 Wiley-Liss, Inc.
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We analyzed the expression profile of two NMDAR1 mRNA isoform subsets. NR1(0xx) and NR1(1xx), in discrete regions of human cerebral cortex. The subsets are characterized by the absence or presence of a 21-amino acid N-terminal cassette. Reverse transcription polymerase chain reaction for NR1 isoforms was performed on total RNA preparations from spared and susceptible regions from 10 pathologically confirmed Alzheimer's disease (AD) cases and 10 matched controls. Primers spanning the splice insert yielded two bands, 342 bp (NR1(0xx)) and 405 bp (NR1(1xx)), on agarose gel electrophoresis. The bands were visualized with ethidium and quantified by densitometry. NR1(1xx) transcript expression was calculated as a proportion of the NR1(1xx) + NR1(0xx) total. Values were significantly lower in AD cases than in controls in mid-cingulate cortex, p < 0.01, superior temporal cortex, p < 0.01 and hippocampus, p similar to 0.05. Cortical proportionate NR1(1xx) transcript expression was invariant over the range of ages acid areas of controls tested, at similar to 50%. This was also true for AD motor and occipital cortex. Proportionate NR1(1xx) expression in AD cingulate and temporal cortex was lower at younger ages and increased with age: this regression was significantly different from that in the homotropic areas of controls. Variations in NR1 N-terminal cassette expression may underlie the local vulnerability to excitotoxic damage of some areas in the AD brain. Alternatively, changes in NR1 mRNA expression may arise as a consequence of the AD disease process.
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The plasma membrane Ca2+ pump is a key regulator of cytosolic free Ca2+. Recent studies have demonstrated the dynamic expression of the plasma membrane Ca2+ pump in a variety of cell types. Furthermore, alterations in plasma membrane calcium pump activity have now been implicated in human disease. In this study, the development of a technique to quantitatively assess mRNA expression of the human plasma membrane Ca2+ ATPase (PMCA1) isoform of the plasma membrane Ca2+ pump, using a real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) assay in a human breast epithelial cell line (MCF-7) is described. The sequences of the PMCA1 primers and probe for real-time RT-PCR are presented. The results also indicate that PMCA1 mRNA can be normalized to both 18S ribosomal RNA (18S rRNA) and human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) in MCF-7 cells. Real-time RT-PCR will be most useful in assessing PMCA1 mRNA expression in cases where only low amounts of RNA are available and/or when numerous samples must be assessed simultaneously. (C) 2001 Elsevier Science Inc. All rights reserved.
Resumo:
Sox18 encodes a member of the Sry-related high mobility group box (SOX) family of developmental transcription factors. Examination of Sox18 expression during embryogenesis has shown that Sox18 is expressed transiently in endothelial cells of developing blood vessels, and mutations in Sox18 have been found to underlie the mouse vascular and hair follicle mutant ragged. In this study we have examined the expression of Sox18 in angiogenesis during wound healing. Full-thickness skin wounds were created in mice, and subsequent expression of vascular endothelial growth factor (VEGF), the VEGF receptor Flk-1, alpha1 (iv) collagen (Col4a1), and Sox18 were studied using in situ hybridization. As has been previously reported, VEGF was expressed predominantly in the keratinocytes at the wound margins. Sox18 expression was found Rye days after wounding during capillary sprouting in granulation tissue and persisted through the proliferative phase of healing, but was not detected in fully epithelialized wounds 21 days after wounding. Sox18 mRNA expression was detected in capillaries within the granulation tissue and showed an identical pattern of distribution to Flk-1 and Col4a1 mRNA expression in endothelial cells. Immunostaining with a polyclonal anti-Sox18 antibody showed SOX18 protein localized in capillary endothelial cells within the granulation tissue. capillaries in the subcutaneous tissue of unwounded skin showed no Sox18 expression. Sox18 may therefore represent a transcription factor involved in the induction of angiogenesis during wound healing and tissue repair, but not in the maintenance of endothelial cells in undamaged tissue.
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In situ hybridization to mRNA in embryo sections or wholemount embryos is one of the most powerful analytical tools available to the molecular developmental biologist. For many workers, this procedure provides the first insights into the function of newly isolated genes, allowing the formulation of hypotheses and setting the course for further research. This paper presents a personal historical perspective of the development of in situ hybridization, looks at the theory and practice of the technique, summarizes the current state of the art, and speculates on possible directions for the future as a tool in functional genomics.
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The aim of this study was to further investigate the mechanism of suppression of natural killer (NK) cell cytotoxic activity In peripheral blood following strenuous exercise. Blood was collected for analysis of NK cell concentration, cytotoxic activity, CD2 surface expression and perforin gene expression from runners (RUN, n = 6) and resting controls (CONTROL, n = 4) pre-exercise, 0, 1.5, 5, and 24 h following a 60-min treadmill run at 80% of VO2 peak. Natural killer cytotoxic activity, measured using a whole blood chromium release assay, fluctuated minimally in the CONTROL group and increased by 63% and decreased by 43% 0 and 1.5 h post-exercise, respectively, in the RUN group (group x time, P < 0.001). Lytic index (cytotoxic activity per cell) did not change. Perforin mRNA, measured using quantitative real-time polymerase chain reaction (ORT-PCR) decreased from pre- to post-exercise and remained decreased through 24 h, The decrease from pre- to 0 In post-exercise was seen predominately in the RUN group and was inversely correlated r = - 0.95) to pre-exercise perform mRNA. The NK cell surface expression of CD2 (lymphocyte function-associated antigen-2) was determined using fluorescent antibodies and flow cytometry, There was no change in the proportion of NK cells expressing CD2 or CD2 density, We conclude that (1) numerical redistribution accounted for most of the change in NK cytotoxic activity following a strenuous run, (2) decrease in perforin gene expression during the run was inversely related to pre-exercise levels but did not parallel changes in cytotoxic activity, and (3) CD2 surface expression was not affected by exercise.
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Isolated systolic hypertension (ISH) occurs predominantly in the elderly, with a considerable morbidity and mortality. Its etiology is unknown but is likely to involve a significant genetic component. The aim of this study was to examine the angiotensinogen gene in ISH. The M235T and G(- 6)A polymorphisms were genotyped by polymerase chain reaction (PCR) in 86 ISH patients and 120 normotensive controls. Plasma angiotensinogen concentration was determined in 198 subjects by an indirect radioimmunoassay technique. Angiotensinogen mRNA concentration was determined by quantitative competitive reverse transcription (RT)-PCR in subcutaneous adipose tissue from a subset of these patients (n = 8) and controls (n = 6). Both the M235T (p = 0.0015) and G(- 6)A (p = 0.029) polymorphisms were associated with ISH. Plasma angiotensinogen concentration was higher in patients than controls (p < 0.0001), but was not associated with genotype. Angiotensinogen mRNA concentration in adipose tissue from ISH subjects was significantly lower than in adipose tissue from normotensive subjects (p = 0.033). The association of angiotensinogen gene variants with ISH and the elevation of plasma angiotensinogen concentration in these patients suggests a role of the angiotensinogen gene in this form of hypertension. Angiotensinogen gene expression may be altered in ISH, but this requires further examination.