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Reverse Transcriptase Polymerase Chain Reaction (reverse + transcriptase_polymerase_chain_reaction)
Kinds of Reverse Transcriptase Polymerase Chain Reaction Selected AbstractsExpression of RNAs encoding for , and , integrin subunits in periodontitis and in cyclosporin A gingival overgrowthJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 11 2003A.-L. Bolcato-Bellemin Abstract Background: Variation of integrin expression in healthy and diseased gingiva revealed a potential biological role for these cell matrix receptors during gingival remodeling. Aim: Here we determined the level of RNA and tissue localization of different integrin subunits in periodontitis and cyclosporin A-induced gingival overgrowth. Methods: The level of expression was determined by Reverse Transcriptase Polymerase Chain Reaction in 12 periodontitis-affected patients, four patients exhibiting severe cyclosporin A-induced gingival overgrowth and seven healthy patients as controls. Results: The RNA encoding for ,1, ,2 and ,5 integrin subunits were reduced in periodontitis gingiva. The reduction observed was stronger in cyclosporin A-treated patients as compared to the healthy controls, while RNA encoding for ,1 subunit was increased. The RNA encoding for ,6 integrin was only reduced in cyclosporin A-treated gingiva. Immunohistochemistry showed that i) integrin ,2 expression is restricted to the gingival epithelium of cyclosporin A-treated patients, ii) the reduction of ,6 integrin expression in cyclosporin A-treated gingiva is due to loss of expression at focal contacts and iii) ,1 integrin is evenly distributed in the three populations with an intensity decrease in periodontitis and cyclosporin A-treated gingiva. Conclusion: Taken together these results showed a role for the integrin receptors in periodontal diseases and cyclosporin A-induced gingival overgrowth. Zusammenfassung Die Variation der Integrin Expression bei gesunder und erkrankter Gingiva zeigt eine potentielle biologische Rolle für diese Zellmatrixrezeptoren während der gingivalen Erneuerung. Wir bestimmten hier die Level von RNA und die Gewebelokalisation von unterschiedlichen Integrin Untereinheiten bei Parodontitis und Cyclosporin A induzierter gingivaler Wucherung. Die Level der Expression wurden mit der reversen Transscriptase Polymerase Kettenreaktion bei 12 Parodontitis-Patienten, 4 Patienten mit schwerer Cyclosporin A induzierter gingivaler Wucherung und sieben gesunden Kontrollpatienten bestimmt. Die kodierende RNA für ,1, ,2 und ,5 Integrin Untereinheiten waren in der Gingiva mit Parodontitis reduziert. Die beobachtete Reduktion war stärker bei den mit Cyclosporin A behandelten Patienten verglichen mit den gesunden Kontrollen, während kodierende RNA für ,1 Untereinheiten erhöht war. Die kodierende RNA für ,6 Integrin war nur bei der Cyclosporin A behandelten Gingiva reduziert. Die Immunhistochemie zeigte (i) die Integrin ,2 Expression ist auf das gingivale Epithel von Cyclosporin A behandelten Patienten beschränkt, (ii) die Reduktion von ,6 Integrin Expression bei Cyclosporin A behandelter Gingiva ist die Folge von fokalen Expressionverlusten und (iii) ,1 Integrin ist gleichmäßig verteilt in den drei Populationen mit einer Intensitätsabnahme bei Parodontitis und Cyclosporin A behandelter Gingiva. Zusammenfassend zeigen die Ergebnisse eine Rolle für die Integrin Rezeptoren bei den parodontalen Erkrankungen und Cyclosporin A induzierter gingivalen Wucherung. Résumé La variation d'expression des intégrines dans les tissus gingivaux sain et pathologique a démontré le rôle biologique potentiel de ces récepteurs de la matrice extracellulaire au cours du remodelage tissulaire gingival. La quantité d'ARN et la localisation tissulaire de certaines sous-unités d'intégrines dans la parodontite et l'hyperplasie gingivale induite par la ciclosporine A ont été déterminées. Le niveau d'expression a étéévalué par transcription inverse des ARN et réaction de polymérisation en chaine chez douze patients atteints de parodontite, quatre patients présentant une hyperplasie gingivale sévère induite par la ciclosporine A et sept patients sains ayant servi de témoins. L'expression des ARN codant pour les sous-unités ,1, ,2 et ,5était diminuée dans le tissu gingival atteint de parodontite. La diminution observée était plus importante chez les patients traités par la ciclosporine A, comparée aux témoins sains alors que l'expression de l'ARN codant pour la sous-unité,1était augmentée. L'expression de l'ARN codant pour la sous-unité,6était diminuée uniquement dans le tissu gingival traité par la ciclosporine A. L'immohistochimie a montré que (1) l'expression de la sous-unité,2 est limitée à l'épithélium gingival des patients traités par la ciclosporine A, (2) la diminution de l'expression de la sous-unité,6 dans le tissu gingival traité par la ciclosporine A est due à une perte des contacts focaux et (3) la sous-unité,1 est répartie de manière uniforme dans les trois groupes avec une diminution de l'intensité dans les cas de parodontite et d'hyperplasie gingivale induite par la ciclosporine A. Ces résultats montrent un rôle des récepteurs de type intégrine dans la pathologie parodontale et l'hyperplasie gingivale induite par la ciclosporine A. [source] Evaluation of a p30 Gene-Based Real-Time Reverse Transcriptase Polymerase Chain Reaction Assay for Detection of Feline CalicivirusesJOURNAL OF VETERINARY INTERNAL MEDICINE, Issue 1 2004Brian A. Scansen This report describes a feline calicivirus (FCV) p30 gene-based real-time SYBR Green I reverse transcriptase polymerase chain reaction (RT-PCR) assay that is capable of detecting low virus concentrations and a broad range of FCV isolates. The assay consisted of a 1-step RT-PCR reaction with primers delineating a 126-base-pair (bp) region of the FCV p30 gene. Sensitivity of the RT-PCR assay was determined to be equivalent to a FCV titer of 1.2 × 101 to 1.2 × 102 TCID50/mL. The assay was linear over a wide range of template concentrations and had a reaction efficiency of 95%. Specific FCV amplification products were detected from 51 wild-type FCV isolates, whereas specific products were not detected from a canine calicivirus, a rabbit calicivirus, and a bovine calicivirus. The primers used in this study amplified a large number of North American FCV isolates and further confirm the diagnostic utility of p30 gene-based real-time RT-PCR for detection of FCV. [source] Cocirculation of and coinfections with hepatitis A virus subgenotypes IIIA and IB in patients from Pune, western IndiaHEPATOLOGY RESEARCH, Issue 2 2007Shobha Chitambar Aim:, During the 1990s, a changing pattern of epidemiology of hepatitis A was reported in different populations of India. The present study was undertaken to investigate the molecular epidemiology of hepatitis A virus (HAV) strains over a period of 10 years. Methods:, Stool/serum samples were collected from hepatitis A patients clinically presenting acute viral hepatitis and hepatic encephalopathy. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to detect HAV-RNA. HAV genomes were examined by sequencing PCR products of VP1/2A junction (168 bp) and RNA polymerase (116 bp) regions. Results:, Subgenotype IIIA and IB were detected in 74.2% and 9.7% of specimens, respectively, while 16.1% of patients had mixed infections. Sewage samples also showed presence of both IIIA (9/10) and IB (1/10) subgenotypes. RNA polymerase region showed two clusters constituting 51.6% and 19.4% strains closer to Nor21 and HM175 strains, respectively, in clinical specimens. Three isolates appeared as discordant subgenotypes in VP1/2A and RNA polymerase regions. Conclusion:, The data revealed cocirculation of and coinfection with subgenotypes IIIA and IB, with predominance of IIIA and genetic heterogeneity of HAV strains in western India. [source] 99mTc-MIBI imaging for prediction of therapeutic effects of second-generation MDR1 inhibitors in malignant brain tumorsINTERNATIONAL JOURNAL OF CANCER, Issue 12 2007Toshio Sasajima Abstract The aim of this study was to explore whether 99mTc-methoxyisobutylisonitrile (99mTc-MIBI) is suitable to elucidate multidrug resistance and prediction of potentiation of antitumor agents by second-generation MDR1 inhibitors (PSC833, MS-209) in malignant brain tumors in rat. Malignant tumor cells (RG2 and C6 gliomas, Walker 256 carcinoma) were incubated with low dose vincristine (VCR) to induce multidrug resistance. MTT assay demonstrated a significant increase of surviving fractions in VCR-resistant sublines compared to those of drug-naive cells. Reverse transcriptase polymerase chain reaction revealed higher expression of MDR1 mRNA in VCR-resistant cells than drug-naive cells in each line. Volume distribution (Vd) of 99mTc-MIBI was negatively correlated with MDR1 mRNA expression among drug-naive and VCR-resistant cells. MDR1 inhibitors decreased surviving fractions and increased Vd of 99mTc-MIBI significantly in VCR-resistant sublines, whereas MDR1 mRNA expression was unchanged. These findings indicate that 99mTc-MIBI efflux was functionally suppressed by MDR1 inhibitors. Autoradiographic images of 99mTc-MIBI revealed higher uptake in drug-naive cells at basal ganglia compared with VCR-resistant cells at the opposite basal ganglia of rats. Oral administration of the second-generation MDR1 inhibitors significantly increased 99mTc-MIBI accumulation of both tumors. Therapeutic effects of VCR with or without the MDR1 inhibitors were also evaluated autoradiographically using 14C-methyl- L -methionine (14C-Met) and MIB-5 index. 14C-Met uptake and MIB-5 index of both tumors treated with VCR following the MDR1 inhibitor treatment significantly decreased compared with tumors treated with VCR alone. Analysis of 99mTc-MIBI accumulation is considered informative for detecting MDR1-mediated drug resistance and for monitoring the therapeutic effects of MDR1 inhibitors in malignant brain tumors. © 2007 Wiley-Liss, Inc. [source] Arabidopsis thaliana acyl-CoA-binding protein ACBP2 interacts with heavy-metal-binding farnesylated protein AtFP6NEW PHYTOLOGIST, Issue 1 2009Wei Gao Summary ,,Arabidopsis thaliana acyl-CoA-binding protein 2 (ACBP2) was observed to interact with farnesylated protein 6 (AtFP6), which has a metal-binding motif (M/LXCXXC). Their interaction and expression in response to heavy metals were investigated. ,,Yeast two-hybrid analysis and in vitro assays showed that an ACBP2 derivative lacking ankyrin repeats did not interact with AtFP6, indicating that the ankyrin repeats mediate protein,protein interaction. Autofluorescence-tagged ACBP2 and AtFP6 transiently co-expressed in tobacco (Nicotiana tabacum) were both targeted to the plasma membrane. ,,Reverse transcriptase polymerase chain reaction and northern blot analyses revealed that AtFP6 mRNA was induced by cadmium (Cd(II)) in A. thaliana roots. Assays using metal-chelate affinity chromatography demonstrated that in vitro translated ACBP2 and AtFP6 bound lead (Pb(II)), Cd(II) and copper (Cu(II)). Consistently, assays using fluorescence analysis confirmed that (His)6 -AtFP6 bound Pb(II), like (His)6 -ACBP2. ,,Arabidopsis thaliana plants overexpressing ACBP2 or AtFP6 were more tolerant to Cd(II) than wild-type plants. Plasma membrane-localized ACBP2 and AtFP6 probably mediate Pb(II), Cd(II) and Cu(II) transport in A. thaliana roots. Also, (His)6 -ACBP2 binds [14C]linoleoyl-CoA and [14C]linolenoyl-CoA, the precursors for phospholipid repair following lipid peroxidation under heavy metal stress at the plasma membrane. ACBP2 -overexpressing plants were more tolerant to hydrogen peroxide than wild-type plants, further supporting a role for ACBP2 in post-stress membrane repair. [source] The disturbance of small RNA pathways enhanced abscisic acid response and multiple stress responses in ArabidopsisPLANT CELL & ENVIRONMENT, Issue 4 2008JIAN-FENG ZHANG ABSTRACT The phytohormone abscisic acid (ABA) regulates plant growth and development as well as stress tolerance. To gain more insights into ABA signalling, a population of chemical-inducible activation-tagged Arabidopsis mutants was screened on the basis of the ABA effect on the inhibition of seed germination. Two novel ABA supersensitive mutants ABA supersensitive during germination1 (absg1) and absg2 were characterized as alleles of Dicer-like1 (DCL1) and HEN1, respectively, as microRNA biogenesis genes, and accordingly, these two mutants were renamed dcl1-11 and hen1-16. The dcl1-11 mutant was an ABA hypersensitive mutant for seed germination and root growth. Reverse transcriptase polymerase chain reaction assays revealed that the expression of ABA- and stress-responsive genes was increased in dcl1-11, as compared with the wild type (WT). Furthermore, the germination assay showed that dcl1-11 was also more sensitive to salt and osmotic stress. The hen1-16 mutant also showed supersensitive to ABA during seed germination. Further analysis showed that, among the microRNA biogenesis genes, all the other mutants were not only enhanced in sensitivity to ABA, salt and osmotic stress, but also enhanced the expression of ABA-responsive genes. In addition to the mutants in the microRNA biogenesis, the interruption of the production of crucial components of other small RNA pathways such as dcl2, dcl3 and dcl4 also caused ABA supersensitive during germination. [source] De novo STXBP1 mutations in mental retardation and nonsyndromic epilepsy,ANNALS OF NEUROLOGY, Issue 6 2009Fadi F. Hamdan PhD We sequenced genes coding for components of the SNARE complex (STX1A, VAMP2, SNAP25) and their regulatory proteins (STXBP1/Munc18-1, SYT1), which are essential for neurotransmission, in 95 patients with idiopathic mental retardation. We identified de novo mutations in STXBP1 (nonsense, p.R388X; splicing, c.169+1G>A) in two patients with severe mental retardation and nonsyndromic epilepsy. Reverse transcriptase polymerase chain reaction and sequencing showed that the splicing mutation creates a stop codon downstream of exon-3. No de novo or deleterious mutations in STXBP1 were found in 190 control subjects, or in 142 autistic patients. These results suggest that STXBP1 disruption is associated with autosomal dominant mental retardation and nonsyndromic epilepsy. Ann Neurol 2009;65:748,753 [source] Small interfering RNA-mediated down-regulation of SPAG9 inhibits cervical tumor growthCANCER, Issue 24 2009Manoj Garg PhD Abstract BACKGROUND: The expression of the SPAG9 is associated with various human malignancies. Earlier work revealed a significant association of SPAG9 expression with the early spread of cervical cancer, making it an attractive therapeutic target. Here, the authors investigated the role of SPAG9 in carcinogenesis of squamous cell carcinoma (SCC) of the cervix. Furthermore, they sought to determine whether ablation of SPAG9 expression reduces the tumor growth of cervical SCC in vivo. METHODS: A plasmid-based small interfering RNA approach was used to specifically knock down the expression of SPAG9 in SiHa cells derived from SCC of the cervix in vitro and in vivo. Reverse transcriptase polymerase chain reaction, immunofluorescence staining, flow cytometry, cellular growth, colony formation, migration, invasion, and wound healing assays were studied to characterize SPAG9 in vitro. Furthermore, a cervical cancer xenograft model in nude mice was established to investigate whether knockdown of SPAG9 reduces the tumor growth of cervical SCC in vivo. RESULTS: The results demonstrated that silencing the SPAG9 by small interfering RNA resulted in inhibition of cell growth, colony formation, migration, and invasion. The authors showed for the first time that the knockdown of SPAG9 expression by small interfering RNA significantly suppressed the tumor growth of cervical SCC in vivo. CONCLUSIONS: These results suggest that SPAG9 expression may play a pivotal role in tumor growth and could contribute to the early spread of cervical cancer. Small interfering RNA-mediated down-regulation of SPAG9 represents a promising therapeutic approach for the treatment of cervical cancer. Cancer 2009. © 2009 American Cancer Society. [source] The effect of deproteinized bovine bone on osteoblast growth factors and proinflammatory cytokine productionCLINICAL ORAL IMPLANTS RESEARCH, Issue 6 2010Paolo Amerio Abstract Objective: To test the ability of Bio-Oss® in inducing growth factors and proinflammatory cytokines that may have a role in inflammation after grafting, bone resorption, remodeling and in the homeostasis of osteoblasts. Material and methods: Normal human osteoblasts were seeded in Petri dishes containing granules of Bio-Oss®, cells were harvested after confluency and RNA was extracted. Reverse transcriptase polymerase chain reaction was performed using specific primers for osteonectin, bone sialoprotein (BSP), bone morphogenetic protein (BMP)-2 and BMP-7, platelet-derived growth factor (PDGF), interleukin 6 (IL-6), tumor necrosis factor , (TNF-,) and integrin ,1. Glycerol-3-phosphate dehydrogenase was used as the housekeeping gene and normal human osteoblasts grown on Petri dishes without Bio-Oss® granules were used as negative controls. Results: Osteoblast grown on Bio-Oss® showed a normal RNA expression of osteonectin, integrin ,1 and PDGF. However, compared with control osteoblasts it showed a reduced expression of BSP, BMP-2 and BMP-7, IL-6 and TNF-,. Conclusions: Our findings further support the evidence that Bio-Oss® is an excellent biomaterial that does not enhance the production of proinflammatory cytokines. To cite this article: Amerio P, Vianale G, Reale M, Muraro R, Tulli A, Piattelli A. The effect of deproteinized bovine bone on osteoblast growth factors and proinflammatory cytokine production. Clin. Oral Impl. Res. 21, 2010; 650,655. doi: 10.1111/j.1600-0501.2009.01881.x [source] Screen for genes differentially expressed during regeneration of the zebrafish caudal finDEVELOPMENTAL DYNAMICS, Issue 3 2004Bhaja K. Padhi Abstract The zebrafish caudal fin constitutes an important model for studying the molecular basis of tissue regeneration. The cascade of genes induced after amputation or injury, leading to restoration of the lost fin structures, include those responsible for wound healing, blastema formation, tissue outgrowth, and patterning. We carried out a systematic study to identify genes that are up-regulated during "initiation" (1 day) and "outgrowth and differentiation" (4 days) of fin regeneration by using two complementary methods, suppression subtraction hybridization (SSH) and differential display reverse transcriptase polymerase chain reaction (DDRT-PCR). We obtained 298 distinct genes/sequences from SSH libraries and 24 distinct genes/sequences by DDRT-PCR. We determined the expression of 54 of these genes using in situ hybridization. In parallel, gene expression analyses were done in zebrafish embryos and early larvae. The information gathered from the present study provides resources for further investigations into the molecular mechanisms of fin development and regeneration. Developmental Dynamics 231:527,541, 2004. © 2004 Wiley-Liss, Inc. [source] Neonatal estrogen exposure inhibits steroidogenesis in the developing rat ovaryDEVELOPMENTAL DYNAMICS, Issue 4 2001Yayoi Ikeda Abstract Treatment of newborn female rats with estrogens significantly inhibits the growth and differentiation of the ovary. To understand the molecular mechanism of estrogen action in the induction of abnormal ovary, we examined the expression profiles of steroidogenic factor 1 (SF-1) and several of its target genes in the developing ovaries after neonatal exposure to synthetic estrogen, estradiol benzoate (EB) by using reverse transcriptase polymerase chain reaction, in situ hybridization, and immunohistochemistry. Morphologic examination indicated inhibitory effects of estrogen on the stratification of follicles and development of theca and interstitial gland during postnatal ovarian differentiation. The expression of the steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage cytochrome P450 (P450SCC), which are both essential for steroid biosynthesis, markedly decreased in theca and interstitial cells throughout the postnatal development of the EB-treated ovary. However, expression of the transcriptional activator of the two genes, SF-1 was unaffected in theca and interstitial cells, although the number of these cells was lower in the EB-treated ovary than in the control ovary. The expression of the estrogen mediator, estrogen receptor-, (ER-,), diminished specifically in theca cells at P6 and recovered by P14 in the EB-treated ovary. These results indicate that the effect of estrogens is mediated by means of ER-, resulting in the down-regulation of StAR and P450SCC genes during early postnatal development of the ovary. These results suggest that the abnormal ovarian development by neonatal estrogen treatment is closely correlated with the reduced steroidogenic activity, and the data obtained by using this animal model may account in part the mechanism for aberrant development and function of the ovary in prenatally estrogen-exposed humans. © 2001 Wiley-Liss, Inc. [source] An evaluation of the etiology of reduced CYP1A1 messenger RNA expression in the Atlantic tomcod from the Hudson River, New York, USA, using reverse transcriptase polymerase chain reaction analysisENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 5 2001Nirmal K. Roy Abstract Adult Atlantic tomcod, Microgadus tomcod, from the Hudson River, New York State, USA, exhibit reduced inducibility of hepatic cytochrome P4501A1 (CYP1A1) mRNA compared with adult tomcod from the cleaner Miramichi River, New Brunswick, Canada, when treated with coplanar polychlorinated biphenyl (PCB) congeners or 2,3,7,8-tetrachlorodibenzo- p -dioxin. In contrast, little difference in CYP1A1 inducibility is observed between tomcod from these two rivers when treated with polycyclic aromatic hydrocarbons (PAHs). We sought to determine if impaired hepatic CYP1A1 inducibility in Hudson River tomcod results from a multigenerational, genetic adaptation or a single generational, physiological acclimation. Embryos and larvae from controlled experimental crosses of Hudson River and Miramichi River parents were exposed for 24 h to water-borne PCB congener 77 (10 ppm), benzo[a]pyrene (BaP; 10 ppm), or dimethysulfoxide, and CYP1A1 expression was assessed in individual larva using competitive reverse transcriptase polymerase chain reaction (RT-PCR) analysis. The CYP1A1 mRNA was significantly induced in larvae from both populations by BaP (47- and 52-fold) and PCB 77 (9- and 22-fold), although levels of expression were higher in offspring of Miramichi matings. Most important, CYP1A1 mRNA was significantly induced by PCB 77 in larvae from Hudson River parents. Concentrations of dioxin, furan, and PCB congeners were measured in livers and eggs of female tomcod from these two locales to quantify the extent of maternal transfer of contaminants. For both rivers, wet-weight contaminant concentrations were significantly higher (4,7 times) in livers than in eggs of the same females, suggesting that a threshold level of contaminants may have to be reached before CYP1A1 transcription is impaired. We conclude that reduced inducibility of hepatic CYP1A1 mRNA in adult tomcod from the Hudson River is most consistent with single-generational acclimation. [source] Expression of ephrin-A2 in the superior colliculus and EphA5 in the retina following optic nerve section in adult ratEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2001J. Rodger Abstract The vertebrate retina projects topographically to visual brain centres. In the developing visual system, gradients of ephrins and Eph receptors play a role in defining topography. At maturity, ephrins but not Ephs are downregulated. Here we show that optic nerve section in adult rat differentially regulates the expression of ephrin-A2 in the superior colliculus (SC) and of EphA5 in the retina. Expression was quantified immunohistochemically; ephrin-A2 levels were also estimated by semiquantitative reverse transcriptase polymerase chain reaction. In the normal SC, ephrin-A2 was expressed at low levels. At 1 month, levels of protein and of mRNA were upregulated across the contralateral SC giving rise to an increasing rostro-caudal gradient. At 6 months, levels had fallen but a gradient remained. In the retina of normal animals, EphA5 was expressed as an increasing naso-temporal gradient. By 1 month, expression was decreased in far temporal retina, resulting in a uniform expression across the naso-temporal axis. We suggest that denervation-induced plastic changes within the SC modify expression of these molecules. [source] Keratinocyte growth factor and scatter factor expression by regionally defined oral fibroblastsEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 1 2003Scott Thomas William McKeown Keratinocyte growth factor (KGF) and hepatocyte growth factor/scatter factor (SF) are two signalling molecules thought to play important roles in regulating epithelial,mesenchymal interactions. Expression of both factors by fibroblasts in subepithelial connective tissue may play a role in maintaining epithelial integrity in health and in the apical migration of junctional epithelium in periodontitis. The aims of this study were (a) to compare expression levels of KGF and SF by periodontal ligament (PDL) and gingival fibroblasts; and (ii) to determine the effects of interleukin (IL)-1,, transforming growth factor (TGF)-,1, platelet-derived growth factor (PDGF)-BB and epidermal growth factor (EGF) on KGF/SF expression by these cell populations. Three paired PDL and gingival fibroblast strains were developed. The KGF and SF protein levels were analysed by enzyme-linked immunosorbent assay. Relative levels of KGF and SF mRNA in cytokine-treated cultures were determined using semiquantitative reverse transcriptase polymerase chain reaction. No differences in the levels of KGF and SF produced by PDL and gingival (SOG) populations were found. In both cell types IL-1, stimulated KGF and SF expression, while TGF-,1 significantly inhibited expression at both the mRNA and protein levels. Epidermal growth factor and PDGF-BB induced differing effects on expression, stimulating SF protein production but inhibiting KGF output in both fibroblast populations. Differences in response to EGF and PDGF were also seen between paired PDL and gingival fibroblasts. [source] Localization of RNAs in oocytes of Eleutherodactylus coqui, a direct developing frog, differs from Xenopus laevisEVOLUTION AND DEVELOPMENT, Issue 6 2003Yvonne M. Beckham SummaryEleutherodactylus coqui develops directly on land to a frog. The large 3.5-mm oocyte of E. coqui has enough yolk to allow development without a feeding tadpole. In the smaller Xenopus laevis oocyte, 1.3 mm in diameter, mRNAs involved in germ layer formation, such as VegT and Vg1, are localized to the vegetal cortex of the oocyte. We hypothesized that an animal shift has occurred in the localization of the E. coqui Orthologs of VegT and Vg1 due to the large egg size. Through a combination of degenerate reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), we cloned 1634 bp of EcVegT and 1377 bp of EcVg1. Northern blot analysis shows that the lengths of these transcripts are 2.5 kb and 1.3 kb, respectively. This result suggests that we have obtained the complete Vg1 transcript, although this transcript has an extremely short 3, untranslated region compared with X. laevis, 256 bp and 1268 bp, respectively. Zygotic expression of EcVegT closely resembles that of VegT, supporting their orthology. Radioactive RT-PCR and in situ hybridization demonstrated the presence of EcVegT and EcVg1 predominantly near the animal pole of the oocyte. RT-PCR showed that the animal blastomeres, formed from the first horizontal cleavage, inherit half of the EcVegT and EcVg1 transcripts, although they contain only about 1% of the embryo volume. Our results indicate major differences between the molecular organization of the eggs of X. laevis and E. coqui. [source] Early evolution of a homeobox gene: the parahox gene Gsx in the Cnidaria and the BilateriaEVOLUTION AND DEVELOPMENT, Issue 4 2003John R. Finnerty Summary Homeobox transcription factors are commonly involved in developmental regulation in diverse eukaryotes, including plants, animals, and fungi. The origin of novel homeobox genes is thought to have contributed to many evolutionary innovations in animals. We perform a molecular phylogenetic analysis of cnox2, the best studied homeobox gene from the phylum Cnidaria, a very ancient lineage of animals. Among three competing hypotheses, our analysis decisively favors the hypothesis that cnox2 is orthologous to the gsx gene of Bilateria, thereby establishing the existence of this specific homeobox gene in the eumetazoan stem lineage, some 650,900 million years ago. We assayed the expression of gsx in the planula larva and polyp of the sea anemone Nematostella vectensis using in situ hybridization and reverse transcriptase polymerase chain reaction. The gsx ortholog of Nematostella, known as anthox2, is expressed at high levels in the posterior planula and the corresponding "head" region of the polyp. It cannot be detected in the anterior planula or the corresponding "foot" region of the polyp. We have attempted to reconstruct the evolution of gsx spatiotemporal expression in cnidarians and bilaterians using a phylogenetic framework. Because of the surprisingly high degree of variability in gsx expression within the Cnidaria, it is currently not possible to infer unambiguously the ancestral cnidarian condition or the ancestral eumetazoan condition for gsx expression. [source] Cell density- and ComE-dependent expression of a group of mutacin and mutacin-like genes in Streptococcus mutansFEMS MICROBIOLOGY LETTERS, Issue 1 2006Jens Kreth Abstract Streptococcus mutans is a major cariogenic inhabitant of the high cell density oral biofilm (dental plaque). In previous studies, we showed that production of one of its virulence factors, the bacteriocin mutacin IV, was regulated by high cell density as well as the competence regulatory system ComED. In this study, we utilized luciferase fusions and real-time reverse transcriptase polymerase chain reaction (RT-PCR), to demonstrate that high cell density and ComED also regulate an uncharacterized group of mutacin and mutacin-like genes. Under high cell density or in the presence of externally added competence-stimulating peptide (CSP), gene expression increased 10- to 30-fold. Interestingly, high cell density was able to bypass the requirement for CSP addition. However, both cell density and CSP-dependent gene expression had a strict requirement for the ComE response regulator. [source] Strain- and region-specific gene expression profiles in mouse brain in response to chronic nicotine treatmentGENES, BRAIN AND BEHAVIOR, Issue 1 2008J. Wang A pathway-focused complementary DNA microarray and gene ontology analysis were used to investigate gene expression profiles in the amygdala, hippocampus, nucleus accumbens, prefrontal cortex (PFC) and ventral tegmental area of C3H/HeJ and C57BL/6J mice receiving nicotine in drinking water (100 ,g/ml in 2% saccharin for 2 weeks). A balanced experimental design and rigorous statistical analysis have led to the identification of 3.5,22.1% and 4.1,14.3% of the 638 sequence-verified genes as significantly modulated in the aforementioned brain regions of the C3H/HeJ and C57BL/6J strains, respectively. Comparisons of differential expression among brain tissues showed that only a small number of genes were altered in multiple brain regions, suggesting presence of a brain region-specific transcriptional response to nicotine. Subsequent principal component analysis and Expression Analysis Systematic Explorer analysis showed significant enrichment of biological processes both in C3H/HeJ and C57BL/6J mice, i.e. cell cycle/proliferation, organogenesis and transmission of nerve impulse. Finally, we verified the observed changes in expression using real-time reverse transcriptase polymerase chain reaction for six representative genes in the PFC region, providing an independent replication of our microarray results. Together, this report represents the first comprehensive gene expression profiling investigation of the changes caused by nicotine in brain tissues of the two mouse strains known to exhibit differential behavioral and physiological responses to nicotine. [source] Transgenic mice expressing tamoxifen-inducible Cre for somatic gene modification in renal epithelial cellsGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 5 2006Irma S. Lantinga-van Leeuwen Abstract Gene inactivation often leads to an embryonic-lethal phenotype. In focal diseases like renal cell carcinomas and polycystic kidney disease, somatic gene inactivation in subsets of cells is likely to occur at later stages. We generated a transgenic mouse line with an inducible form of Cre recombinase for conditional gene modifications in kidney epithelial cells. To this end a 1.4-kb promoter fragment of the kidney-specific cadherin gene (KspCad) was cloned upstream of a tamoxifen-inducible Cre recombinase (CreERT2) encoding sequence. Expression and activity of Cre was evaluated using reverse transcriptase polymerase chain reaction (RT-PCR) analysis and by crossbreeding to Z/EG reporter mice. One KspCad-CreERT2 line showed kidney-specific Cre expression and mediated recombination upon tamoxifen treatment in Z/EG reporter mice. No reporter gene expression was detected in untreated animals or in extrarenal tissues upon treatment. Within the kidneys, enhanced green fluorescent protein (EGFP) fluorescence was observed in epithelial cells in several nephronic segments. In addition, the system successfully recombined a floxed Pkd1 gene. genesis 44:225,232, 2006. © 2006 Wiley-Liss, Inc. [source] Helicobacter pylori Infection in the Cat: Evaluation of Gastric Colonization, Inflammation and FunctionHELICOBACTER, Issue 1 2001Kenneth W. Simpson Background. Further elucidation of the consequences of Helicobacter pylori infection on gastric mucosal inflammation and gastric secretory function would be facilitated by an animal model that is susceptible to infection with H. pylori, is broadly similar in gastric physiology and pathology to people, and is amenable to repeated non-invasive evaluation. The goal of this study was to examine the interrelationship of bacterial colonization, mucosal inflammation and gastric secretory function in cats with naturally acquired H. pylori infection. Materials and Methods. Twenty clinically healthy cats with naturally acquired H. pylori infection (cagA,, picB) and 19 Helicobacter -free cats were evaluated. Gastric colonization was determined by tissue urease activity, light microscopy, culture and PCR. The mucosal inflammatory response was evaluated by light microscopy, and by RT-PCR of the pro-inflammatory cytokines IL-1,, IL-1,, IL-8 and TNF-, in gastric mucosa. Gastric secretory function was assessed by measuring pentagastrin-stimulated acid secretion, fasting plasma gastrin, and antral mucosal gastrin and somatostatin immunoreactivity. Results. H. pylori colonized the pylorus, fundus and cardia in similar density. Bacteria were observed free in the lumen of gastric glands and were also tightly adherent to epithelial cells where they were associated with microvillus effacement. Mononuclear inflammation, lymphoid follicle hyperplasia, atrophy and fibrosis were observed primarily in H. pylori -infected cats, with the pylorus most severely affected. Neutrophilic and eosinophilic infiltrates, epithelial dysplasia, and up-regulation of mucosal IL-1, and IL-8 were observed solely in infected cats. Fasting plasma gastrin concentrations and pentagastrin-stimulated acid output were similar in both infected and uninfected cats. There was no relationship of bacterial colonization density or gastric inflammation to plasma gastrin concentrations or gastric acid output. Conclusions. The pattern of colonization and the mucosal inflammatory response in cats with naturally acquired H. pylori are broadly similar to those in infected people, particularly children, and non-human primates. The upregulation of IL-8 in infected cats was independent of cagA and picB. Our findings argue against a direct acid-suppressing effect of H. pylori on the gastric secretory-axis in chronically infected cats. Abbreviations: RT-PCR, reverse transcriptase polymerase chain reaction, HLO; Helicobacter -like organisms. [source] Effect of porto-systemic shunting on NOS expression after extended hepatectomy in ratsHEPATOLOGY RESEARCH, Issue 1 2009Hironori Hayashi Aim:, Several surgical procedures have been developed for reducing portal vein pressure to prevent postoperative liver injury. Nitric oxide synthase expression (NOS) induced by elevation of portal vein pressure is thought to play an important role in liver regeneration, but the details are not well understood. Methods:, Rats in the control group and in the subcutaneous splenic transposition (SST) group underwent 90% partial hepatectomy. Survival and portal vein pressure were analyzed. The serum IL-6 and TNF-, levels were measured by enzyme-linked immunosorbent assay (ELISA). Hepatocyte proliferation and apoptosis 12 hours after hepatectomy were analyzed immunohistochemically. The protein and messenger RNA expression of inducible and endothelial NOS were analyzed using Western blotting and quantitative reverse transcriptase polymerase chain reaction, respectively. Results:, The survival rate of the SST group was significantly higher. Portal vein pressure, TNF-, level and the apoptotic index were significantly lower in the SST group. Twelve hours after surgery, liver inducible NOS (iNOS) protein expression was significantly lower in the SST group. However, protein expression of endothelial NOS was not significantly different between the groups. Conclusion:, Inducible NOS expression after extended hepatectomy is related to the effects of porto-systemic shunting on the splanchnic circulation. Also, iNOS induction and concomitant nitric oxide generation appear to participate in the cytotoxicity of excessive portal pressure after extended hepatectomy. [source] Preventive effects of ME3738 on hepatic fibrosis induced by bile duct ligation in ratsHEPATOLOGY RESEARCH, Issue 7 2008Kazunori Maeda Aim:, The aim of this study was to examine the preventive effects of ME3738 on hepatic fibrosis induced by bile duct ligation (BDL) in rats. Methods:, ME3738 (20 mg/day) was administered orally for 21 days immediately after BDL. Fibrosis was assessed by measuring hepatic hydroxyproline (Hyp) content. Activated hepatic stellate cells (HSCs) were assessed by ,-smooth muscle actin (,-SMA) immunostaining. Hepatic thiobarbituric acid-reactive substance (TBARS), 4-hydroxy-2-nonenal (4-HNE) and 8-hydroxy-2-deoxyguanosine (8-OHdG) immunostaining were used to analyze oxidative stress. The gene expressions of collagen-I, transforming growth factor-,1 (TGF-,1), tissue inhibitor of metalloproteinases-1 (TIMP-1), interleukin-6 (IL-6) and heme oxygenase-1 (HO-1) in the liver were examined by real-time reverse transcriptase polymerase chain reaction (RT,PCR). Results:, Hepatic Hyp content and the area of hepatic fibrosis in BDL rats treated with ME3738 were reduced by 24% and 39% compared with non-treated BDL rats (hepatic Hyp, 9.40 ± 2.85 vs. 12.39 ± 3.91 mg/liver; P = 0.036; area of hepatic fibrosis, 13.1 ± 3.8 vs. 21.5 ± 10.9; P = 0.045). Furthermore, ,-SMA-positive cells were significantly reduced by 40% (22.3 ± 14.8 vs. 37.6 ± 14.2; P = 0.011), collagen-I mRNA by 83% (6.5 ± 2.2 vs. 38.3 ± 9.1; P = 0.002), HO-1 mRNA by 58% (4.13 ± 1.22 vs. 9.73 ± 1.80; P = 0.018) and hepatic HO-1 content by 26% (2.13 ± 0.80 vs. 2.87 ± 0.19; P = 0.01) following ME3738 treatment. The hepatic expression of TBARS, 4-HNE, 8-OHdG and mRNA levels of TGF-,1, TIMP-1 and IL-6 in the liver were unchanged by ME3738 treatment. Conclusion:, Oral ME3738 administration may prevent the progression of hepatic fibrosis in BDL rats through suppression of the activation and collagen synthesis of HSC and, in part, oxidative stress. ME3738 has potential as a therapeutic drug for cholestatic liver fibrosis. [source] Molecular characterization of a prophenoloxidase cDNA from the malaria mosquito Anopheles stephensiINSECT MOLECULAR BIOLOGY, Issue 2 2000L. Cui Abstract Some refractory anopheline mosquitoes are capable of killing Plasmodium, the causative agent of malaria, by melanotic encapsulation of invading ookinetes. Phenoloxidase (PO) appears to be involved in the formation of melanin and toxic metabolites in the surrounding capsule. A cDNA encoding Anopheles stephensi prophenoloxidase (Ans-proPO) was isolated from a cDNA library screened with an amplimer produced by reverse transcriptase polymerase chain reaction (RT-PCR) with degenerate primers designed against conserved proPO sequences. The 2.4-kb-long cDNA has a 2058 bp open reading frame encoding Ans-proPO of 686 amino acids. The deduced amino acid sequence shows significant homology to other insect proPO sequences especially at the two putative copper-binding domains. In A. stephensi, Ans-proPO expression was detected in larval, pupal and adult stages. The Ans-proPO mRNA was detected by RT-PCR and in situ hybridization in haemocytes, fat body and epidermis of adult female mosquitoes. A low level of expression was detected in the ovaries, whereas no expression was detected in the midguts. Semi-quantitative RT-PCR analysis of Ans-proPO mRNA showed that its expression was similar in adult female heads, thoraxes and abdomens. No change in the level of Ans-proPO expression was found in adult females after blood feeding, bacterial challenge or Plasmodium berghei infection. However, elevated PO activity was detected in P. berghei -infected mosquitoes, suggesting that in non-selected permissive mosquitoes PO may be involved in limiting parasite infection. Genomic Southern blot and immunoblots suggest the presence of more than one proPO gene in the A. stephensi genome, which is consistent with the findings in other Diptera and Lepidoptera species. The greatest similarity in sequence and expression profile between Ans-proPO and A. gambiae proPO6 suggests that they might be homologues. Our results demonstrate that Ans-proPO is constitutively expressed through different developmental stages and under different physiological conditions, implying that other factors in the proPO activation cascade regulate melanotic encapsulation. [source] Expression and immunocytochemical analysis of Autographa californica nucleopolyhedrovirus (AcMNPV) orf74 geneINSECT SCIENCE, Issue 5 2006SHI-HENG AN Abstract Autographa californica nucleopolyhedrovirus orf74 (Ac74) is located between 62 311 and 63 108bp in the AcMNPV genome, which encodes 265 amino acid residues with a predicted 31 kDa molecular weight. The homologues of Ac74 were searched using BLASTP in protein databases, GenBank/EMBL and SWISS-PROT. The result revealed that deduced Ac74 protein was homologous to the predicted products from 10 lepidoptera NPV ORFs. The multiple sequence alignments of Ac74 and its 10 homologues manifested only one amino acid residue was completely conserved. The transcript analysis revealed that the transcript of Ac74 was detected from 24,72 hours post-infection (hpi). The product of Ac74 was detected at 24 hpi and lasted until 72 hpi by Western blot using anti-Ac74 antiserum, consistent with reverse transcriptase polymerase chain reaction results. These results suggested Ac74 was expressed during the later stages of infection. The product of Ac74 was 31 kDa in size, consistent with predicted molecular weight. The subcellular localization of Ac74 proteins manifested Ac74 protein in the cytoplasm, and was hardly present in the nucleus at 24 hpi. The fluorescence was also observed in polyhedra, except cytoplasm at 72 hpi. Together, Ac74 is a functional protein with 3 1kDa molecular weight and is located in the cytoplasm and the polyhedra. [source] Prostaglandin F2, stimulates MEK-ERK signalling but decreases the expression of alkaline phosphatase in dental pulp cellsINTERNATIONAL ENDODONTIC JOURNAL, Issue 6 2010M. C. Chang Chang MC, Chen YJ, Lee MY, Lin LD, Wang TM, Chan CP, Tsai YL, Wang CY, Lin BR, Jeng JH. Prostaglandin F2, stimulates MEK-ERK signalling but decreases the expression of alkaline phosphatase in dental pulp cells. International Endodontic Journal, 43, 461,468, 2010. Abstract Aim, To study prostaglandin F2, (PGF2,) receptor expression and downstream signalling in cultured human dental pulp cells and the effect of PGF2, on the alkaline phosphatase (ALP) activity of dental pulp cells. Methodology, Human dental pulp cells were cultured and exposed to PGF2,. The expression of PGF2, (FP) receptors was analysed by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. The activation of extracellular regulated kinase (ERK) and cAMP responsive element binding protein/activating transcription factor-1 (CREB/ATF-1) signalling was determined by Western blotting. The expression of ALP in pulp cells after exposure to PGF2, was evaluated by ALP staining and PCR. Results, Dental pulp cells expressed FP receptor mRNA and protein. Exposure to PGF2, revealed little cytotoxicity to pulp cells. PGF2, induced both ERK and CREB/ATF-1 phosphorylation in pulp cells. Exposure to PGF2, (>1 ,mol L,1) further decreased the ALP activity and mRNA expression. However, U0126 (an inhibitor of MEK1) showed little preventive effect on the decline of ALP activity in dental pulp cells by PGF2,. Conclusion, PGF2, may potentially activate FP receptors leading to ERK/CREB-ATF-1 activation during its production in inflamed dental pulp. PGF2, attenuated the ALP activity of pulp cells possibly via pathways not solely by MEK/ERK activation. PGF2, is a contributing factor of pulpal inflammation by regulating the activities of pulp cells. [source] MicroRNA-10b is overexpressed in malignant glioma and associated with tumor invasive factors, uPAR and RhoCINTERNATIONAL JOURNAL OF CANCER, Issue 6 2009Takashi Sasayama Abstract MicroRNAs (miRNAs) are effective post-transcriptional regulators of gene expression and are important in many biological processes. Although the oncogenic and tumor suppressive functions of several miRNAs have been characterized, the role of miRNAs in mediating tumor invasion and migration remains largely unexplored. Recently, miR-10b was identified as an miRNA highly expressed in metastatic breast cancer, promoting cell migration and invasion. Here, we performed real-time reverse transcriptase polymerase chain reaction (RT-PCR) assays on 43 glioma samples (17 glioblastoma, 6 anaplastic astrocytoma, 10 low-grade astrocytoma, 6 oligodendroglioma and 4 ependymoma) and 6 glioma cell lines. We found that miR-10b expression was upregulated in all glioma samples compared to non-neoplastic brain tissues. The expression levels of miR-10b were associated with higher grade glioma. In addition, mRNA expressions of RhoC and urokinase-type plasminogen activator receptor (uPAR), which were thought to be regulated by miR-10b via HOXD10, were statistically significantly correlated with the expression of miR-10b (p < 0.001, p = 0.001, respectively). Also, protein expression levels of RhoC and uPAR were associated with expression levels of miR-10b (p = 0.009, p = 0.014, respectively). Finally, multifocal lesions on enhanced MRI of 7 malignant gliomas were associated with higher expression levels of miR-10b (p = 0.02). Our data indicated that miR-10b might play some role in the invasion of glioma cells. © 2009 UICC [source] BCR/ABL p210, p190 and p230 fusion genes in 250 Mexican patients with chronic myeloid leukaemia (CML)INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 3 2002R.M. Arana-Trejo There are two major forms of the BCR/ABL fusion gene, involving ABL exon 2, but including different exons of BCR gene. The transcripts b2a2 or b3a2 code for a p210 protein. Another fusion gene leads to the expression of an e1a2 transcript, which codes for a p190 protein. Another, less common fusion gene is c3a2[e19a2], which encodes a p230 protein. The incidence of one or the other rearrangement in chronic myeloid leukaemia (CML) patients varies in different reported series. This study was designed to determine the frequency of coexpresion of the p210, p190 and p230 transcripts in 250 Mexican patients with CML. We performed nested and multiplex reverse transcriptase polymerase chain reaction (RT-PCR) on bone marrow samples from adult patients and found that all cases were positive for some type of BCR/ABL rearrangement. In 226 (90.4%) patients it was p210, while the remaining 9.6% showed coexpression or one of the transcripts of p190/p210/p230. In 7% of patients with p210 expression there are both isoforms (b3a2/b2a2), presumably the result of alternative splicing. The rate of coexpression of the p190/p210 transcripts was 5%, which is much lower than in other reports. This may be due to the technical factors. These patients had high platelet counts, marked splenomegaly and chromosomal abnormalities in addition to Ph,. Other types of coexpression seen were p210/p230 and p190/p210/p230, in patients with high-risk clinical factors. Our study confirms the occurrence of coexpression of different BCR/ABL transcripts, although the rate (9.6%) was much lower than has been reported in other populations. This may reflect either the sensitivity of the detection techniques used or the possibility of genetic differences between the populations studied. Coexpression may be due to alternative splicing or to phenotypic variation, with clinical courses different from classical CML. [source] Assessment of Growth, Physiological and Biochemical Parameters and Activities of Antioxidative Enzymes in Salinity Tolerant and Sensitive Basmati Rice VarietiesJOURNAL OF AGRONOMY AND CROP SCIENCE, Issue 6 2007M. P. Singh Abstract This investigation was undertaken to compare the level of salinity tolerance of the newly bred CSR-30 basmati rice variety with that of the salinity sensitive HBC-19 and Pokkali rice varieties. Twenty-one-day-old hydroponically raised seedlings at 6 and 12 dS m,1 were investigated for growth, photosynthetic rate, chlorophyll content, ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) activity, relative water content (RWC), membrane stability index (MSI), lipid peroxidation, Na/K ratio and activities and gene expression of various isoforms of antioxidative enzymes. Salinity stress led to reduction in shoot length, leaf area, dry weight, RWC, MSI, rate of photosynthesis, chlorophyll content and Rubisco activity in all the three rice varieties. The levels of reduction in these parameters were maximal in HBC-19 followed by those in CSR-30 and Pokkali respectively. Cumulative superoxide dismutase (SOD) activity increased in Pokkali and CSR-30 in consonance with increase in salinity stress while it decreased in HBC-19. The Mn-SOD activity however, was enhanced in all three varieties in the presence of salinity stress while the activities of Fe-SOD, Cu/Zn-SOD and ascorbate peroxidase were decreased in HBC-19 when compared with CSR-30 and Pokkali. The activity of catalase (CAT) was higher in HBC-19 when compared with its activity in CSR-30 and Pokkali. The levels of gene expressions of the three isoforms of SOD ascertained by reverse transcriptase polymerase chain reaction were not necessarily indicative of the activities of the corresponding enzymes. Thus, despite the maximal enhancement in gene expression of Fe-SOD in HBC-19 in response to salinity stress, the activity of this enzyme in HBC-19 remained low. Similarly, despite a marginal increase in gene expression of Cu-Zn SOD in the three varieties, its activity was significantly higher in Pokkali and CSR-30 when compared with that in HBC-19. A significant enhancement in the activity of CAT at 12 dS m,1 in HBC-19 when compared with CSR-30 and Pokkali might confer a degree of tolerance to H2O2 stress in this variety in the presence of higher levels of NaCl at the seedling stage. [source] Quantification of the expression and inducibility of 12 rat cytochrome P450 isoforms by quantitative RT,PCRJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 6 2006Etienne Caron Abstract The administration of xenobiotics may significantly alter the expression of cytochromes P450 (CYPs), thereby leading to potentially toxic cellular, physiologic, and pharmacologic responses. Indeed, an important task in the development of new therapeutic entities is to evaluate efficiently and quantitatively their potential effects on the expression level of different CYPs. In this report, reverse transcriptase polymerase chain reaction (RT,PCR) was used to measure basal and induced mRNA of a wide range of rat CYP isoforms. Rats (n = 3 per treatment) were treated with five prototype inducers of CYP isoforms or with vehicle only. RT and PCR efficiencies were determined using appropriate RNA and DNA standards. Messenger RNA was quantified by PicoGreen standard curves and normalized to cyclophilin. Quantitative RT,PCR was used successfully to demonstrate that CYP isoforms were induced at the mRNA level following drug administration. Notably, phenobarbital resulted in significant induction of CYP2B1, CYP2B2, CYP2C6, CYP2C13, CYP2E1, CYP3A1, and CYP3A2. 3-Methylcholanthrene induced CYP1A1, CYP1A2, and CYP1B1. CYP2C11 expression was highly variable and suppressed by pyridine, whereas the expression of CYP2E1 was suppressed by dexamethasone. We demonstrated that quantitative RT,PCR can be used to evaluate efficiently the effect of compounds on the expression of a wide range of CYP isoforms. The technique is advantageous over others in that it is very sensitive, efficient and applicable to highly homologous CYP isoforms. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:368,378, 2005; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20103 [source] Different apoptosis ratios and gene expressions in two human cell lines after sevoflurane anaesthesiaACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 9 2009S. KVOLIK Background: The aim of this study was to determine the effect of a single exposure of carcinoma cells (Caco-2 and HEp-2) to an anaesthetic gas mixture containing sevoflurane 3%, applied for a period of either 1 or 2 h, on the induction of apoptosis, propapototic gene expression and sphingomyelinase activity. Methods: Apoptosis was determined by flow cytometry. p53, caspase 3 and CYP2E1 gene expression was determined using reverse transcriptase polymerase chain reaction. Activities of acid (aSMase) and neutral sphingomyelinases (nSMase) were measured using methyl- 14C sphingomyeline, and for de novo ceramide and lipid synthesis [3H] palmitic acid was used. All results were compared with controls and analysed by Mann,Whitney and Kruskal,Wallis tests. Results: In the treated Caco-2 cells, the apoptotic ratio increased 24 h after anaesthesia (16.9%; P=0.04). The expression of both p53 and caspase-3 genes increased in Caco-2 and decreased in HEp-2 cells. The CYP2E1 gene expression was observed only in the Caco-2 cells. In control cells, the catalytic activity of aSMase was 2.3 times higher than that of nSMase activity. Decreased aSMase and nSMase activities were observed in Caco-2 cells 24 h after exposition. aSMase activity was halved (54.2%; P=0.06) in HEp-2 cells 24 h after anaesthesia. De novo ceramide synthesis correlated with SMase activity in Caco-2 cells. Conclusion: Sevoflurane anaesthesia induces late apoptosis in the colonic and laryngeal cancer cells investigated. Although the results obtained may indicate that an anaesthetic gas mixture containing sevoflurane induces p53-dependent apoptosis in the Caco-2 cells, the mechanism of apoptosis induction is unclear and remains to be elucidated. [source] | ||||||||||||||||||||||||||||||||||||||||