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Molecular Effects (molecular + effects)
Selected AbstractsAdvanced fluorescence in situ hybridization to localize and quantify gene expression in Japanese medaka (Oryzias latipes) exposed to endocrine-disrupting compoundsENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 9 2009June-Woo Park Abstract In an earlier study, we described the development of fluorescence in situ hybridization (FISH) using confocal microscopy to localize and quantify gene expression in fish. Here, we report the results of FISH application to investigate effects of model endocrine-disrupting chemicals (EDCs), 17,-ethinylestradiol (EE2) and 17,-trenbolone (TB), on expressions of EDC-responsive genes in Japanese medaka (Oryzias latipes) at the cellular/tissue level paired with histological observation. Gene expressions of vitellogenin-II (Vit-II), androgen receptor (AR), and cytochrome P450 gonadal aromatase (CYP19a) were determined after exposure to 5, 50, or 500 ng/L of EE2 or 50, 500, or 5,000 ng/L of TB for 7 d. Exposure to the greatest concentration of EE2 or TB significantly reduced fecundity and caused histological alterations in gonads. 17,-Ethinylestradiol induced Vit-II expression in both male gonads and liver relative to controls and resulted in greater intensity of hematoxylin staining in hepatocytes, which was significantly correlated with Vit-II induction in liver. When exposed to EE2 at less than 50 ng/L, CYP19a expression associated with early stage oocytes was greater than that in controls. However, at 500 ng/L, this trend was reversed. The greater Vit-II expression in testis from all EE2 groups, and the lesser expression of CYP19a in ovaries from the 500 ng/L group, likely is related to changes in the number of cells in which these genes are predominantly expressed rather than to an increase in expression per cell. 17,-Trenbolone significantly induced AR expression in ovaries but did not alter AR expression in female liver. It was concluded that FISH combined with histology enables advanced elucidation of molecular effects of chemicals by associating changes in gene expression with certain tissues and/or cell types and allows these changes to be related to histological effects. [source] Development and validation of a 2,000-gene microarray for the fathead minnow (Pimephales promelas)ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 7 2007Patrick Larkin Abstract Gene microarrays provide the field of ecotoxicology new tools to identify mechanisms of action of chemicals and chemical mixtures. Herein we describe the development and application of a 2,000-gene oligonucleotide microarray for the fathead minnow Pimephales promelas, a species commonly used in ecological risk assessments in North America. The microarrays were developed from various cDNA and subtraction libraries that we constructed. Consistency and reproducibility of the microarrays were documented by examining multiple technical replicates. To test application of the fathead minnow microarrays, gene expression profiles of fish exposed to 17,-estradiol, a well-characterized estrogen receptor (ER) agonist, were examined. For these experiments, adult male fathead minnows were exposed for 24 h to waterborne 17,-estradiol (40 or 100 ng/L) in a flow-through system, and gene expression in liver samples was characterized. Seventy-one genes were identified as differentially regulated by estradiol exposure. Examination of the gene ontology designations of these genes revealed patterns consistent with estradiol's expected mechanisms of action and also provided novel insights as to molecular effects of the estrogen. Our studies indicate the feasibility and utility of microarrays as a basis for understanding biological responses to chemical exposure in a model ecotoxicology test species. [source] Differential functional effects of novel mutations of the transcription factor FOXL2 in BPES patients,HUMAN MUTATION, Issue 8 2008Jeyabalan Nallathambi Abstract Mutations of the transcription factor FOXL2, involved in cranio-facial and ovarian development lead to the Blepharophimosis-Ptosis-Epicanthus Inversus Syndrome (BPES) in human. Here, we describe nine mutations in the open reading frame of FOXL2. Six of them are novel: c.292T>A (p.Trp98Arg), c.323T>C (p.Leu108Pro), c.650C>G (p.Ser217Cys) and three frameshifts. We have performed localization and functional studies for three of them. We have observed a strong cytoplasmic mislocalization induced by the missense mutation p.Leu108Pro located in the forkhead (FKH) domain of FOXL2. In line with this, transcriptional activity assays confirmed the loss-of-function induced by this variant. Interestingly, the novel mutation p.Ser217Cys, mapping between the FKH and the polyalanine domain of FOXL2 and producing a mild eyelid phenotype, led to normal localization and transactivation. We have also modeled the structure of the FKH domain to explore the potential structural impact of the mutations reported here and other previously reported ones. This analysis shows that mutants can be sorted into two classes: those that potentially alter protein-protein interactions and those that might disrupt the interactions with DNA. Our findings reveal new insights into the molecular effects of FOXL2 mutations, especially those affecting the FKH binding domain. © 2008 Wiley-Liss, Inc. [source] Molecular modifiers of T cell antigen receptor triggering threshold: the mechanism of CD28 costimulatory receptorIMMUNOLOGICAL REVIEWS, Issue 1 2003Oreste Acuto Summary:, CD28 was thought to represent a prototypic membrane receptor responsible for delivering the classically defined ,second signal' needed to avoid T cell paralysis when recognizing antigen presented by appropriate antigen presenting cells (APCs). Almost two decades after its molecular identification, the mechanism by which this ,second receptor' facilitates clonal expansion and differentiation upon antigen encounter is still not fully elucidated. There may be at least two reasons for this partially gray picture: the use of nonphysiological experimental conditions to study it and the fact that the action of CD28 may be partly masked by the presence of additional T cell surface receptors that also provide some costimulatory signals, although not equivalent to the one delivered through CD28. Thus, instead of aging, the study of CD28 is still a topical subject. What is appearing through work of recent years is that far from being purely qualitative, the CD28 signal provides a key quantitative contribution to potently boost the T cell antigen receptor (TCR) signal. In other words, CD28 is in part a signaling ,sosia' of the TCR. Also, it is clear now that CD28 operates via multiple molecular effects. Still, what we do not understand is the ,qualitative' part of this signal, perhaps due to lack of identification of unique signaling components and/or pathways activated by CD28 only. Here we review a series of recent findings pointing towards novel avenues to better understand the molecular basis of CD28 function. [source] Predicting the tautomeric equilibrium of acetylacetone in solution.JOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 4 2010Abstract This study investigates how the various components (method, basis set, and treatment of solvent effects) of a theoretical approach influence the relative energies between keto and enol forms of acetylacetone, which is an important model system to study the solvent effects on chemical equilibria from experiment and theory. The computations show that the most popular density functional theory (DFT) approaches, such as B3LYP overestimate the stability of the enol form with respect to the keto form by ,10 kJ mol,1, whereas the very promising SCS-MP2 approach is underestimating it. MP2 calculations indicate that in particular the basis set size is crucial. The Dunning Huzinaga double , basis (D95z(d,p)) used in previous studies overestimates the stability of the keto form considerably as does the popular split-valence plus polarization (SVP) basis. Bulk properties of the solvent included by continuum approaches strongly stabilize the keto form, but they are not sufficient to reproduce the reversal in stabilities measured by low-temperature nuclear magnetic resonance experiments in freonic solvents. Enthalpic and entropic effects further stabilize the keto form, however, the reversal is only obtained if also molecular effects are taken into account. Such molecular effects seem to influence only the energy difference between the keto and the enol forms. Trends arising due to variation in the dielectric constant of the solvent result from bulk properties of the solvent, i.e., are already nicely described by continuum approaches. As such this study delivers a deep insight into the abilities of various approaches to describe solvent effects on chemical equilibria. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2010 [source] Risk assessment of genetically modified crops for nutrition and healthNUTRITION REVIEWS, Issue 1 2009Javier A Magaña-Gómez The risk assessment of genetically modified (GM) crops for human nutrition and health has not been systematic. Evaluations for each GM crop or trait have been conducted using different feeding periods, animal models, and parameters. The most common result is that GM and conventional sources induce similar nutritional performance and growth in animals. However, adverse microscopic and molecular effects of some GM foods in different organs or tissues have been reported. Diversity among the methods and results of the risk assessments reflects the complexity of the subject. While there are currently no standardized methods to evaluate the safety of GM foods, attempts towards harmonization are on the way. More scientific effort is necessary in order to build confidence in the evaluation and acceptance of GM foods. [source] Phase I study of decitabine with doxorubicin and cyclophosphamide in children with neuroblastoma and other solid tumors: A children's oncology group study,,PEDIATRIC BLOOD & CANCER, Issue 4 2010MRCP, Rani E. George MD Abstract Background Demethylating agents may alter the expression of genes involved in chemotherapy resistance. We conducted a phase I trial to determine the toxicity and molecular effects of the demethylating agent, decitabine, followed by doxorubicin and cyclophosphamide in children with refractory solid tumors. Procedure Stratum A included children with any solid tumor; Stratum B included neuroblastoma patients only. Patients received a 1-hr decitabine infusion for 7 days, followed by doxorubicin (45,mg/m2) and cyclophosphamide (1,g/m2) on day 7. Pharmacokinetic studies were performed after the first dose of decitabine. Biological studies included methylation and gene expression analyses of caspase-8, MAGE-1 and fetal hemoglobin (HbF), and expression profiling of pre- and post-treatment peripheral blood and bone marrow cells. Results The maximum-tolerated dose of decitabine was 5,mg/m2/day for 7 days. Dose-limiting toxicities at 10,mg/m2/day were neutropenia and thrombocytopenia. Decitabine exhibited rapid clearance from plasma. Three of 9 patients in Stratum A and 4/12 patients in Stratum B had stable disease for ,4 months. Sustained MAGE-1 demethylation and increased HbF expression were observed in the majority of patients post-treatment (12/20 and 14/16, respectively). Caspase-8 promoter demethylation and gene expression were seen in 2/7 bone marrow samples. Differentially expressed genes were identified by microarray analysis. Conclusion Low-dose decitabine when combined with doxorubicin/cyclophosphamide has tolerable toxicity in children. However, doses of decitabine capable of producing clinically relevant biologic effects were not well tolerated with this combination. Alternative strategies of combining demethylating agents with non-cytotoxic, biologically targeted agents such as histone deactelyase inhibitors should be explored. Pediatr Blood Cancer. 2010;55:629,638. © 2010 Wiley-Liss, Inc. [source] Jr-ZFP2, encoding a Cys2/His2-type transcription factor, is involved in the early stages of the mechano-perception pathway and specifically expressed in mechanically stimulated tissues in woody plantsPLANT CELL & ENVIRONMENT, Issue 6 2008NATHALIE LEBLANC-FOURNIER ABSTRACT Plants respond to environmental mechanical stimulation, such as wind, by modifying their growth and development. To study the molecular effects of stem bending on 3-week-old walnut trees, a cDNA-AFLP approach was developed. This study allowed the identification of a cDNA, known as Jr-ZFP2, encoding a Cys2/His2-type two-zinc-fingered transcription factor. Reverse transcriptase-polymerase chain reaction analysis confirmed that Jr-ZFP2 mRNA accumulation is rapidly and transiently induced after mechanical stimulation. After bending, Jr-ZFP2 transcript increase was restricted to the stem, the organ where the mechanical solicitation was applied. Furthermore, other abiotic factors, such as cold or salt, did not modify Jr-ZFP2 mRNA accumulation in walnut stems under our experimental conditions, whereas growth studies demonstrated that salt stress was actually perceived by the plants. These results suggest that the regulation of Jr-ZFP2 expression is more sensitive to mechanical stimulus. This gene will be a good marker for studying the early stages of mechanical perception in woody plants. [source] Analysis of Testosterone Effects on Sonic Hedgehog Signaling in Juvenile, Adolescent and Adult Sprague Dawley Rat PenisTHE JOURNAL OF SEXUAL MEDICINE, Issue 3 2010Christopher W. Bond MS ABSTRACT Introduction., Smooth muscle apoptosis is a major contributing factor to erectile dysfunction (ED) development in prostatectomy and diabetic patients and animal models. A critical regulator of penile smooth muscle and apoptosis is Sonic hedgehog (SHH). The SHH protein is decreased in ED models and SHH treatment of cavernous nerve (CN) injured rats prevents smooth muscle apoptosis. A close association between androgen deficiency and ED has been suggested in the literature, but few studies have examined the molecular effects on penile smooth muscle and on known signaling mechanisms that regulate morphology. Aim., Examine testosterone and SHH interaction in eugonadal adult, adolescent and juvenile rats by performing castration studies and treatment with supraphysiological testosterone. Methods., The eugonadal adult Sprague Dawley rats were either treated with testosterone for 7 or 14 days (N = 14) or were castrated for 4 or 7 days (N = 12). The juvenile rats were treated with testosterone for 8 days (N = 7). The adolescent rats were castrated and sacrificed at P88 (N = 8). The control rats had empty vehicle (N = 22) or sham surgery (N = 20). Main Outcome Measures., The active form of SHH protein and mRNA were quantified by semi-quantitative immunohistochemical analysis and real-time reverse transcriptase polymerase chain reaction (RT-PCR). Results., Testosterone treatment did not alter SHH signaling in juvenile rats. Shh mRNA increased 3.2-fold and SHH protein increased 1.2-fold in rats castrated during puberty. In adult rats, castration decreased Shh mRNA 3.2-fold but did not alter SHH protein. Testosterone supplement in adult rats increased Shh mRNA 2.3-fold and decreased SHH protein 1.3-fold. Conclusions., SHH signaling is independent of testosterone in normal juvenile rats and is sensitive to testosterone during adolescence, while testosterone supplement in the adult adversely impacts SHH signaling in a very similar manner to that observed with CN injury. Bond CW, Angeloni NL, and Podlasek CA. Analysis of testosterone effects on sonic hedgehog signaling in juvenile, adolescent and adult Sprague Dawley rat penis. J Sex Med 2010;7:1116,1125. [source] Chronic Administration of Ketamine Elicits Antidepressant-Like Effects in Rats without Affecting Hippocampal Brain-Derived Neurotrophic Factor Protein LevelsBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 6 2008Lêda S. Garcia The present study was aimed to evaluate behavioural and molecular effects of the chronic treatment with ketamine and imipramine in rats. To this aim, rats were 14 days treated once a day with ketamine (5, 10 and 15 mg/kg) and imipramine (10, 20 and 30 mg/kg) and then subjected to the forced swimming and open-field tests. Ketamine and imipramine, at the all doses tested, reduced immobility time, and increased both climbing and swimming time of rats compared to the saline group, without affecting spontaneous locomotor activity. Brain-derived neurotrophic factor (BDNF) hippocampal levels were assessed in imipramine- and ketamine-treated rats by ELISA sandwich assay. Chronic administration of both drugs, ketamine and imipramine, did not modify BDNF protein levels in the rat hippocampus. In conclusion, our findings demonstrate for the first time that chronic administration of acute inactive doses of ketamine (5 mg/kg) becomes active after chronic treatment, while no signs of tolerance to the behavioural effects of ketamine were observed after chronic administration of acute active doses (10 and 15 mg/kg). Finally, these findings further support the hypothesis that NMDA receptor could be a new pharmacological target for the treatment of mood disorders. [source] An in ovo chicken model to study the systemic and localized teratogenic effects of valproic acidBIRTH DEFECTS RESEARCH, Issue 4 2002Amy I. Whitsel Background The antiepileptic valproic acid (VPA) is a teratogen whose embryopathic mechanism(s) remain uncertain. Elucidating potential cellular and molecular effects of VPA is complicated by systemic application paradigms. We developed an in ovo model to reproduce the teratogenic effects of VPA and a localized VPA application procedure to determine whether VPA can selectively effect abnormal development in one region of the embryo. Methods VPA was applied topically to chicken embryos in ovo at different embryonic stages. Embryos were later evaluated for gross and skeletal anomalies. Pax-2 and Pax-6 protein expression in the developing eye was also evaluated because VPA-induced eye anomalies are similar to those seen by the disruption of Pax-2 and Pax-6. For localized application, a thin sheet of the synthetic polymer Elvax was impregnated with VPA. A small piece of the VPA-impregnated polymer was applied directly to the presumptive wing bud region in Stage 10,17 embryos. Embryos were examined for gross and skeletal anomalies. Sham controls were employed for all experiments. Results Chicken embryos exposed to VPA in ovo demonstrated increased mortality, growth delay and anomalies similar to ones previously seen in humans: neural tube, cardiovascular, craniofacial, limb and skeletal. Pax-2 and Pax-6 protein expression was qualitatively diminished in the eye. Localized wing bud VPA exposure caused structural abnormalities in the developing wing in the absence of other anomalies in the embryos. These wing defects were similar to those observed after topical whole-embryo VPA application. Conclusions These results indicate that at least one mechanism for the teratogenicity of VPA involves a direct effect on developing tissue. The nature of the abnormalities observed implies that this effect may be mediated by disruption of genes that regulate pattern formation. Teratology 66:153,163, 2002. © 2002 Wiley-Liss, Inc. [source] Adalimumab therapy rapidly inhibits p38 mitogen-activated protein kinase activity in lesional psoriatic skin preceding clinical improvementBRITISH JOURNAL OF DERMATOLOGY, Issue 6 2010L. Soegaard-Madsen Summary Background, The pathogenesis of psoriasis and the mechanisms of action of antitumour necrosis factor (TNF)-, therapies are incompletely understood. Objectives, To investigate the early molecular effects of adalimumab in psoriatic skin. Methods, Biopsies taken from patients with psoriasis were examined before and after the onset of adalimumab therapy. TNF-, protein level and mRNA expression were measured by enzyme-linked immunosorbent assay and quantitative reverse transcription,polymerase chain reaction, respectively. The activities of p38 mitogen-activated protein kinase (MAPK) and extracellular regulated kinase 1 and 2 (ERK1/2) as well as the downstream kinases MAPK-activated protein kinase 2 (MK2) and mitogen- and stress-activated protein kinase 1 and 2 (MSK1/2) were measured by Western blot analyses. Results, We demonstrated that clinical and histological improvements were detected from day 14. The increased activity of p38 MAPK in lesional psoriatic skin was significantly inhibited by adalimumab already at day 4. The activities of ERK1/2, MSK1/2 and MK2 were reduced at the end of study (day 84) when the level of TNF-, in lesional psoriatic skin reached the nonlesional level, and the Psoriasis Area and Severity Index score was reduced. Conclusions, The rapid inhibition of p38 MAPK by adalimumab in lesional psoriatic skin preceded clinical and histological improvements, demonstrating an association between TNF-, neutralization and p38 MAPK inhibition. Thus, inhibition of p38 MAPK may be a novel mechanism by which adalimumab mediates its antipsoriatic effect. [source] | |||||||||||||