Senescence evasion in melanoma progression: uncoupling of DNA‐damage signaling from p53 activation and p21 expression

The best‐established function of the melanoma‐suppressor p16 is mediation of cell senescence, a permanent arrest following cell proliferation or certain stresses. The importance of p16 in melanoma suggests indolence of the other major senescence pathway through p53. Little or no p53 is expressed in senescent normal human melanocytes, but p16‐deficient melanocytes can undergo p53‐mediated senescence. As p16 expression occurs in nevi but falls with progression toward melanoma, we here investigated whether p53‐dependent senescence occurs at some stage and, if not, what defects were detectable in this pathway, using immunohistochemistry. Phosphorylated checkpoint kinase 2 (CHEK2) can mediate DNA‐damage signaling, and under some conditions senescence, by phosphorylating and activating p53. Remarkably, we detected no prevalent p53‐mediated senescence in any of six classes of lesions. Two separate defects in p53 signaling appeared common: in nevi, lack of p53 phosphorylation by activated CHEK2, and in melanomas, defective p21 upregulation by p53 even when phosphorylated.     

LC-MS/MS Confirms That COX-1 Drives Vascular Prostacyclin Whilst Gene Expression Pattern Reveals Non-Vascular Sites of COX-2 Expression

There are two schools of thought regarding the cyclooxygenase (COX) isoform active in the vasculature. Using urinary prostacyclin markers some groups have proposed that vascular COX-2 drives prostacyclin release. In contrast, we and others have found that COX-1, not COX-2, is responsible for vascular prostacyclin production. Our experiments have relied on immunoassays to detect the prostacyclin breakdown product, 6-keto-PGF_1α and antibodies to detect COX-2 protein. Whilst these are standard approaches, used by many laboratories, antibody-based techniques are inherently indirect and have been criticized as limiting the conclusions that can be drawn. To address this question, we measured production of prostanoids, including 6-keto-PGF_1α, by isolated vessels and in the circulation in vivo using liquid chromatography tandem mass spectrometry and found values essentially identical to those obtained by immunoassay. In addition, we determined expression from the Cox2 gene using a knockin reporter mouse in which luciferase activity reflects Cox2 gene expression. Using this we confirm the aorta to be essentially devoid of Cox2 driven expression. In contrast, thymus, renal medulla, and regions of the brain and gut expressed substantial levels of luciferase activity, which correlated well with COX-2-dependent prostanoid production. These data are consistent with the conclusion that COX-1 drives vascular prostacyclin release and puts the sparse expression of Cox2 in the vasculature in the context of the rest of the body. In doing so, we have identified the thymus, gut, brain and other tissues as target organs for consideration in developing a new understanding of how COX-2 protects the cardiovascular system.     

Non‐native mangroves support carbon storage,sediment carbon burial,and accretion of coastal ecosystems

Mangrove forests play an important role in climate change adaptation and mitigation by maintaining coastline elevations relative to sea level rise, protecting coastal infrastructure from storm damage, and storing substantial quantities of carbon (C) in live and detrital pools. Determining the efficacy of mangroves in achieving climate goals can be complicated by difficulty in quantifying C inputs (i.e., differentiating newer inputs from younger trees from older residual C pools), and mitigation assessments rarely consider potential offsets to CO₂ storage by methane (CH₄) production in mangrove sediments. The establishment of non‐native Rhizophora mangle along Hawaiian coastlines over the last century offers an opportunity to examine the role mangroves play in climate mitigation and adaptation both globally and locally as novel ecosystems. We quantified total ecosystem C storage, sedimentation, accretion, sediment organic C burial and CH₄ emissions from ~70 year old R. mangle stands and adjacent uninvaded mudflats. Ecosystem C stocks of mangrove stands exceeded mudflats by 434 ± 33 Mg C/ha, and mangrove establishment increased average coastal accretion by 460%. Sediment organic C burial increased 10‐fold (to 4.5 Mg C ha^?1 year^?1), double the global mean for old growth mangrove forests, suggesting that C accumulation from younger trees may occur faster than previously thought, with implications for mangrove restoration. Simulations indicate that increased CH₄ emissions from sediments offset ecosystem CO₂ storage by only 2%–4%, equivalent to 30–60 Mg CO₂‐eq/ha over mangrove lifetime (100 year sustained global warming potential). Results highlight the importance of mangroves as novel systems that can rapidly accumulate C, have a net positive atmospheric greenhouse gas removal effect, and support shoreline accretion rates that outpace current sea level rise. Sequestration potential of novel mangrove forests should be taken into account when considering their removal or management, especially in the context of climate mitigation goals.     

Evaluating ecosystem effects of climate change on tropical island streams using high spatial and temporal resolution sampling regimes

Climate change is expected to alter precipitation patterns worldwide, which will affect streamflow in riverine ecosystems. It is vital to understand the impacts of projected flow variations, especially in tropical regions where the effects of climate change are expected to be one of the earliest to emerge. Space‐for‐time substitutions have been successful at predicting effects of climate change in terrestrial systems by using a spatial gradient to mimic the projected temporal change. However, concerns have been raised that the spatial variability in these models might not reflect the temporal variability. We utilized a well‐constrained rainfall gradient on Hawaii Island to determine (a) how predicted decreases in flow and increases in flow variability affect stream food resources and consumers and (b) if using a high temporal (monthly, four streams) or a high spatial (annual, eight streams) resolution sampling scheme would alter the results of a space‐for‐time substitution. Declines in benthic and suspended resource quantity (10‐ to 40‐fold) and quality (shift from macrophyte to leaf litter dominated) contributed to 35‐fold decreases in macroinvertebrate biomass with predicted changes in the magnitude and variability in the flow. Invertebrate composition switched from caddisflies and damselflies to taxa with faster turnover rates (mosquitoes, copepods). Changes in resource and consumer composition patterns were stronger with high temporal resolution sampling. However, trends and ranges of results did not differ between the two sampling regimes, indicating that a suitable, well‐constrained spatial gradient is an appropriate tool for examining temporal change. Our study is the first to investigate resource to community wide effects of climate change on tropical streams on a spatial and temporal scale. We determined that predicted flow alterations would decrease stream resource and consumer quantity and quality, which can alter stream function, as well as biomass and habitat for freshwater, marine, and terrestrial consumers dependent on these resources.     

W323S variant of Xiap-Bir3 binds to SMAC but not caspase-9

The ability of the wild-type XIAP BIR3 domain as well as its Trp323Ser variant in inhibition of human caspase-9, binding to AVPFVASLPN (SMAC-peptide), SMAC protein, and mature caspase-9 was investigated. In order to investigate the role of W323 on these interactions, this residue was mutated to Serine. Circular dichroism as well as thermal denaturation studies showed that W323S mutation did not hamper proper folding of the protein. The dissociation constants for the interaction of the wild type BIR3 as well as its mutant to Smac-type peptide were found to be 1.8 and 27 muM, respectively. The inhibition of and binding to caspase-9 by wild-type BIR3 and its mutant were also compared. While the wild-type protein potently inhibited the enzyme, the mutant failed to do so. The lack of caspase-9 inhibition was due to absence of interaction of the mutant BIR3 with mature caspase-9. These results indicate that Trp323 of BIR3 plays a pivotal role both in maintaining necessary conformation for caspase-9 interaction and to a lesser extent, recognition of Smac-type peptide. Moreover, decreased stability of the mutant compared with the wild type indicates that W323 is essential for maintaining the stability BIR3-Smac-peptide complex.     

Binding of plasminogen and tissue plasminogen activator to plasmin-modulated factor X and factor Xa

Previous work in our laboratory has suggested that the fibrinolytic enzyme plasmin (Pn) inactivates coagulation factors X (FX) and Xa (FXa) in the presence of Ca(2+) and anionic phospholipid (aPL), producing fragments which bind plasminogen (Pg) and accelerate tissue plasminogen activator (t-PA). Our goals here were to determine if the Pn-mediated fragments of FX or FXa remain associated, whether they directly bind t-PA, and to quantify their interaction with Pg. Binding to aPL, benzamidine-Sepharose, or the active-site inhibitor dansyl-Glu-Gly-Arg-chloromethyl ketone demonstrated that Pn cleavage yielded noncovalent heterodimers of a fragment containing the aPL-binding domain (FXgamma(47) or FXagamma(33)) and a 13-kDa fragment (FXgamma(13) or FXagamma(13)). Both ligand blotting and surface plasmon resonance (SPR) showed that Pn-cleaved FX and FXa bound t-PA directly when Pn-treatment was effected in the presence of aPL and Ca(2+). Using SPR, apparent K(d) values of 1-3 microM and 0.3-0.4 microM were measured directly and by competition for the FXgamma(47/13)-Pg and FXagamma(33/13)-Pg interactions, respectively. For the first time, Pg-binding to a receptor was shown to be Ca(2+) enhanced, although primarily mediated by C-terminal lysine residues. Mathematical modeling of kinetic data suggesting two Pg per FXgamma(47/13) or FXagamma(33/13) was consistent with our conclusion that each subunit of FXgamma(47/13) or FXagamma(33/13) contains a C-terminal lysine. Earlier X-ray structures show that these Lys residues are distal from each other and the membrane, supporting the model where each interacts with a separate Pg. t-PA acceleration by FXgamma(47/13) or FXagamma(33/13) may therefore involve simultaneous presentation of two substrate molecules.     

Clonal variation in phenotype and life span of human embryonic fibroblasts (MRC-5) transduced with the catalytic component of telomerase (hTERT).

Expression of telomerase (hTERT) in certain cell types has been shown to extend cellular life span without malignant transformation. We studied the phenotype of 26 telomerase-transduced fibroblast clones (TTFC) generated from a mass culture of hTERT retrovirally transduced MRC-5 cells. About two-thirds of the transduced clones senesced at the expected time or shortly thereafter, despite high levels of expression of telomerase and telomere length maintenance. The remaining one-third of the clones were "immortalized" (followed for over 200 cumulative population doublings). All clones maintained a nontransformed phenotype: contact inhibition, anchorage dependency, lack of tumor formation in nude mice, dose dependency to serum and growth factors, low expression of a matrix metalloproteinase associated with metastatic invasion (MMP-9) and high expression of its inhibitor TIMP-1, and no cytogenetic abnormalities by G-banding. In addition, fibroblast-specific biological parameters, such as colony size, production of collagenase, and response to MMC and gamma radiation were tightly regulated at the clonal and subclonal levels.