Mast Cell Survival and Mediator Secretion in Response to Hypoxia

Tissue hypoxia is a consequence of decreased oxygen levels in different inflammatory conditions, many associated with mast cell activation. However, the effect of hypoxia on mast cell functions is not well established. Here, we have investigated the effect of hypoxia per se on human mast cell survival, mediator secretion, and reactivity. Human cord blood derived mast cells were subjected to three different culturing conditions: culture and stimulation in normoxia (21% O2); culture and stimulation in hypoxia (1% O2); or 24 hour culture in hypoxia followed by stimulation in normoxia. Hypoxia, per se, did not induce mast cell degranulation, but we observed an increased secretion of IL-6, where autocrine produced IL-6 promoted mast cell survival. Hypoxia did not have any effect on A23187 induced degranulation or secretion of cytokines. In contrast, cytokine secretion after LPS or CD30 treatment was attenuated, but not inhibited, in hypoxia compared to normoxia. Our data suggests that mast cell survival, degranulation and cytokine release are sustained under hypoxia. This may be of importance for host defence where mast cells in a hypoxic tissue can react to intruders, but also in chronic inflammations where mast cell reactivity is not inhibited by the inflammatory associated hypoxia

Nuclear Entry of Activated MAPK Is Restricted in Primary Ovarian and Mammary Epithelial Cells

The MAPK/ERK1/2 serine kinases are primary mediators of the Ras mitogenic signaling pathway. Phosphorylation by MEK activates MAPK/ERK in the cytoplasm, and phospho-ERK is thought to enter the nucleus readily to modulate transcription.Here, however, we observe that in primary cultures of breast and ovarian epithelial cells, phosphorylation and activation of ERK1/2 are disassociated from nuclear translocalization and transcription of downstream targets, such as c-Fos, suggesting that nuclear translocation is limited in primary cells. Accordingly, in import assays in vitro, primary cells showed a lower import activity for ERK1/2 than cancer cells, in which activated MAPK readily translocated into the nucleus and activated c-Fos expression. Primary cells express lower levels of nuclear pore complex proteins and the nuclear transport factors, importin B1 and importin 7, which may explain the limiting ERK1/2 import found in primary cells. Additionally, reduction in expression of nucleoporin 153 by siRNA targeting reduced ERK1/2 nuclear activity in cancer cells.ERK1/2 activation is dissociated from nuclear entry, which is a rate limiting step in primary cells and in vivo, and the restriction of nuclear entry is disrupted in transformed cells by the increased expression of nuclear pores and/or nuclear transport factors

Ifuzzer: An evolutionary interpreter fuzzer using genetic programming

We present an automated evolutionary fuzzing technique to find bugs in JavaScript interpreters. Fuzzing is an automated black box testing technique used for finding security vulnerabilities in the software by providing random data as input. However, in the case of an interpreter, fuzzing is challenging because the inputs are piece of codes that should be syntactically/semantically valid to pass the interpreter’s elementary checks. On the other hand, the fuzzed input should also be uncommon enough to trigger exceptional behavior in the interpreter, such as crashes, memory leaks and failing assertions. In our approach, we use evolutionary computing techniques, specifically genetic programming, to guide the fuzzer in generating uncommon input code fragments that may trigger exceptional behavior in the interpreter. We implement a prototype named IFuzzer to evaluate our technique on real-world examples. IFuzzer uses the language grammar to generate valid inputs. We applied IFuzzer first on an older version of the JavaScript interpreter of Mozilla (to allow for a fair comparison to existing work) and found 40 bugs, of which 12 were exploitable. On subsequently targeting the latest builds of the interpreter, IFuzzer found 17 bugs, of which four were security bugs

Provasopressin expression by breast cancer cells: implications for growth and novel treatment strategies

The arginine vasopressin (AVP) gene is expressed in certain cancers such as breast cancer, where it is believed to act as an autocrine growth factor. However, little is known about the regulation of the AVP protein precursor (proAVP) or AVP-mediated signaling in breast cancer and this study was undertaken to address some of the basic issues. The cultured cell lines examined (Mcf7, Skbr3, BT474, ZR75, Mcf10a) and human breast cancer tissue extract were found to express proAVP mRNA. Western analysis revealed multiple forms of proAVP protein were present in cell lysates, corresponding to those detected in human hypothalamus extracts. Monoclonal antibodies directed against different regions of proAVP bound to intact live Mcf7 and Skbr3 cells. Dexamethasone increased the amount of proAVP-associated glycopeptide (VAG) secreted by Skbr3 cells and a combination of dexamethasone, IBMX and 8br-cAMP increased cellular levels of VAG. Exogenous AVP (1, 10, and 100 nM) elevated phospho-ERK1/2 levels, and increased cell proliferation was observed in the presence of 10 nM AVP. Concurrent treatment with the V1a receptor antagonist SR49059 reduced the effects of AVP on proliferation in Mcf7 cells, and abolished it in Skbr3 cells. Results here show that proAVP components are found at the surface of Skbr3 and Mcf7 cells and are also secreted from these cells. In addition, they show that AVP promotes cancer cell growth, apparently through a V1-type receptor-mediated pathway and subsequent ERK1/2 activation. Thus, strategies for targeting proAVP should be examined for their effectiveness in diagnosing and treating breast cancer

Nitrones for understanding and ameliorating the oxidative stress associated with aging

Oxidative damage from reactive oxygen species (ROS) and the carbon-centred radicals arising from them is important to the process of aging, and age-related diseases are generally caused, exacerbated or mediated by oxidative stress. Nitrones can act as spin traps to detect, identify, quantify and locate the radicals responsible using electron paramagnetic resonance (EPR or ESR) spectroscopy, and a new carnitine-derived nitrone, CarnDOD-7C, designed to accumulate in mitochondria is reported. Nitrones also have potential as therapeutic antioxidants, e.g. for slowing cellular aging, and as tools for chemical biology. Two low-molecular weight nitrones, DIPEGN-2 and DIPEGN-3, are reported, which combine high water-solubility with high lipophilicity and obey Lipinski's rule of five

Evaluation of the fullerene compound DF-1 as a radiation protector

<p>Abstract</p> <p>Background</p> <p>Fullerene compounds are known to possess antioxidant properties, a common property of chemical radioprotectors. DF-1 is a dendrofullerene nanoparticle with antioxidant properties previously found to be radioprotective in a zebrafish model. The purpose of this study was to evaluate the radioprotective effects of DF-1 in a murine model of lethal total body irradiation and to assess for selective radioprotection of normal cells versus tumor cells.</p> <p>Methods</p> <p><it>In vitro </it>radioresponse was evaluated with clonogenic assays with human tumor cells and fibroblast lines in the presence of varying concentrations of DF-1 or vehicle. DNA double strand break induction and repair was evaluated with immunocytochemistry for γH2AX. Lethal total body irradiation was delivered with 137Cs after intraperitoneal delivery of DF-1 or vehicle control. Bone marrow hypoxia was evaluated with piminidazole uptake assessed by flow cytometry.</p> <p>Results</p> <p>DF-1 provided modest radioprotection of human cancer cell lines and fibroblast cell lines when delivered prior to irradiation (dose modifying factor or 1.1). There was no evidence of selective protection of fibroblasts versus tumor cells. Cells treated with DF-1 at radioprotective doses were found to have fewer γH2AX foci at 1 and 6 hours after irradiation compared to vehicle treated controls. The LD50/30 for C57Bl6/Ncr mice treated with a single 300 mg/kg dose of DF-1 pre-irradiation was 10.09 Gy (95% CI 9.58-10.26) versus 8.29 Gy (95% CI, 8.21-8.32) for control mice. No protective effects were seen with a single 200 mg/kg dose. No increase in pimonidazole uptake was appreciated in bone marrow of mice treated with DF-1 compared to vehicle controls.</p> <p>Conclusions</p> <p>DF-1 has modest activity as a radiation protector <it>in vivo</it>. There was no evidence of selective protection from irradiation of normal versus tumor cells with DF-1.</p