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Ribonuclease Protection (ribonuclease + protection)
Selected AbstractsLead-induced alterations of apoptosis and neurotrophic factor mRNA in the developing rat cortex, hippocampus, and cerebellumJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 5 2007Shirley L. Chao Abstract Previous reports have recently shown the prototypic neurotoxicant, lead, to induce apoptosis in the brains of developing organisms. In the current study, timed-pregnant rats were exposed to lead acetate (0.2% in the drinking water) 24 h following birth at postnatal day 1 (PND 1). Dams and pups were continuously exposed to lead through the drinking water of the dam until PND 20. Postnatal exposure in the pups resulted in altered mRNA levels of the following apoptotic and neurotrophic factors: caspase 2 and 3, bax, bcl-x, brain-derived neurotrophic factor (BDNF). Ribonuclease protection assays were conducted to measure the factors simultaneously at the following postnatal time points: 9, 12, 15, 20, 25, days. Our results suggest a brain region- and time-specific response following lead acetate exposure. The region most vulnerable to alterations occurs in the hippocampus with alterations beginning at PND 12, in which caspase 3, bcl-x, BDNF increase with lead exposure. Significant treatment effects were not observed for both the cortex and cerebellum. © 2007 Wiley Periodicals, Inc. J Biochem Mol Toxicol 21:265,272, 2007; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20191 [source] Steroid hormone receptors and coregulators in endocrine-resistant and estrogen-independent breast cancer cellsINTERNATIONAL JOURNAL OF CANCER, Issue 4 2006Nanna Sarvilinna Abstract Resistance to hormonal therapy is often a problem in the treatment of breast cancer patients. It has been suggested that resistance could be explained by altered nuclear hormone receptor or coregulator levels or inappropriately increased agonist activity of selective estrogen receptor modulator (SERM). To test these hypotheses, we have established novel MCF-7 cell line-derived in vitro models of anti-estrogen- and progestin-resistant and estrogen-independent breast cancer by long-term culture in the presence of toremifene and medroxyprogesterone acetate (MPA) and in the absence of estradiol, respectively. Using cell growth and multiprobe ribonuclease protection assays, the expression of 5 nuclear hormone receptors and 9 coregulators as well as the alterations in the cell proliferation and target gene transcription in response to hormonal treatments were studied. Progesterone receptor (PR) expression was decreased and silencing mediator for retinoid acid and thyroid hormone receptors (SMRT) and amplified in breast cancer-1 (AIB1) expression increased in anti-estrogen-resistant cells. Estrogen caused PR and ER, upregulation in all cell lines, but we did not observe increased agonist activity of anti-estrogen measured by regulation of these estrogen target genes. Basal ER, levels and estrogenic growth response were decreased and p300/CBP-associated factor (pCAF) and AIB1 upregulated by estrogen in progestin-resistant cells, but coregulator levels were unchanged. Estrogen-independent cells were still estrogen-responsive and PR, nuclear receptor corepressor (N-CoR) and SMRT expression was increased whereas steroid receptor coactivator-1 (SRC-1a) and CBP-related protein p300 (p300) expression decreased. Their growth was inhibited by toremifene, but estradiol was able to abrogate this effect, which might have interesting clinical implications concerning the use of postmenopausal hormone replacement therapy. © 2005 Wiley-Liss, Inc. [source] Alcohol Exposure Alters the Expression Pattern of Neural Cell Adhesion Molecules During Brain DevelopmentJOURNAL OF NEUROCHEMISTRY, Issue 3 2000R. Miñana Abstract: Neural cell adhesion molecules (NCAMs) play critical roles during development of the nervous system. The aim of this study is to investigate the possible effect of ethanol exposure on the pattern of expression and sialylation of NCAM isoforms during postnatal rat brain development because alterations in NCAM content and distribution have been associated with defects in cell migration, synapse formation, and memory consolidation, and deficits in these processes have been observed after in utero alcohol exposure. The expression of NCAM isoforms in the developing cerebral cortex of pups from control and alcohol-fed mothers was assessed by western blotting, ribonuclease protection assay, and immunocytochemistry. The highly sialylated form of NCAM [polysialic acid (PSA)-NCAM] is mainly expressed during the neonatal period and then is down-regulated in parallel with the appearance of NCAM 180 and NCAM 140. Ethanol exposure increases PSA-NCAM levels during the neonatal period, delays the loss of PSA-NCAM, decreases the amount of NCAM 180 and NCAM 140 isoforms, and reduces sialyltransferase activity during postnatal brain development. Neuraminidase treatment of ethanol-exposed neonatal brains leads to more intense band degradation products, suggesting a higher content of NCAM polypeptides carrying PSA in these samples. However, NCAM mRNA levels are not changed by ethanol. Immunocytochemical analysis demonstrates that ethanol triggers an increase in PSA-NCAM immunolabeling in the cytoplasm of astroglial cells, accompanied by a decrease in immunogold particles over the plasma membrane. These findings indicate that ethanol exposure during brain development alters the pattern of NCAM expression and suggest that modification of NCAM could affect neuronal-glial interactions that might contribute to the brain defects observed after in utero alcohol exposure. [source] Inhibition of hepatitis B virus by lentiviral vector delivered antisense RNA and hammerhead ribozymesJOURNAL OF VIRAL HEPATITIS, Issue 4 2005K. L. Nash Summary., Chronic hepatitis B virus (HBV) infection is an important cause of cirrhosis and hepatocellular carcinoma. Current treatments are limited and may be ineffective. Nucleic acid-mediated targeting of viral mRNA is an attractive and specific approach for viral infection and lentiviral vectors provide a means to express antisense sequences or ribozymes stably in target cells permitting continuous production within that cell and its progeny. To demonstrate long-term gene expression by lentiviral vectors in hepatocytes and to introduce lentiviral vectors expressing anti-HBV genes to assess their effect against HBV, lentiviral vectors expressing a reporter gene were assessed for longevity of gene expression in hepatocytes in vitro. Hammerhead ribozymes and antisense sequences targeting the HBV encapsidation signal (,), X or surface antigen on mRNAs were cloned into lentiviral vectors and used to transduce HBV expressing hepatocytes where the effect on HBV mRNA level was assessed using ribonuclease protection. Gene expression in hepatocytes from integrated vectors continued for over 4 months without selection. Antisense RNA targeting HBs mRNA reduced this transcript, whilst antisense RNA to HBX mRNA was ineffective. Sense RNAs corresponding to , and HBX mRNA also reduced HBV mRNA levels. Ribozymes targeting HBs and HBX mRNA effectively reduced HBV mRNA levels compared with inactive constructs indicating their effect to be enzymatic rather than antisense. Lentiviral vectors can produce long-term gene expression in hepatocytes and thus permit prolonged expression of antiviral genes targeting the HBV encapsidation signal, surface and X mRNAs as treatments for chronic HBV infection. [source] Increased circulating and intrahepatic T-cell-specific chemokines in chronic hepatitis C: relationship with the type of virological response to peginterferon plus ribavirin combination therapyALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 5 2004A. Apolinario Summary Aims :,To determine the serum and intrahepatic levels of T-helper-1-associated chemokines in patients with chronic hepatitis C before, during and after peginterferon plus ribavirin combination therapy and to search for correlations with baseline characteristics of hepatitis C virus-related chronic liver disease and type of therapeutic response. Methods :,Serum chemokine levels were determined by enzyme-linked immunosorbent assays and intrahepatic chemokine messenger RNA and protein levels were tested by ribonuclease protection assay and immunohistochemistry. Results :,Serum and intrahepatic chemokine levels were elevated in all patients with chronic hepatitis C and showed a marked decrease in patients who obtained a virological response vs. non-responders. Increased serum interferon-,-inducible protein-10 levels at baseline in genotype 1-infected patients were significantly associated with greater degrees of intrahepatic inflammation and fibrosis (P = 0.0046 and P = 0.02, respectively) and with virological non-response (P = 0.01). In patients with genotype 1, basal serum interferon-,-inducible protein-10 levels greater than 299 pg/mL identified 80% of non-responders and lower than 299 pg/mL identified 63% of responders. Conclusions :,Circulating and intrahepatic T-helper-1-associated chemokines are abnormally elevated in patients with chronic hepatitis C. Increased baseline serum interferon-,-inducible protein-10 levels in genotype 1-infected patients are associated with virological non-response to peginterferon plus ribavirin combination therapy. [source] Oxygen control of ethylene biosynthesis during seed development in Arabidopsis thaliana (L.) HeynhPLANT CELL & ENVIRONMENT, Issue 6 2002K. M. Ramonell Abstract An unforeseen side-effect on plant growth in reduced oxygen is the loss of seed production at concentrations around 25% atmospheric (50 mmol mol,1 O2). In this study, the model plant Arabidopsis thaliana (L.) Heynh. cv. ,Columbia' was used to investigate the effect of low oxygen on ethylene biosynthesis during seed development. Plants were grown in a range of oxygen concentrations (210 [equal to ambient], 160, 100, 50 and 25 mmol mol,1) with 0·35 mmol mol,1 CO2 in N2. Ethylene in full-sized siliques was sampled using gas chromatography, and viable seed production was determined at maturity. Molecular analysis of ethylene biosynthesis was accomplished using cDNAs encoding 1-aminocyclopropane -1-carboxylic acid (ACC) synthase and ACC oxidase in ribonuclease protection assays and in situ hybridizations. No ethylene was detected in siliques from plants grown at 50 and 25 mmol mol,1 O2. At the same time, silique ACC oxidase mRNA increased three-fold comparing plants grown under the lowest oxygen with ambient controls, whereas ACC synthase mRNA was unaffected. As O2 decreased, tissue-specific patterning of ACC oxidase and ACC synthase gene expression shifted from the embryo to the silique wall. These data demonstrate how low O2 modulates the activity and expression of the ethylene biosynthetic pathway during seed development in Arabidopsis. [source] Detection of Coconut cadang-cadang viroid sequences in oil and coconut palm by ribonuclease protection assayANNALS OF APPLIED BIOLOGY, Issue 1 2009G. Vadamalai Abstract A ribonuclease protection assay (RPA) has been developed for detecting Coconut cadang-cadang viroid (CCCVd) sequences. An RNA probe complementary to full-length CCCVd246 was used, terminating at nucleotide 65 in the upper conserved region, and linked to a non-viroid 5, sequence, which acted as an internal control for ribonuclease activity. Extracts from CCCVd-infected coconut (Cocos nucifera) and African oil (Elaeis guineensis) palms protected three major fragments of approximately 250, 125 and 50 nt and a variable number of minor fragments. Extracts of healthy coconut palms, Potato spindle tuber viroid -infected tomato and transfer RNA did not protect the probe. The approximately 250 nt fragment is predicted to indicate the presence of monomers and dimers of circular CCCVd246, linear CCCVd246 with the same termini as the probe and point mutants of these forms. The origin of smaller protected fragments is discussed. RPA-detected CCCVd sequences in 13 of 18 oil palms surveyed in a commercial plantation in Malaysia. Signal intensity varied between the positive oil palms and was generally lower than in coconut palms infected with CCCVd. An infection phenotype was implied but not confirmed by the observation that in a group of 10 oil palms with orange leaf spotting, 9 contained CCCVd, whereas in a group of 8 palms without orange spotting, the viroid was detected in 4. Of four coconut palms in Sri Lanka shown by dot-blot assay to contain CCCVd-related RNA, one was shown by RPA to be positive for the CCCVd246 sequence. RPA is therefore a robust and sensitive test for CCCVd sequences, and our results show that sequences closely related to CCCVd246 are not confined to the Philippines. [source] Gene expression of cytokine receptors in HL60 cells exposed to a 50 Hz magnetic fieldBIOELECTROMAGNETICS, Issue 5 2002Jiliang Zhou Abstract The effects of a 50 Hz extremely low frequency (ELF) sinusoidal magnetic field (MF) on the expression of genes relating to cytokine receptors were studied in HL60 cells. Transcription levels of tumor necrosis factor receptor (TNFR) p55 and p75, interleukin-6 receptor-, (IL-6R,) and transforming growth factor-, receptor 1 (TGF,R1) were quantified in cells exposed to an intensity of 0.1 or 0.8 mT for periods ranging from 30 min to 72 h. Cells treated with 10 nM of phorbol 12-myristate 13-acetate (PMA) for 8 h served as a positive control. Gene expression values were assessed by the ribonuclease protection assay (RPA) and normalized to those of the noninducible gene GAPDH. The results showed that MF exposure at 0.1 and 0.8 mT for 72 h increased TNFR p75 and IL-6R, mRNA expression in HL60 cells. No significant change in gene expression levels of TNFR p55 and TGF,R1 was observed under any of the exposure conditions. In addition, we report here for the first time that IL-6R, mRNA expression can be suppressed by PMA in HL60 cells. Bioelectromagnetics 23:339,346, 2002. © 2002 Wiley-Liss, Inc. [source] 111In-labelled octreotide binding by the somatostatin receptor subtype 2 in neuroendocrine tumours,BRITISH JOURNAL OF SURGERY (NOW INCLUDES EUROPEAN JOURNAL OF SURGERY), Issue 5 2003S. H. Hashemi Background: The aim of this study was to investigate the importance of somatostatin receptor subtype 2 (SSTR2) expression for 111In-labelled diethylenetriamine-pentaacetic acid (DTPA)- D -Phe1 -octreotide binding and uptake of 111In in neuroendocrine tumours. Methods: 111In activity concentrations in surgical biopsies from neuroendocrine tumours (midgut carcinoid and medullary thyroid carcinoma), breast carcinoma and blood were determined 1,8 days after intravenous injection of 111In-labelled DTPA- D -Phe1 -octreotide (140,350 MBq). The ratio of 111In activity concentrations between tumour tissue and blood (T/B value) was calculated. The expression of SSTR2 messenger RNA (mRNA) in tumour biopsies was quantitated by ribonuclease protection assay and SSTR2 protein was localized by immunocytochemistry. Results: T/B values were highest for tumour biopsies from midgut carcinoids (mean 160 (range 4,1200); n = 65) followed by medullary thyroid carcinoma (mean 38 (range 2,350); n = 88) and breast carcinoma (mean 18 (range 4,41); n = 4). The expression of SSTR2 mRNA (relative to the NCI-H69 cell line) was highest in tumour biopsies from midgut carcinoids (mean 2·5 (range 0·83,6·0); n = 40) followed by medullary thyroid carcinoma (mean 1·3 (range 0·20,6·0); n = 7) and breast carcinoma (mean 0·66 (range 0·29,1·0); n = 9). In tumour biopsies SSTR2 protein was localized exclusively to tumour cells. Conclusion: Midgut carcinoid tumours showed a much higher level of SSTR2 expression than medullary thyroid carcinoma in accordance with superior tumour imaging by octreotide scintigraphy. The high SSTR2 mRNA values and T/B values observed in midgut carcinoid tumours were positively correlated. Copyright © 2003 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd. [source] | |||||||||||||