Dynamic modulation of phosphoprotein expression in ovarian cancer xenograft models

The authors thank Medical Research Scotland and the Scottish Funding Council. This work was su pported by Medical Research Scotland [FRG353 to V.A.S.]; the FP7 -­‐ Directorate -­‐ General for Research and Innovation of the European Commission [EU HEALTH -­‐ F4 -­‐ 2012 -­‐ 305033 to Coordinating Action Systems Medicine -­‐ D.J.H.]; the Chief Scientist Office of Scotland [D.J.H.], the Scottish Funding Council [D.J.H. and S.P.L.]. Health Canada Scholarship (Indspire) [KEF], Scottish Overseas Research Student Award Scheme (University of Edinburgh)[KEF] and the Three Fires Award (Wikwemikong Board of Education)[KEF].Background: The dynamic changes that occur in protein expression after treatment of a cancer in vivo are poorly described. In this study we measure the effect of chemotherapy over time on the expression of a panel of proteins in ovarian cancer xenograft models. The objective was to identify phosphoprotein and other protein changes indicative of pathway activation that might link with drug response. Methods: Two xenograft models, platinum-responsive OV1002 and platinum-unresponsive HOX424, were used. Treatments were carboplatin and carboplatin-paclitaxel. Expression of 49 proteins over 14 days post treatment was measured by quantitative immunofluorescence and analysed by AQUA . Results: Carboplatin treatment in the platinum-sensitive OV1002 model triggered up-regulation of cell cycle, mTOR and DDR pathways, while at late time points WNT, invasion , EMT and MAPK pathways were modulated. Estrogen receptor-alpha (ESR1) and ERBB pathways were down-regulated early, within 24h from treatment administration. Combined carboplatin-paclitaxel treatment triggered a more extensive response in the OV1002 model modulating expression of 23 of 49 proteins. Therefore the cell cycle and DDR pathways showed similar or more pronounced changes than with carboplatin alone . In addition to expression of pS6 and pERK increasing, components of the AKT pathway were modulated with pAKT increasing while its regulator PTEN was down-regulated early. WNT signaling, EMT and invasion markers were modulated at later time points. Additional pathways were also observed with the NFκB and JAK/STAT pathways being up-regulated. ESR1 was down-regulated as was HER4, while further protein members of the ERB B pathway were upregulated late. By contrast, in the carboplatin-unresponsive HOX 424 xenograft, carboplatin only modulated expression of MLH1 while carboplatin-paclitaxel treatment modulated ESR1 and pMET.Publisher PDFPeer reviewe

Immunological characterization of chromogranins A and B and secretogranin II in the bovine pancreatic islet

Antisera against chromogranin A and B and secretogranin II were used for analysing the bovine pancreas by immunoblotting and immunohistochemistry. All three antigens were found in extracts of fetal pancreas by one dimensional immunoblotting. A comparison with the soluble proteins of chromaffin granules revealed that in adrenal medulla and in pancreas antigens which migrated identically in electrophoresis were present. In immunohistochemistry, chromogranin A was found in all pancreatic endocrine cell types with the exception of most pancreatic polypeptide-(PP-) producing cells. For chromogranin B, only a faint immunostaining was obtained. For secretorgranin II, A-and B-cells were faintly positive, whereas the majority of PP-cells exhibited a strong immunostaining for this antigen. These results establish that chromogranins A and B and secretogranin II are present in the endocrine pancreas, but that they exhibit a distinct cellular localization

Discrimination of conventional and organic white cabbage from a long-term field trial study using untargeted LC-MS-based metabolomics

The influence of organic and conventional farming practices on the content of single nutrients in plants is disputed in the scientific literature. Here, large-scale untargeted LC-MS-based metabolomics was used to compare the composition of white cabbage from organic and conventional agriculture, measuring 1,600 compounds. Cabbage was sampled in 2 years from one conventional and two organic farming systems in a rigidly controlled long-term field trial in Denmark. Using Orthogonal Projection to Latent Structures-Discriminant Analysis (OPLS-DA), we found that the production system leaves a significant (p = 0.013) imprint in the white cabbage metabolome that is retained between production years. We externally validated this finding by predicting the production system of samples from one year using a classification model built on samples from the other year, with a correct classification in 83% of cases. Thus, it was concluded that the investigated conventional and organic management practices have a systematic impact on the metabolome of white cabbage. This emphasizes the potential of untargeted metabolomics for authenticity testing of organic plant products

Defending the genome from the enemy within:mechanisms of retrotransposon suppression in the mouse germline

The viability of any species requires that the genome is kept stable as it is transmitted from generation to generation by the germ cells. One of the challenges to transgenerational genome stability is the potential mutagenic activity of transposable genetic elements, particularly retrotransposons. There are many different types of retrotransposon in mammalian genomes, and these target different points in germline development to amplify and integrate into new genomic locations. Germ cells, and their pluripotent developmental precursors, have evolved a variety of genome defence mechanisms that suppress retrotransposon activity and maintain genome stability across the generations. Here, we review recent advances in understanding how retrotransposon activity is suppressed in the mammalian germline, how genes involved in germline genome defence mechanisms are regulated, and the consequences of mutating these genome defence genes for the developing germline

Utilization of a deoxynucleoside diphosphate substrate by HIV reverse transcriptase

Background: Deoxynucleoside triphosphates (dNTPs) are the normal substrates for DNA sysnthesis is catalyzed by polymerases such as HIV-1 reverse transcriptase (RT). However, substantial amounts of deoxynucleoside diphosphates (dNDPs) are also present in the cell. Use of dNDPs in HIV-1 DNA sysnthesis could have significant implications for the efficacy of nucleoside RT inhibitors such as AZT which are first line therapeutics fro treatment of HIV infection. Our earlier work on HIV-1 reverse transcriptase (RT) suggested that the interaction between the γ phosphate of the incoming dNTP and RT residue K65 in the active site is not essential for dNTP insertion, implying that this polymerase may be able to insert dNPs in addition to dNTPs. Methodology/Principal Findings: We examined the ability of recombinant wild type (wt) and mutant RTs with substitutions at residue K65 to utilize a dNDP substrate in primer extension reactions. We found that wild type HIV-1 RT indeed catalyzes incorporation of dNDP substrates whereas RT with mutations of residue K645 were unable to catalyze this reaction. Wild type HIV-1 RT also catalyzed the reverse reaction, inorganic phosphate-dependent phosphorolysis. Nucleotide-mediated phosphorolytic removal of chain-terminating 3′-terminal nucleoside inhibitors such as AZT forms the basis of HIV-1 resistance to such drugs, and this removal is enhanced by thymidine analog mutations (TAMs). We found that both wt and TAM-containing RTs were able to catalyze Pi-mediated phosphorolysis of 3′-terminal AZT at physiological levels of Pi with an efficacy similar to that for ATP-dependent AZT-excision. Conclusion: We have identified two new catalytic function of HIV-1 RT, the use of dNDPs as substrates for DNA synthesis, and the use of Pi as substrate for phosphorolytic removal of primer 3′-terminal nucleotides. The ability to insert dNDPs has been documented for only one other DNA polymerase The RB69 DNA polymerase and the reverse reaction employing inorganic phosphate has not been documented for any DNA polymerase. Importantly, our results show that Pi-mediated phosphorolysis can contribute to AZT resistance and indicates that factors that influence HIV resistance to AZT are more complex than previously appreciated. © 2008 Garforth et al

Denitrification and nitrous oxide emissions from riparian forests soils exposed to prolonged nitrogen runoff

Compared to upland forests, riparian forest soils have greater potential to remove nitrate (NO3) from agricultural run-off through denitrification. It is unclear, however, whether prolonged exposure of riparian soils to nitrogen (N) loading will affect the rate of denitrification and its end products. This research assesses the rate of denitrification and nitrous oxide (N2O) emissions from riparian forest soils exposed to prolonged nutrient run-off from plant nurseries and compares these to similar forest soils not exposed to nutrient run-off. Nursery run-off also contains high levels of phosphate (PO4). Since there are conflicting reports on the impact of PO4 on the activity of denitrifying microbes, the impact of PO4 on such activity was also investigated. Bulk and intact soil cores were collected from N-exposed and non-exposed forests to determine denitrification and N2O emission rates, whereas denitrification potential was determined using soil slurries. Compared to the non-amended treatment, denitrification rate increased 2.7- and 3.4-fold when soil cores collected from both N-exposed and non-exposed sites were amended with 30 and 60 μg NO3-N g-1 soil, respectively. Net N2O emissions were 1.5 and 1.7 times higher from the N-exposed sites compared to the non-exposed sites at 30 and 60 μg NO3-N g-1 soil amendment rates, respectively. Similarly, denitrification potential increased 17 times in response to addition of 15 μg NO3-N g-1 in soil slurries. The addition of PO4 (5 μg PO4–P g-1) to soil slurries and intact cores did not affect denitrification rates. These observations suggest that prolonged N loading did not affect the denitrification potential of the riparian forest soils; however, it did result in higher N2O emissions compared to emission rates from non-exposed forests

Free backbone carbonyls mediate rhodopsin activation

Conserved prolines in the transmembrane helices of G-protein-coupled receptors (GPCRs) are often considered to function as hinges that divide the helix into two segments capable of independent motion. Depending on their potential to hydrogen-bond, the free C=O groups associated with these prolines can facilitate conformational flexibility, conformational switching or stabilization of the receptor structure. To address the role of conserved prolines in family A GPCRs through solid-state NMR spectroscopy, we focus on bovine rhodopsin, a GPCR in the visual receptor subfamily. The free backbone C=O groups on helices H5 and H7 stabilize the inactive rhodopsin structure through hydrogen-bonds to residues on adjacent helices. In response to light-induced isomerization of the retinal chromophore, hydrogen-bonding interactions involving these C=O groups are released, thus facilitating repacking of H5 and H7 onto the transmembrane core of the receptor. These results provide insights into the multiple structural and functional roles of prolines in membrane proteins

Tendinopathy—from basic science to treatment

Chronic tendon pathology (tendinopathy), although common, is difficult to treat. Tendons possess a highly organized fibrillar matrix, consisting of type I collagen and various 'minor' collagens, proteoglycans and glycoproteins. The tendon matrix is maintained by the resident tenocytes, and there is evidence of a continuous process of matrix remodeling, although the rate of turnover varies at different sites. A change in remodeling activity is associated with the onset of tendinopathy. Major molecular changes include increased expression of type III collagen, fibronectin, tenascin C, aggrecan and biglycan. These changes are consistent with repair, but they might also be an adaptive response to changes in mechanical loading. Repeated minor strain is thought to be the major precipitating factor in tendinopathy, although further work is required to determine whether it is mechanical overstimulation or understimulation that leads to the change in tenocyte activity. Metalloproteinase enzymes have an important role in the tendon matrix, being responsible for the degradation of collagen and proteoglycan in both healthy patients and those with disease. Metalloproteinases that show increased expression in painful tendinopathy include ADAM (a disintegrin and metalloproteinase)-12 and MMP (matrix metalloproteinase)-23. The role of these enzymes in tendon pathology is unknown, and further work is required to identify novel and specific molecular targets for therapy