Influence of ARHGEF3 and RHOA Knockdown on ACTA2 and Other Genes in Osteoblasts and Osteoclasts

Osteoporosis is a common bone disease that has a strong genetic component. Genome-wide linkage studies have identified the chromosomal region 3p14-p22 as a quantitative trait locus for bone mineral density (BMD). We have previously identified associations between variation in two related genes located in 3p14-p22, ARHGEF3 and RHOA, and BMD in women. In this study we performed knockdown of these genes using small interfering RNA (siRNA) in human osteoblast-like and osteoclast-like cells in culture, with subsequent microarray analysis to identify genes differentially regulated from a list of 264 candidate genes. Validation of selected findings was then carried out in additional human cell lines/cultures using quantitative real-time PCR (qRT-PCR). The qRT-PCR results showed significant down-regulation of the ACTA2 gene, encoding the cytoskeletal protein alpha 2 actin, in response to RHOA knockdown in both osteoblast-like (P<0.001) and osteoclast-like cells (P = 0.002). RHOA knockdown also caused up-regulation of the PTH1R gene, encoding the parathyroid hormone 1 receptor, in Saos-2 osteoblast-like cells (P<0.001). Other findings included down-regulation of the TNFRSF11B gene, encoding osteoprotegerin, in response to ARHGEF3 knockdown in the Saos-2 and hFOB 1.19 osteoblast-like cells (P = 0.003– 0.02), and down-regulation of ARHGDIA, encoding the Rho GDP dissociation inhibitor alpha, in response to RHOA knockdown in osteoclast-like cells (P<0.001). These studies identify ARHGEF3 and RHOA as potential regulators of a number of genes in bone cells, including TNFRSF11B, ARHGDIA, PTH1R and ACTA2, with influences on the latter evident in both osteoblast-like and osteoclast-like cells. This adds further evidence to previous studies suggesting a role for the ARHGEF3 and RHOA genes in bone metabolism

Neurotensin Receptor 1 Gene (NTSR1) Polymorphism Is Associated with Working Memory

BACKGROUND: Recent molecular genetics studies showed significant associations between dopamine-related genes (including genes for dopamine receptors, transporters, and degradation) and working memory, but little is known about the role of genes for dopamine modulation, such as those related to neurotensin (NT), in working memory. A recent animal study has suggested that NT antagonist administration impaired working memory in a learning task. The current study examined associations between NT genes and working memory among humans. METHODS: Four hundred and sixty healthy undergraduate students were assessed with a 2-back working memory paradigm. 5 SNPs in the NTSR1 gene were genotyped. 5 ANOVA tests were conducted to examine whether and how working memory differed by NTSR1 genotype, with each SNP variant as the independent variable and the average accuracy on the working memory task as the dependent variable. RESULTS: ANOVA results suggested that two SNPs in the NTSR1 gene (rs4334545 and rs6090453) were significantly associated with working memory. These results survived corrections for multiple comparisons. CONCLUSIONS: Our results demonstrated that NTSR1 SNP polymorphisms were significantly associated with variance in working memory performance among healthy adults. This result extended previous rodent studies showing that the NT deficiency impairs the working memory function. Future research should replicate our findings and extend to an examination of other dopamine modulators

Antimutagenic and antigenotoxic effects of vegetable matrices on the activity of pesticides

Two in vitro tests, one to detect bacterial mutagenicity (Ames test) on Salmonella typhimurium TA98, TA100, and TA1535 and the other the primary DNA damage (SOS Chromotest) on Escherichia coli PQ37, were applied to determine the overall genotoxic activity of 12 pesticides (azinphos methyl, chlorothalonil, chlorphyriphos ethyl, chlorphyriphos methyl, λ-cyhalothrin, cypermethrin, cyprodinil, fenazaquin, fludioxonil, indoxacarb, iprodione and penconazol). These were detected by gas chromatography (GC) analysis with electron capture (ECD) and nitrogen phosphorus detection (NPD) in 18 samples of vegetables. Some extracts of vegetables, found positive for pesticides with GC, were subjected to the Ames test and SOS Chromotest to evaluate the possible antimutagenic and/or antigenotoxic effects of vegetable matrices. The same bioassays were also performed on the mixtures of pesticides found in these samples to evaluate whether interactions could occur between pesticides and be responsible for the possible antimutagenic and/or antigenotoxic effects of the contaminated matrices. Experiments were also carried out to compare the results found for contaminated vegetables with their content of antioxidant components. Significant differences in mutagenicity and genotoxicity were found among the pesticides selected for this study. Of the 12 pesticides tested, only azinphos methyl, cyprodinil, fludioxonil and iprodione were found to be positive for both S. typhimurium and E. coli. No mutagenic/genotoxic activity was found in the extracts of vegetables contaminated by pesticides. S. typhimurium TA1535 showed a strong positive mutagenic effect for the mixtures of pesticides while they were not able to induce the SOS system. The data concerning the content of polyphenols and the total reducing activity of the contaminated vegetables indicated high amounts of antioxidants that could explain the inhibitory effect on the activity of pesticides shown by vegetables. © 2009 Taylor & Francis

Endometriosi profonda e disfunzioni urinarie dopo chirurgia laparoscopica: modulazione della dissezione chirurgica e riscontro anatomo-istologico

Scopo dello studio. Valutazione della relazione tra dolore ed endometriosi, ed in particolare la possibile correlazione tra disturbi vescico-urinari e la quantità di fibre nervose e cellule gangliari resecate con l’intervento chirurgico laparoscopico per endometriosi profonda. Materiali e metodi. Studio retrospettivo su 230 donne con endometriosi severa e sottoposte ad intervento di chirurgia laparoscopica. Sede dello studio: Hôpital de Hautpierre, Strasburgo e Seconda Università di Napoli. Risultati. L’incidenza di ritenzione urinaria è stata del 4.5% (n=10). Assenza di differenze quantitative nel pattern di distribuzione di fibre nervose e cellule gangliari nei campioni resecati di entrambi i gruppi. Netto miglioramento della sintomatologia dolorosa in entrambi i gruppi, con peggioramento della disuria nelle pazienti con ritenzione urinaria post-operatoria. Conclusioni. Con la tecnica chirurgica nerve-sparing si è avuta una bassa incidenza di ritenzione urinaria; tuttavia non sono state riscontrate differenze significative nel pattern di distribuzione di fibre nervose e cellule gangliari nei due gruppi in esame

Expression of CPI-17 and myosin phosphatase correlates with Ca2+ sensitivity of protein kinase C-induced contraction in rabbit smooth muscle

Various smooth muscles have unique contractile characteristics, such as the degree of Ca2+ sensitivity induced by physiological and pharmacological agents. Here we evaluated six different rabbit smooth muscle tissues for protein kinase C (PKC)-induced Ca2+ sensitization. We also examined the expression levels of myosin light chain phosphatase (MLCP), the MLCP inhibitor phosphoprotein CPI-17, and the thin filament regulator h-calponin.Immunohistochemical and Western blot analyses indicated that CPI-17 was found primarily in smooth muscle, although expression varied among different tissues. Vascular muscles contained more CPI-17 than visceral muscles, with further distinction existing between tonic and phasic subtypes. For example, the tonic femoral artery possessed approximately 8 times the cellular CPI-17 concentration of the phasic vas deferens.In contrast to CPI-17 expression patterns, phasic muscles contained more MLCP myosin-targeting subunit than tonic tissues. Calponin expression was not statistically different.Addition of phorbol ester to α-toxin-permeabilized smooth muscle caused an increase in contraction and phosphorylation of both CPI-17 and myosin light chain (MLC) at submaximal [Ca2+]i. These responses were several-fold greater in femoral artery as compared to vas deferens.We conclude that the expression ratio of CPI-17 to MLCP correlates with the Ca2+ sensitivities of contraction induced by a PKC activator. PKC stimulation of arterial smooth muscle with a high CPI-17 and low MLCP expression generated greater force and MLC phosphorylation than stimulation of visceral muscle with a relatively low CPI-17 and high MLCP content. This implicates CPI-17 inhibition of MLCP as an important component in modulating vascular muscle tone