The function of BK channels extracted and purified within SMALPs.

Human BK channels are large voltage and Ca2+-activated K+ channels, involved in several important functions within the body. The core channel is a tetramer of α subunits, and its function is modulated by the presence of ß and γ accessory subunits. BK channels composed of α subunits, as well as BK channels composed of α and ß1 subunits, were successfully solubilised from HEK cells with styrene maleic acid (SMA) polymer and purified by nickel affinity chromatography. Native SMA-PAGE analysis of the purified proteins showed the α subunits were extracted as a tetramer. In the presence of ß1 subunits, they were co-extracted with the α subunits as a heteromeric complex. Purified SMA lipid particles (SMALPs) containing BK channel could be inserted into planar lipid bilayers (PLB) and single channel currents recorded, showing a high conductance (≈260 pS), as expected. The open probability was increased in the presence of co-purified ß1 subunits. However, voltage-dependent gating of the channel was restricted. In conclusion, we have demonstrated that SMA can be used to effectively extract and purify large, complex, human ion channels, from low expressing sources. That these large channels can be incorporated into PLB from SMALPs and display voltage-dependent channel activity. However, the SMA appears to reduce the voltage dependent gating of the channels.

Glucocorticoids induce production of reactive oxygen species/reactive nitrogen species and DNA damage through an iNOS mediated pathway in breast cancer.

BACKGROUND: Psychological stress increases the circulating levels of the stress hormones cortisol and norepinephrine (NE). Chronic exposure to elevated stress hormones has been linked to a reduced response to chemotherapy through induction of DNA damage. We hypothesize that stress hormone signalling may induce DNA damage through the production of reactive oxygen species (ROS)/reactive nitrogen species (RNS) and interference in DNA repair processes, promoting tumourigenesis. METHODS: Breast cancer cell lines were incubated with physiological levels of cortisol and NE in the presence and absence of receptor antagonists and inducible nitric oxide synthase (iNOS) inhibitors and DNA damage measured using phosphorylated γ-H2AX. The rate of DNA repair was measured using comet assays and electrochemical sensors were used to detect ROS/RNS in the cell lysates from cells exposed to stress hormones. A syngeneic mouse model was used to assess the presence of iNOS in mammary tumours in stressed versus control animals and expression of iNOS was examined using western blotting and qRT-PCR. RESULTS: Acute exposure to cortisol and NE significantly increased levels of ROS/RNS and DNA damage and this effect was diminished in the presence of receptor antagonists. Cortisol induced DNA damage and the production of RNS was further attenuated in the presence of an iNOS inhibitor. An increase in the expression of iNOS in response to psychological stress was observed in vivo and in cortisol-treated cells. Inhibition of glucocorticoid receptor-associated Src kinase also produced a decrease in cortisol-induced RNS. CONCLUSION: These results demonstrate that glucocorticoids may interact with iNOS in a non-genomic manner to produce damaging levels of RNS, thus allowing an insight into the potential mechanisms by which psychological stress may impact breast cancer.

Glucocorticoid induced loss of oestrogen receptor alpha gene methylation and restoration of sensitivity to fulvestrant in triple negative breast cancer.

The response to psychological stress can differ depending on the type and duration of the stressor. Acute stress can facilitate a "fight or flight response" and aid survival, whereas chronic long-term stress with the persistent release of stress hormones such as cortisol has been shown to be detrimental to health. We are now beginning to understand how this stress hormone response impacts important processes such as DNA repair and cell proliferation processes in breast cancer. However, it is not known what epigenetic changes stress hormones induce in breast cancer. Epigenetic mechanisms include modification of DNA and histones within chromatin that may be involved in governing the transcriptional processes in cancer cells in response to changes by endogenous stress hormones. The contribution of endogenous acute or long-term exposure of glucocorticoid stress hormones, and exogenous glucocorticoids to methylation patterns in breast cancer tissues with different aetiologies remains to be evaluated. In vitro and in vivo models were developed to investigate the epigenetic modifications and their contribution to breast cancer progression and aetiology. A panel of triple negative breast cancer cell lines were treated with the glucocorticoid, cortisol which resulted in epigenetic alteration characterised by loss of methylation on promoter regions of tumour suppressor genes including ESR1, and loss of methylation on LINE-1 repetitive element used as a surrogate marker for global methylation. This was verified in vivo in MDA-MB-231 xenografts; the model verified the loss of methylation on ESR1 promoter, and subsequent increase in ESR1 expression in primary tumours in mice subjected to restraint stress. Our study highlights that DNA methylation landscape in breast cancer can be altered in response to stress and glucocorticoid treatment.

Stress hormone-mediated acceleration of breast cancer metastasis is halted by inhibition of nitric oxide synthase.

Stress hormones have been shown to be important mediators in driving malignant growth and reducing treatment efficacy in breast cancer. Glucocorticoids can induce DNA damage through an inducible nitric oxide synthase (iNOS) mediated pathway to increase levels of nitric oxide (NO). Using an immune competent mouse breast cancer model and 66CL4 breast cancer cells we identified a novel role of NOS inhibition to reduce stress-induced breast cancer metastasis. On a mechanistic level we show that the glucocorticoid cortisol induces expression of keys genes associated with angiogenesis, as well as pro-tumourigenic immunomodulation. Transcriptomics analysis confirmed that in the lungs of tumour-bearing mice, stress significantly enriched pathways associated with tumourigenesis, some of which could be regulated with NOS inhibition. These results demonstrate the detrimental involvement of NOS in stress hormone signalling, and the potential future benefits of NOS inhibition in highly stressed patients.

Artificial transmembrane ion channels from commercial surfactants.

Sodium ion transport across a phospholipid bilayer has been demonstrated by a new class of transmembrane ion channel mimetic compounds in which the filtering effect of a calixarene has been coupled to the membrane penetrating qualities of a commercial surfactant.

Bridging the gap: ageing, pharmacokinetics and pharmacodynamics.

Changes in pharmacokinetics and pharmacodynamics in elderly patients generally result in an increase in the incidence of drug toxicity and adverse drug reactions. Molecular alterations associated with ageing could bring about biological changes, a consequence of which is an altered response to pharmacological agents. Unfortunately, research in this area has yet to progress beyond the cataloguing of the pharmacokinetic and pharmacodynamic changes observed in the elderly. Therefore, real progress in our understanding of pharmacogerontology could be achieved if it were possible to merge pharmacokinetic and pharmacodynamic studies with recent advances in our understanding of the causal processes bringing about ageing changes at the cellular level. Therefore, this review will focus on the mechanisms of ageing in the hope that the information will be of value to those planning independent studies.

Characterization of cellular senescence mechanisms in human corneal endothelial cells.

The human cornea is a tri-laminar structure composed of several cell types with substantial mitotic potential. Age-related changes in the cornea are associated with declining visual acuity and the onset of overt age-related corneal diseases. Corneal transplantation is commonly used to restore vision in patients with damaged or diseased corneas. However, the supply of donor tissue is limited, and thus there is considerable interest in the development of tissue-engineered alternatives. A major obstacle to these approaches is the short replicative lifespan of primary human corneal endothelial cells (HCEC). Accordingly, a comprehensive investigation of the signalling pathways and mechanisms underpinning proliferative lifespan and senescence in HCEC was undertaken. The effects of exogenous human telomerase reverse transcriptase expression, p53 knockdown, disruption of the pRb pathway by over-expression of CDK4 and reduced oxygen concentration on the lifespan of primary HCEC were evaluated. We provide proof-of-principle that forced expression of telomerase, when combined with either p53 knockdown or CDK4 over-expression, is sufficient to produce immortalized HCEC lines. The resultant cell lines express an HCEC-specific transcriptional fingerprint, and retain expression of the corneal endothelial temperature-sensitive potassium channel, suggesting that significant dedifferentiation does not occur as a result of these modes of immortalization. Exploiting these insights into proliferative lifespan barriers in HCEC will underpin the development of novel strategies for cell-based therapies in the human cornea.