The Evaluation and Quantitation of Dihydrogen Metabolism Using Deuterium Isotope in Rats

Purpose: Despite the significant interest in molecular hydrogen as an antioxidant in the last eight years, its quantitative metabolic parameters in vivo are still lacking, as is an appropriate method for determination of hydrogen effectivity in the mammalian organism under various conditions.

Basic Procedures: Intraperitoneally-applied deuterium gas was used as a metabolic tracer and deuterium enrichment was determined in the body water pool. Also, in vitro experiments were performed using bovine heart submitochondrial particles to evaluate superoxide formation in Complex I of the respiratory chain.

Main Findings: A significant oxidation of about 10% of the applied dose was found under physiological conditions in rats, proving its antioxidant properties. Hypoxia or endotoxin application did not exert any effect, whilst pure oxygen inhalation reduced deuterium oxidation. During in vitro experiments, a significant reduction of superoxide formation by Complex I of the respiratory chain was found under the influence of hydrogen. The possible molecular mechanisms of the beneficial effects of hydrogen are discussed, with an emphasis on the role of iron sulphur clusters in reactive oxygen species generation and on iron species-dihydrogen interaction.

Principal Conclusions: According to our findings, hydrogen may be an efficient, non-toxic, highly bioavailable and low-cost antioxidant supplement for patients with pathological conditions involving ROS-induced oxidative stress.

In situ catalyst activity control in a novel membrane reactor-Reaction driven wireless electrochemical promotion of catalysis

A dual chamber membrane reactor was used in order to study the effect of macroscopically applied oxygen chemical potential differences to a platinum catalyst supported on a mixed oxygen ion and electronic conducting membrane. It is believed that the oxygen chemical potential difference imposed by the use of an oxygen sweep in one of the reactor chambers causes the back-spillover of oxygen species from the support onto the catalyst surface, resulting in the modification of the catalytic activity. The use of different sweep gases, such as ethylene and hydrogen was investigated as the means to reverse the rate modification by removing the spilt over species from the catalyst surface and returning the system to its initial state. Oxygen sweep in general had a positive effect on the reaction rate with rate increases up to 20% measured. Experimental results showed that hydrogen is a more potent sweep gas than ethylene in terms of the ability to reverse rate modification. A 10% rate loss was observed when using an ethylene sweep as compared with an almost 60% rate decrease when hydrogen was used as the sweep gas. © 2009 Elsevier Ltd. All rights reserved.

In situ modification of a Pt catalyst supported on a mixed ionic-electronic conducting membrane

The electrochemical promotion of a platinum catalyst for ethylene oxidation on a dual chamber membrane reactor was studied. The catalyst was supported on a La_0.6Sr_0.4Co_0.2Fe_0.803 membrane. Due the supporting membrane's electronic conductivity it is possible to promote the reaction by controlling the oxygen chemical potential difference across the membrane. Upon establishment of an oxygen potential difference across the membrane, oxygen species can migrate and spillover onto the catalyst surface, modifying the catalytic activity. Initial experiments showed an overall promotion of approximately one order of magnitude of the reaction rate of ethylene, under an oxygen atmosphere on the sweep side of the membrane reactor, as compared with the rate under an inert sweep gas. The reaction rate can keep its promoted state even after the flow of oxygen on the sweep side was interrupted. This behavior caused further promotion with every experiment cycle. The causes of permanent promotion and on demonstrating controllable promotion of the catalytic activity are presented. This is an abstract of a paper presented at the AIChE Annual Meeting (San Francisco, CA 11/12-17/2006).

Hypoxia in prostate cancer:a powerful shield against tumour destruction?

Tumour hypoxia is progressively emerging as a common feature of prostate tumours associated with poor prognosis. While the molecular basis of disease progression is increasingly well documented, the potential role of hypoxia in these processes remains poorly evaluated. By dissecting the impact of hypoxia-inducible factor 1 alpha on molecular responses, this review provides evidence for a powerful protecting role of oxygen deprivation against oxidative stress injury, androgen deprivation, chemotherapeutic and radiation cytotoxicity. We propose hypoxia as a potent tumour-induced shield against destruction and suggest its targeting may need to be routinely addressed in the management of prostate cancer.

Pyrrolo-1,5-benzoxazepines induce apoptosis in chronic myelogenous leukemia (CML) cells by bypassing the apoptotic suppressor bcr-abl

Expression of the transforming oncogene bcr-abl in chronic myelogenous leukemia (CML) cells is reported to confer resistance against apoptosis induced by many chemotherapeutic agents such as etoposide, ara-C, and staurosporine. In the present study some members of a series of novel pyrrolo-1,5-benzoxazepines potently induce apoptosis, as shown by cell shrinkage, chromatin condensation, DNA fragmentation, and poly(ADP-ribose) polymerase (PARP) cleavage, in three CML cell lines, K562, KYO.1, and LAMA 84. Induction of apoptosis by a representative member of this series, PBOX-6, was not accompanied by either the down-regulation of Bcr-Abl or by the attenuation of its protein tyrosine kinase activity up to 24 h after treatment, when approximately 50% of the cells had undergone apoptosis. These results suggest that down-regulation of Bcr-Abl is not part of the upstream apoptotic death program activated by PBOX-6. By characterizing the mechanism in which this novel agent executes apoptosis, this study has revealed that PBOX-6 caused activation of caspase 3-like proteases in only two of the three CML cell lines. In addition, inhibition of caspase 3-like protease activity using the inhibitor z-DEVD-fmk blocked caspase 3-like protease activity but did not prevent the induction of apoptosis, suggesting that caspase 3-like proteases are not essential in the mechanism by which PBOX-6 induces apoptosis in CML cells. In conclusion, this study demonstrates that PBOX-6 can bypass Bcr-Abl-mediated suppression of apoptosis, suggesting an important potential use of these compounds in the treatment of CML.

Thiol isomerases negatively regulate the cellular shedding activity of ADAM17

ADAM17 (where ADAM is 'a disintegrin and metalloproteinase') can rapidly modulate cell-surface signalling events by the proteolytic release of soluble forms of proligands for cellular receptors. Many regulatory pathways affect the ADAM17 sheddase activity, but the mechanisms for the activation are still not clear. We have utilized a cell-based ADAM17 assay to show that thiol isomerases, specifically PDI (protein disulfide isomerase), could be responsible for maintaining ADAM17 in an inactive form. Down-regulation of thiol isomerases, by changes in the redox environment (for instance as elicited by phorbol ester modulation of mitochondrial reactive oxygen species) markedly enhanced ADAM17 activation. On the basis of ELISA binding studies with novel fragment antibodies against ADAM17 we propose that isomerization of the disulfide bonds in ADAM17, and the subsequent conformational changes, form the basis for the modulation of ADAM17 activity. The shuffling of disulfide bond patterns in ADAMs has been suggested by a number of recent adamalysin crystal structures, with distinct disulfide bond patterns altering the relative orientations of the domains. Such a mechanism is rapid and reversible, and the role of thiol isomerases should be investigated further as a potential factor in the redox regulation of ADAM17.

Protective role of glycerol against benzene stress: insights from the Pseudomonas putida proteome

Chemical activities of hydrophobic substances can determine the windows of environmental conditions over which microbial systems function and the metabolic inhibition of microorganisms by benzene and other hydrophobes can, paradoxically, be reduced by compounds that protect against cellular water stress (Bhaganna et al. in Microb Biotechnol 3:701-716, 2010; Cray et al. in Curr Opin Biotechnol 33:228-259, 2015a). We hypothesized that this protective effect operates at the macromolecule structure-function level and is facilitated, in part at least, by genome-mediated adaptations. Based on proteome profiling of the soil bacterium Pseudomonas putida, we present evidence that (1) benzene induces a chaotrope-stress response, whereas (2) cells cultured in media supplemented with benzene plus glycerol were protected against chaotrope stress. Chaotrope-stress response proteins, such as those involved in lipid and compatible-solute metabolism and removal of reactive oxygen species, were increased by up to 15-fold in benzene-stressed cells relative to those of control cultures (no benzene added). By contrast, cells grown in the presence of benzene + glycerol, even though the latter grew more slowly, exhibited only a weak chaotrope-stress response. These findings provide evidence to support the hypothesis that hydrophobic substances induce a chaotropicity-mediated water stress, that cells respond via genome-mediated adaptations, and that glycerol protects the cell's macromolecular systems. We discuss the possibility of using compatible solutes to mitigate hydrocarbon-induced stresses in lignocellulosic biofuel fermentations and for industrial and environmental applications.

EFFECTS OF ENVIRONMENTAL ANTIOXIDANTS ON PRO-ANGIOGENIC ROS SIGNALLING IN ENDOTHELIAL COLONY FORMING CELLS IN VITRO

Introduction. Endothelial colony-forming cells (ECFCs) hold great cytotherapeutic potential for ischaemic disease. Whilst increasing evidence supports a key role for reactive oxygen species (ROS), specifically those derived from Nox NADPH oxidases, in the underlying angiogenic processes of these and other endothelial cells, such studies investigating the role of redox signalling may be hampered by the standard inclusion of antioxidant agents in endothelial cell media, such as phenol red. Aims. To study the effects of antioxidants present in culture media on pro-angiogenic function of ECFCs in vitro. Methods. Human ECFCs isolated from umbilical cord blood were maintained in media with and without antioxidant components (EGM2 and phenol red-free DMEM, respectively) prior to treatment with pro-oxidant PMA and assessment of their in vitro migratory capacity using a scratch-wound assay to measure pro-angiogenic activity. Results. Our previous work in our group indicated that PMA (500nM) increased ECFC migration in a both a superoxide and NADPH oxidase-dependent manner (control 18.6±2.8, PMA 32.7±6.6% wound closure; n=6, P<0.05), as indicated by attenuation with PEG-SOD and VAS2870. However, inconsistencies in the data generated under varying experimental conditions led us to hypothesise that antioxidant agents in the standard ECFC media may be influencing these effects. Indeed, a direct comparison of cell migration between ECFCs incubated in EGM2 DMEM demonstrated a clear trend towards higher migration in the latter (EGM2 9.0±4.5, DMEM 22.7±6.4%; n=3, P=NS). Similar to our previous EGM2 studies, cell migration was potentiated by PMA (control 11.6±1.6, PMA 25.1±2.8%; n=3, P<0.05), but at a lower dose (100nM), which is consistent with a reduction in media antioxidants. Notably, this response was attenuated by VAS2870 (PMA 37.6±7.3, PMA+VAS2870 10.3±2.9%; n=6, P<0.05), underlining a likely role for Nox NADPH oxidases. Conclusion. Taken together, these data indicate that ECFC migration is sensitive to different endothelial cell growth media, which appears to be dependent upon their antioxidant content. Although further experiments, such as quantification of cellular superoxide generation by dihydroethidium fluorescence may be required to confirm a specific role for antioxidants, such blunting of ROS signalling in vitro is clearly an important consideration which may significantly impact upon data interpretation.

Retinal pigment epithelial cell multinucleation in the aging eye - a mechanism to repair damage and maintain homoeostasis

Retinal pigment epithelial (RPE) cells are central to retinal health and homoeostasis. Dysfunction or death of RPE cells underlies many age-related retinal degenerative disorders particularly age-related macular degeneration. During aging RPE cells decline in number, suggesting an age-dependent cell loss. RPE cells are considered to be postmitotic, and how they repair damage during aging remains poorly defined. We show that RPE cells increase in size and become multinucleate during aging in C57BL/6J mice. Multinucleation appeared not to be due to cell fusion, but to incomplete cell division, that is failure of cytokinesis. Interestingly, the phagocytic activity of multinucleate RPE cells was not different from that of mononuclear RPE cells. Furthermore, exposure of RPE cells in vitro to photoreceptor outer segment (POS), particularly oxidized POS, dose-dependently promoted multinucleation and suppressed cell proliferation. Both failure of cytokinesis and suppression of proliferation required contact with POS. Exposure to POS also induced reactive oxygen species and DNA oxidation in RPE cells. We propose that RPE cells have the potential to proliferate in vivo and to repair defects in the monolayer. We further propose that the conventionally accepted 'postmitotic' status of RPE cells is due to a modified form of contact inhibition mediated by POS and that RPE cells are released from this state when contact with POS is lost. This is seen in long-standing rhegmatogenous retinal detachment as overtly proliferating RPE cells (proliferative vitreoretinopathy) and more subtly as multinucleation during normal aging. Age-related oxidative stress may promote failure of cytokinesis and multinucleation in RPE cells.

A BRCA1 deficient, NFκB driven immune signal predicts good outcome in triple negative breast cancer

Triple negative (TNBCs) and the closely related Basal-like (BLBCs) breast cancers are a loosely defined collection of cancers with poor clinical outcomes. Both show strong similarities with BRCA1-mutant breast cancers and BRCA1 dysfunction, or 'BRCAness', is observed in a large proportion of sporadic BLBCs. BRCA1 expression and function has been shown in vitro to modulate responses to radiation and chemotherapy. Exploitation of this knowledge in the treatment of BRCA1-mutant patients has had varying degrees of success. This reflects the significant problem of accurately detecting those patients with BRCA1 dysfunction. Moreover, not all BRCA1 mutations/loss of function result in the same histology/pathology or indeed have similar effects in modulating therapeutic responses. Given the poor clinical outcomes and lack of targeted therapy for these subtypes, a better understanding of the biology underlying these diseases is required in order to develop novel therapeutic strategies.We have discovered a consistent NFκB hyperactivity associated with BRCA1 dysfunction as a consequence of increased Reactive Oxygen Species (ROS). This biology is found in a subset of BRCA1-mutant and triple negative breast cancer cases and confers good outcome. The increased NFκB signalling results in an anti-tumour microenvironment which may allow CD8+ cytotoxic T cells to suppress tumour progression. However, tumours lacking this NFκB-driven biology have a more tumour-promoting environment and so are associated with poorer prognosis. Tumour-derived gene expression data and cell line models imply that these tumours may benefit from alternative treatment strategies such as reprogramming the microenvironment and targeting the IGF and AR signalling pathways.

Beneficial Effects of Berberine on Oxidized LDL-induced Cytotoxicity in Human Retinal Müller Cells

PURPOSE. Limited mechanistic understanding of diabetic retinopathy (DR) has hindered therapeutic advances. Berberine, an isoquinolone alkaloid, has shown favorable effects on glucose and lipid metabolism in animal and human studies, but effects on DR are unknown. We previously demonstrated intraretinal extravasation and modification of LDL in human diabetes, and toxicity of modified LDL to human retinal M¨uller cells. We now explore pathogenic effects of modified LDL on M¨uller cells, and the efficacy of berberine in mitigating this cytotoxicity. METHODS. Confluent human M¨uller cells were exposed to in vitro–modified ‘highly oxidized, glycated (HOG-) LDL versus native-LDL (N-LDL; 200 mg protein/L) for 6 or 24 hours, with/ without pretreatment with berberine (5 lM, 1 hour) and/or the adenosine monophosphate (AMP)-activated protein kinase (AMPK) inhibitor, Compound C (5 lM, 1 hour). Using techniques including Western blots, reactive oxygen species (ROS) detection assay, and quantitative real-time PCR, the following outcomes were assessed: cell viability (CCK-8 assay), autophagy (LC3, Beclin-1, ATG-5), apoptosis (cleaved caspase 3, cleaved poly-ADP ribose polymerase), oxidative stress (ROS, nuclear factor erythroid 2-related factor 2, glutathione peroxidase 1, NADPH oxidase 4), angiogenesis (VEGF, pigment epithelium-derived factor), inflammation (inducible nitric oxide synthase, intercellular adhesion molecule 1, IL-6, IL-8, TNF-a), and glial cell activation (glial fibrillary acidic protein). RESULTS. Native-LDL had no effect on cultured human M¨uller cells, but HOG-LDL exhibited marked toxicity, significantly decreasing viability and inducing autophagy, apoptosis, oxidative stress, expression of angiogenic factors, inflammation, and glial cell activation. Berberine attenuated all the effects of HOG-LDL (all P < 0.05), and its effects were mitigated by AMPK inhibition (P < 0.05). CONCLUSIONS. Berberine inhibits modified LDL-induced M¨uller cell injury by activating the AMPK pathway, and merits further study as an agent for preventing and/or treating DR.