1α,25-Dihydroxyvitamin D3 Stimulates Activator Protein 1 DNA-Binding Activity by a Phosphatidylinositol 3-Kinase/Ras/MEK/Extracellular Signal Regulated Kinase 1/2 and c-Jun N-Terminal Kinase 1-Dependent Increase in c-Fos, Fra1, and c-Jun Expression in Human Keratinocytes

1α,25-Dihydroxyvitamin D3 added to human keratinocytes increases differentiation through an activation of the transcription factor activator protein 1. We have previously reported that the 1α,25-dihydroxyvitamin D3-induced increase of activator protein 1 DNA binding activity is mediated by a protein kinase C-independent mechanism. The purpose of this study was to investigate further the mechanisms by which 1α,25-dihydroxyvitamin D3 modulates activator protein 1 DNA binding activity in cultured normal human keratinocytes. Western blotting experiments revealed that 1α,25-dihydroxyvitamin D3 caused a rapid and transient activation of the mitogen-activated protein kinases, extracellular signal regulated kinase 1/2 and c-Jun N-terminal kinase 1. 1α,25-Dihydroxyvitamin D3 also enhanced the expression of the activator protein 1 subunits, c-Fos, Fra1, and c-Jun as determined by northern and western blotting. The 1α,25-dihydroxyvitamin D3-induced activator protein 1 DNA binding activity was completely blocked by the MEK inhibitor PD 98059 indicating that the MEK/extracellular signal regulated kinase pathway is involved in the activation of activator protein 1. Transfection experiments showed that 1α,25-dihydroxyvitamin D3 also increased the activator protein 1-dependent transactivation, which was completely blocked by expression of a dominant negative Ras, suggesting that the 1α,25-dihydroxyvitamin D3-induced activator protein 1 activity involves Ras-dependent signaling. Furthermore, preincubation of the keratinocytes with the specific phosphatidylinositol 3-kinase inhibitors, Wortmannin and LY294002, demonstrated that the 1α,25-dihydroxyvitamin D3-induced activation of extracellular signal regulated kinase 1/2 and c-Jun N-terminal kinase 1 required phosphatidylinositol 3-kinase activity. Finally, preincubation of keratinocytes with a polyclonal antibody against the membrane receptor annexin II, blocked the 1α,25-dihydroxyvitamin D3-induced activation of extracellular signal regulated kinase 1/2 and c-Jun N-terminal kinase 1. Taken together, our results indicate that 1α,25-dihydroxyvitamin D3, via binding to the membrane receptor annexin II, induces activation of the phos-phatidylinositol 3-kinase/Ras/MEK/extracellular signal regulated kinase 1/2 and c-Jun N-terminal kinase 1 signal transduction pathway resulting in increased expression of c-Fos, Fra1, and c-Jun, and subsequently increased activator protein 1 DNA binding activity and gene transcription

In-situ remediation of TCE by ERD in clay tills:Feasibility and performance of full-scale application insights gained through an integrated investigative approach for 2 sites

Background/Objectives. Remediation of trichloroethene (TCE) in clay and other low permeabil-ity geologic media, where groundwater flow occurs preferentially in higher permeability sand lenses or fractures, is a significant challenge. At older sites, much of the contaminant mass is pre-sent as a sorbed phase in the matrix due to matrix diffusion. The principal challenge for in situ remediation in clay is to achieve effective contact between contaminant and bioremediation addi-tives (e.g., organic electron donors and bioaugmentation cultures). The feasibility and perfor-mance of full-scale applications of ERD in clay tills were investigated in a research project in-cluding 2 sites in Denmark undergoing remediation since 2006.Site remediation approach. At the Sortebrovej site an emulsified oil donor (EOS) and a bio-augmentation culture (KB1®) with specific degraders Dehalococcoides were injected in a net-work of screened wells and spread in natural sand stringers embedded in the clay till. At the Gl. Kongevej site organic molasses donor and Bioclear Dechlorinating bioaugmentation culture with specific degraders Dehalococcoides were injected with a drive-point probe (Geoprobe) at 25 cm spaced vertical intervals in the clay till in a closely spaced network. Investigative activities. An integrated investigative approach consisting of water and clay core sample analysis, including stable isotopes and specific degraders, as well as analysis for chlorin-ated solvents, degradation products, donor fermentation products and redox sensitive parameters combined with modelling was applied. Groundwater monitoring of selected wells was performed 2-3 times per year, and very detailed subsampling (on 0.25-5 cm scale) of the intact clay cores for matrix profile analysis was performed after 2 and 4 years. The transport including matrix diffu-sion and degradation in fractures/sand stringers and in bioactive zones in the clay till adjacent to the fractures/sand stringers was modelled to gain insight on the effects of sand stringer/fracture /injection spacing, thickness of bioactive zones, density/numbers of specific degraders, donor longevity, etc., on remediation efficiency and timeframes. Results/Lessons learned. The results showed that the chlorinated solvent TCE was converted into its daughter products (DCE, VC and ethene) but complete conversion of contaminants to ethene (as expected) was not achieved in 4 years. Large variation in the effect of ERD in the clay matrix between sites, boreholes and even between cores was observed. After 4 years, the mass removal at the 2 sites varied between &lt;5% and 50% within the treated zones. The limited effi-ciency of the bioremediation in terms of mass removal is due to the limited spatial extent of dechlorination. If degradation is restricted to narrow bioactive zones of a few cm developing around fractures and sand stringers, contaminants in the remaining part of the matrix are not de-graded and remediation efficiency is low due to the mass transfer limitations. However, the bio-active zones may expand in zones where both donor and chlorinated compounds are present. And in some cores TCE was depleted (degraded to DCE) in zones up to 1.8 m thick, an extent, which could not be explained by diffusive loss to narrow bioactive zones. Hence, biomass migration in the clay matrix appears to play an important role in terms of contaminants mass reduction. <br/

FSH isoform composition of commercial gonadotrophin preparations: a neglected aspect?

The clinical efficacy of commercial gonadotrophin preparations has been the subject of an intense debate during recent years. Arguments have primarily focused on the origin of FSH activity (urine versus recombinant derived) and whether the preparation included LH-like activity. FSH isoform composition has received little or no attention, and is usually considered to have negligible effect on clinical effectiveness. By presenting the available data on the FSH isoform composition of commercial gonadotrophin preparations, the present paper challenges this assumption. To evaluate whether the FSH isoform composition affected the efficacy of a product, a meta-analysis was performed that compared a preparation expressing an acidic isoform profile (urinary-derived Metrodin-HP) with a preparation rich in less acidic isoforms (recombinant derived Gonal F). A total of five randomized clinical trials that specifically compared these two preparations was identified and included in the analysis. All parameters relating to the direct effect of FSH on the follicle differed significantly in favour of the product rich in less acidic isoforms, while data on pregnancy outcome did not reach significance. The importance of the FSH isoform profile and whether the FSH is derived from urine or by recombinant technique is discussed in relation to clinical efficacy. It is suggested that the FSH isoform profile of commercial gonadotrophin preparations is of clinical importance and should be taken into account when evaluating efficac