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RNase Protection (rnase + protection)
Selected AbstractsReciprocal chemokine receptor and ligand expression in the human placenta: Implications for cytotrophoblast differentiationDEVELOPMENTAL DYNAMICS, Issue 4 2004Penelope M. Drake Abstract At the onset of pregnancy, the human placenta, which forms the interface between the embryo/fetus and the mother, must rapidly develop into a life-sustaining organ. The many unusual processes entailed in placental development include the poorly understood phenomenon of maternal tolerance of the hemiallogeneic cells of the conceptus, including, most remarkably, placental trophoblasts that invade the uterine wall. To investigate whether this fetal organ exerts control over the maternal immune system at the level of leukocyte trafficking, we examined placental expression of chemokines, well-known cytokine regulators of leukocyte movements. In situ hybridization revealed abundant expression of 13 chemokines in the stromal but not the trophoblast compartment of chorionic villi. Potential roles for these molecules include recruitment of the resident macrophage (Hofbauer cell) population to the villi. In parallel, cytotrophoblast production of a panel of nine chemokine receptors was assessed by using RNase protection assays. The numerous receptors detected suggested the novel possibility that the paracrine actions of chemokine ligands derived from either the villous stroma or the decidua could mediate general aspects of placental development, with specific contributions to cytotrophoblast differentiation along the pathway that leads to uterine invasion. Developmental Dynamics 229:877,885, 2004. © 2004 Wiley-Liss, Inc. [source] Differential effects of acute and chronic exercise on plasticity-related genes in the rat hippocampus revealed by microarrayEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2002Raffaella Molteni Abstract Studies were performed to determine the effects of acute and chronic voluntary periods of exercise on the expression of hippocampal genes. RNAs from rodents exposed to a running wheel for 3, 7 and 28 days were examined using a microarray with 1176 cDNAs expressed primarily in the brain. The expression of selected genes was quantified by Taqman RT-PCR or RNase protection assay. The largest up-regulation was observed in genes involved with synaptic trafficking (synapsin I, synaptotagmin and syntaxin); signal transduction pathways (Ca2+/calmodulin-dependent protein kinase II, CaM-KII; mitogen-activated/extracellular signal-regulated protein kinase, MAP-K/ERK I and II; protein kinase C, PKC-,) or transcription regulators (cyclic AMP response element binding protein, CREB). Genes associated with the glutamatergic system were up-regulated (N -methyl- d -aspartate receptor, NMDAR-2A and NMDAR-2B and excitatory amino acid carrier 1, EAAC1), while genes related to the gamma-aminobutyric acid (GABA) system were down-regulated (GABAA receptor, glutamate decarboxylase GAD65). Brain-derived neurotrophic factor (BDNF) was the only trophic factor whose gene was consistently up-regulated at all timepoints. These results, together with the fact that most of the genes up-regulated have a recognized interaction with BDNF, suggest a central role for BDNF on the effects of exercise on brain plasticity. The temporal profile of gene expression seems to delineate a mechanism by which specific molecular pathways are activated after exercise performance. For example, the CaM-K signal system seems to be active during acute and chronic periods of exercise, while the MAP-K/ERK system seems more important during long-term exercise. [source] Cyclin D1 gene contains a cryptic promoter that is functional in human cancer cellsGENES, CHROMOSOMES AND CANCER, Issue 3 2001Alessandro Terrinoni A novel cyclin D1 (CCND1),TROP2 fusion oncogene has been isolated from human cancer cells. Unexpectedly, the chimeric cDNA was found to express TROP2 in the absence of exogenous promoters. Mutagenesis of the TROP2 and CCND1 sequences and in vitro transcription/translation show that a cryptic promoter is present in the 3, coding region of CCND1. The CCND1 cryptic promoter is functional in luciferase assays, where it augments the basal expression levels by eightfold and efficiently cooperates with an SV-40 enhancer. The transcription start sites of the cryptic promoter map at bases 797 and 935 of CCND1, as determined by RNase protection assays. The cryptic promoter possesses canonical binding sites for ubiquitous transcription factors and W/S, X1, and CAAT/Y boxes that are characteristic of major histocompatibility complex class II gene promoters. Remarkably, the cryptic CCND1 promoter is active in human cancer cells and generates a truncated transcript that contains CCND1 instability sequences. Thus, this novel CCND1 transcription unit may play a role in the regulation of the expression of cyclin D1 and in tumor cell growth. © 2001 Wiley-Liss, Inc. [source] Downregulation of KiSS-1 expression is responsible for tumor invasion and worse prognosis in gastric carcinomaINTERNATIONAL JOURNAL OF CANCER, Issue 6 2004Dipok Kumar Dhar Abstract KiSS-1 is a promising candidate tumor-suppressor gene and may play a key role in the metastatic cascade. The expression profile and the role of KiSS-1 in cancer progression are largely unknown in most of the cancers, including gastric cancer. In this study, KiSS-1 expression was evaluated by RNase protection assay and localization was done by in situ hybridization in 40 gastric cancers and their adjacent normal gastric mucosa. For comparison with clinicopathologic characteristics and patient prognosis, all patients were divided into 2 groups having high and low KiSS-1 expression by using the median as the cutoff value of KiSS-1 expression as determined by the RNase protection assay. Gastric cancers with low KiSS-1 had frequent venous invasion, distant metastasis and tumor recurrence. Accordingly, patients with low KiSS-1 -expressing tumors had a significantly worse overall and disease-free survival. In multivariate analysis, KiSS-1 became the strongest independent prognostic factor among the conventional prognosticators for gastric cancer patients. Collectively, these findings suggest that KiSS-1 may play a crucial role in gastric cancer invasion and could be a useful target for therapeutic intervention. © 2004 Wiley-Liss, Inc. [source] Enhanced Chondrogenesis and Wnt Signaling in PTH-Treated Fractures,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 12 2007Sanjeev Kakar Abstract Studies have shown that systemic PTH treatment enhanced the rate of bone repair in rodent models. However, the mechanisms through which PTH affects bone repair have not been elucidated. In these studies we show that PTH primarily enhanced the earliest stages of endochondral bone repair by increasing chondrocyte recruitment and rate of differentiation. In coordination with these cellular events, we observed an increased level of canonical Wnt-signaling in PTH-treated bones at multiple time-points across the time-course of fracture repair, supporting the conclusion that PTH responses are at least in part mediated through Wnt signaling. Introduction: Since FDA approval of PTH [PTH(1,34); Forteo] as a treatment for osteoporosis, there has been interest in its use in other musculoskeletal conditions. Fracture repair is one area in which PTH may have a significant clinical impact. Multiple animal studies have shown that systemic PTH treatment of healing fractures increased both callus volume and return of mechanical competence in models of fracture healing. Whereas the potential for PTH has been established, the mechanism(s) by which PTH produces these effects remain elusive. Materials and Methods: Closed femoral fractures were generated in 8-wk-old male C57Bl/6 mice followed by daily systemic injections of either saline (control) or 30 ,g/kg PTH(1,34) for 14 days after fracture. Bones were harvested at days 2, 3, 5, 7, 10, 14, 21, and 28 after fracture and analyzed at the tissue level by radiography and histomorphometry and at the molecular and biochemical levels level by RNase protection assay (RPA), real-time PCR, and Western blot analysis. Results: Quantitative ,CT analysis showed that PTH treatment induced a larger callus cross-sectional area, length, and total volume compared with controls. Molecular analysis of the expression of extracellular matrix genes associated with chondrogenesis and osteogenesis showed that PTH treated fractures displayed a 3-fold greater increase in chondrogenesis relative to osteogenesis over the course of the repair process. In addition, chondrocyte hypertrophy occurred earlier in the PTH-treated callus tissues. Analysis of the expression of potential mediators of PTH actions showed that PTH treatment significantly induced the expression of Wnts 4, 5a, 5b, and 10b and increased levels of unphosphorylated, nuclear localized ,-catenin protein, a central feature of canonical Wnt signaling. Conclusions: These results showed that the PTH-mediated enhancement of fracture repair is primarily associated with an amplification of chondrocyte recruitment and maturation in the early fracture callus. Associated with these cellular effects, we observed an increase in canonical Wnt signaling supporting the conclusion that PTH effects on bone repair are mediated at least in part through the activation of Wnt-signaling pathways. [source] Relationship of in vivo and ex vivo levels of TH1 and TH2 cytokines with viremia in HAART patients with and without opportunistic infectionsJOURNAL OF MEDICAL VIROLOGY, Issue 4 2006Sardar Sindhu Abstract TH1/TH2 cytokines' imbalance is critical to HIV-1 progression and pathogenesis. Opportunistic infections-related cytokine perturbations in the setting of highly active antiretroviral therapy (HAART) are unclear. The objective of this cross-sectional study was to identify the relationship between TH1/TH2 cytokines and viremia in HAART patients with/without opportunistic infections. Sera from 17 HAART patients with and 43 without opportunistic infections, and 20 HIV-seronegative controls were used to measure the levels of IL-2, IFN-,, IL-4, and IL-10 proteins and mRNAs by ELISA and RNase protection assays, respectively. Ex vivo cytokine production by the CD4+/CD8+ T cells from four low and four high viremia patients randomly selected from non-opportunistic infection group was also evaluated. Serum IL-2 and IFN-, levels were lower (P,<,0.05) in patients than controls; this reduction was more pronounced for IFN-, in non-opportunistic infection patients. IL-4 and IL-10 were higher in patients than controls; this elevation was more remarkable in patients with opportunistic infections. Serum TH1/TH2 cytokine levels correlated with viremia. In vitro cytokine production assays showed that CD4+ T cells from low viremia patients mainly produced IL-2 and IFN-,, CD8+ T cells from high viremia patients produced IL-4, and both subsets comparably produced IL-10 in patients with similar viremia. Positive correlations between sera/supernatant proteins and cellular mRNAs were also found statistically significant (P,<,0.05). It was therefore concluded that in vivo TH1/TH2 cytokine levels in HAART patients and their ex vivo production by the CD4+/CD8+ T cells correlated with viremia and were also modulated by the presence of opportunistic infections in these patients. J. Med. Virol. 78:431,439, 2006. © 2006 Wiley-Liss, Inc. [source] Acute Alcohol Intoxication Increases REDD1 in Skeletal MuscleALCOHOLISM, Issue 5 2008Charles H. Lang Background:, The mechanism by which acute alcohol (EtOH) intoxication decreases basal muscle protein synthesis via inhibition of the Ser/Thr kinase mammalian target of rapamycin (mTOR) is poorly defined. In this regard, mTOR activity is impaired after over expression of the regulatory protein REDD1. Hence, the present study assessed the ability of REDD1 as a potential mediator of the EtOH-induced decrease in muscle protein synthesis. Methods:, The effect of acute EtOH intoxication on REDD1 mRNA and protein was determined in striated muscle of rats and mouse myocytes using an RNase protection assay and Western blotting, respectively. Other components of the mTOR signaling pathway were also assessed by immunoblotting. For comparison, REDD1 mRNA/protein was also determined in the muscle of rats chronically fed an alcohol-containing diet for 14 weeks. Results:, Intraperitoneal (IP) injection of EtOH increased gastrocnemius REDD1 mRNA in a dose- and time-dependent manner, and these changes were associated with reciprocal decreases in the phosphorylation of 4E-BP1, which is a surrogate marker for mTOR activity and protein synthesis. No change in REDD1 mRNA was detected in the slow-twitch soleus muscle or heart. Acute EtOH produced comparable increases in muscle REDD1 protein. The EtOH-induced increase in gastrocnemius REDD1 was independent of the route of EtOH administration (oral vs. IP), the nutritional state (fed vs. fasted), gender, and age of the rat. The nonmetabolizable alcohol tert -butanol increased REDD1 and the EtOH-induced increase in REDD1 was not prevented by pretreatment with the alcohol dehydrogenase inhibitor 4-methylpyrazole. In contrast, REDD1 mRNA and protein were not increased in the isolated hindlimb perfused with EtOH or in C2C12 myocytes incubated with EtOH, under conditions previously reported to decrease protein synthesis. Pretreatment with the glucocorticoid receptor antagonist RU486 failed to prevent the EtOH-induced increase in REDD1. Finally, the EtOH-induced increase in REDD1 was not associated with altered formation of the TSC1,TSC2 complex or the phosphorylation of TSC2 which is down stream in the REDD1 stress response pathway. In contradistinction to the changes observed with acute EtOH intoxication, REDD1 mRNA/protein was not changed in gastrocnemius from chronic alcohol-fed rats despite the reduction in 4E-BP1 phosphorylation. Conclusions:, These data indicate that in fast-twitch skeletal muscle (i) REDD1 mRNA/protein is increased in vivo by acute EtOH intoxication but not in response to chronic alcohol feeding, (ii) elevated REDD1 in response to acute EtOH appears due to the production of an unknown secondary mediator which is not corticosterone, and (iii) the EtOH-induced decrease in protein synthesis can be dissociated from a change in REDD1 suggesting that the induction of this protein is not responsible for the rapid decrease in protein synthesis after acute EtOH administration or for the development of alcoholic myopathy in rats fed an alcohol-containing diet. [source] Sex-dimorphic effects of progesterone and its reduced metabolites on gene expression of myelin proteins by rat Schwann cellsJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 2 2006Valerio Magnaghi Abstract Data obtained in our and other laboratories have indicated that progesterone (P) and its derivatives, dihydroprogesterone (DHP) and tetrahydroprogesterone (THP), stimulate the expression of two myelin proteins of the peripheral nervous system (PNS) [i.e., glycoprotein zero (P0) and peripheral myelin protein 22 (PMP22)]. We have now considered the effects of P and its derivatives on these and other myelin proteins [i.e., myelin-associated glycoprotein (MAG) and myelin and lymphocyte protein (MAL)] in sex-specific cultures of rat Schwann cells. Gene expression of myelin proteins was assessed by RNase protection assay. Treatment with P or DHP induced a stimulatory effect on P0 mRNA levels in male but not in female Schwann cells. In contrast, treatment with THP increased gene expression of P0 exclusively in female Schwann cells. A similar sex-difference was also evident for other myelin proteins. Indeed, PMP22 expression was stimulated by treatment with P in male cultures, whereas THP induced an increase of mRNA levels in female cultures. Moreover, MAG was stimulated by THP treatment in male cultures only, whereas MAL expression was unaffected by neuroactive steroid treatment in both male and female cultures. In conclusion, the present observations indicate that the effects of neuroactive steroids on myelin proteins are sexually dimorphic. This finding might represent an important background for sex-specific therapies of acquired and inherited peripheral neuropathies. [source] Thalidomide inhibits UVB-induced mouse keratinocyte apoptosis by both TNF-,-dependent and TNF-,-independent pathwaysPHOTODERMATOLOGY, PHOTOIMMUNOLOGY & PHOTOMEDICINE, Issue 6 2003Kurt Q. Lu Background: Thalidomide is an anti-inflammatory pharmacologic agent that has been utilized as a therapy for a number of dermatologic diseases. Its anti-inflammatory properties have been attributed to its ability to antagonize tumor necrosis factor-alfa (TNF-,) production by monocytes. However, its mechanism of action in the skin is not known. Purpose: To test our hypothesis that thalidomide may antagonize TNF-, production in the skin, we used a mouse model for acute ultraviolet-B (UVB) exposure, a known stimulus for inducing this cytokine. Results: A single bolus dose of thalidomide (either 100 or 400 mg/kg) given immediately before UVB exposure (40,120 mJ/cm2) inhibited, in a dose-dependent manner, sunburn cell formation (i.e. keratinocyte (KC) apoptosis as defined by histologic appearance and confirmed by terminal transferase mediated biotinylated dUTP nick end labelling staining) in mouse skin biopsy specimens. However, this agent did not affect the formation of cyclobutane pyrimidine dimers, a measure of UVB-induced DNA damage, which is an early event associated with apoptosis. RNase protection assays confirmed that high (400 mg/kg), but not low (100 mg/kg), doses of thalidomide inhibited the UVB-induced increase in steady-state TNF-, mRNA. Additionally, our in vitro data using neonatal mouse KCs showed that thalidomide prevented UVB-induced cell death (JAM assay). The antiapoptotic effects of thalidomide can be reversed by the addition of exogenous recombinant mouse TNF-, and hence reconstituting UVB-induced programmed cell death. The inhibition of sunburn cell formation by low-dose thalidomide in the absence of TNF-, inhibition suggests that other, unidentified mechanisms of apoptosis inhibition are active. Conclusions: These data suggest that the anti-inflammatory effects of thalidomide can affect UVB injury, and may, in part, explain its action in photosensitivity diseases such as cutaneous lupus erythematosus. [source] Brevican in the developing hippocampal fimbria: Differential expression in myelinating oligodendrocytes and adult astrocytes suggests a dual role for brevican in central nervous system fiber tract developmentTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 3 2001Tokiko Ogawa Abstract Brevican is one of the most abundant extracellular matrix proteoglycans in the mammalian brain. We have previously shown that brevican produced by gray matter astrocytes constitutes a major component of perineuronal extracellular matrix in the adult brain. In this paper, we investigate the expression of brevican in the postnatal hippocampal fimbria to explore the role of the proteoglycan in central nervous system fiber tract development. We demonstrate that brevican is expressed by both oligodendrocytes and white matter astrocytes in the fimbria, but the expression of brevican in these two glial cell types is differently regulated during development. At P14, brevican immunoreactivity was observed throughout the fimbria, with particularly strong immunoreactivity in the developing interfascicular glial rows. In situ hybridization showed that oligodendrocytes in the glial rows strongly express brevican during the second and third postnatal weeks. Expression in oligodendrocytes was then down-regulated after P21. In the adult fimbria, no brevican expression was observed in oligodendrocytes. The time window of brevican expression coincides with the phase in which immature oligodendrocytes actively extend membrane processes and enwrap axon fibers. In contrast, the expression in astrocytes started around P21 as oligodendrocytes began to down-regulate the expression. In the adult fimbria, brevican expression was restricted to astrocytes. In situ hybridization with isoform-specific probes and RNase protection assays showed that the authentic, secreted form of brevican, not the glycosylphosphatidylinositol-anchored variant, is the predominant species expressed in the developing fimbria. Our results suggest that brevican plays a dual role in developing and adult fiber tracts. J. Comp. Neurol. 432:285,295, 2001. © 2001 Wiley-Liss, Inc. [source] Modulation of body fluids and angiotensin II receptors in a rat model of intra-uterine growth restrictionTHE JOURNAL OF PHYSIOLOGY, Issue 3 2005Sophie Bédard We previously reported that sodium restriction during pregnancy reduces plasma volume expansion and promotes intra-uterine growth restriction (IUGR) in rats while it activates the renin,angiotensin,aldosterone system (RAAS). In the present study, we proceeded to determine whether expression of the two angiotensin II (ANGII) receptor subtypes (AT1 and AT2) change in relation to maternal water,electrolyte homeostasis and fetal growth. To this end, pregnant (gestation day 15) and non-pregnant Sprague-Dawley rats were randomly assigned to two groups fed either normal, or Na+ -restricted diets for 7 days. At the end of the treatment period, plasma aldosterone and renin activity as well as plasma and urine electrolytes were measured. Determinations for AT1 and AT2 mRNA and protein were made by RNase protection assay and photoaffinity labelling, respectively, using a number of tissues implicated in volume regulation and fetal growth. In non-pregnant rats, Na+ restriction decreases Na+ excretion without altering plasma volume, plasma Na+ concentration or the expression of AT1 and AT2 mRNA or protein in the tissues examined. In normally fed pregnant rats when compared to non-pregnant controls, AT1 mRNA increases in the hypothalamus as well as pituitary and declines in uterine arteries, while AT1 protein decreases in the kidney and AT2 mRNA declines in the adrenal cortex. In pregnant rats, Na+ restriction induces a decrease in plasma Na+, an increase in plasma urea, as well as a decline in renal urea and creatinine clearance rates. Protein levels for both AT1 and AT2 in the pituitary and AT2 mRNA in the adrenal cortex are lower in the Na+ -restricted pregnant group when compared to normally fed pregnant animals. Na+ restriction also induces a decrease in AT1 protein in the placenta. In conclusion, these results suggest that pregnancy may increase sensitivity to Na+ depletion by the tissue-specific modulation of ANGII receptors. Finally, these receptors may be implicated in the IUGR response to low Na+. [source] Induction of CCL13 expression in synovial fibroblasts highlights a significant role of oncostatin M in rheumatoid arthritisARTHRITIS & RHEUMATISM, Issue 7 2009Christoph Hintzen Objective To investigate the molecular mechanisms of CCL13/monocyte chemoattractant protein 4 (MCP-4) chemokine expression through proinflammatory cytokines in different primary human fibroblasts and the contribution of CCL13 to monocyte migration. Methods Using RNase protection assays and enzyme-linked immunosorbent assays, we quantified the expression of CCL13 compared with that of CCL2/MCP-1 in primary human fibroblasts. Boyden chamber assays were performed to determine the importance of CCL13 for migration of primary monocytes. Pharmacologic inhibitors as well as small interfering RNA knockdown approaches were used to investigate the signaling pathways regulating CCL13 expression. Results The interleukin-6 (IL-6),type cytokine oncostatin M (OSM) was a powerful inducer of CCL13 expression in primary synovial fibroblasts from patients with rheumatoid arthritis (RA) as well as those from healthy control subjects but not in other types of fibroblasts. Neither IL-6 nor tumor necrosis factor , could stimulate the expression of CCL13 in synovial fibroblasts; IL-1, was a very weak inducer. Synovial fibroblasts from patients with RA constitutively produced low amounts of CCL13, which was partially dependent on constitutive production of OSM. By investigating the underlying molecular mechanism, we identified STAT-5, ERK-1/2, and p38 as critical factors involved in OSM-dependent transcription and messenger RNA stabilization of CCL13. Conclusion In contrast to other prominent cytokines involved in the pathogenesis of RA, OSM can strongly up-regulate the expression of CCL13, a chemokine recently identified in the synovial fluid of patients with RA. Despite potent OSM-induced signal transduction in all types of fibroblasts analyzed, only synovial fibroblasts secreted CCL13, which might be indicative of tissue-specific imprinting of different fibroblasts during development. [source] Elevated Expression of Interleukins in Lung Adenocarcinomas Induced by N -Nitrosobis(2-hydroxypropyl)amine in RatsCANCER SCIENCE, Issue 10 2000Toshifumi Tsujiuchi The expression of interleukins (ILs) in lung adenocarcinomas induced by N -nitrosobis(2-hydroxypropyl)amine (BHP) in rats was investigated using a multiprobe RNase protection assay (RPA) followed by densitometric quantification. Male Wistar rats, 6 weeks old, were given 2000 ppm BHP in their drinking water for 12 weeks and maintained without further treatment until they were killed at week 25. Total RNAs were extracted from 14 individual adenocarcinomas and 2 specimens of normal lung tissue of untreated rats. In adenocarcinomas, elevated expression of IL-1, (6/14), IL-1, (14/14), IL-3 (7/14), IL-4 (11/14), IL-5 (9/14), IL-6 (11/14) and IL-10 (8/14) was observed, compared with normal lung tissues. In contrast, no expression of IL-2 was detected in any case. The results suggest that preferential expression of these ILs and their complex networks may contribute to the development and progression of lung adenocarcinomas induced by BHP in rats. [source] | |||||||||||||