Accumulation of lipophilic dications by mitochondria and cells

Lipophilic monocations can pass through phospholipid bilayers and accumulate in negatively-charged compartments such as the mitochondrial matrix, driven by the membrane potential. This property is used to visualize mitochondria, to deliver therapeutic molecules to mitochondria and to measure the membrane potential. In theory, lipophilic dications have a number of advantages over monocations for these tasks, as the double charge should lead to a far greater and more selective uptake by mitochondria, increasing their therapeutic potential. However, the double charge might also limit the movement of lipophilic dications through phospholipid bilayers and little is known about their interaction with mitochondria. To see whether lipophilic dications could be taken up by mitochondria and cells, we made a series of bistriphenylphosphonium cations comprising two triphenylphosphonium moieties linked by a 2-, 4-, 5-, 6- or 10-carbon methylene bridge. The 5-, 6- and 10-carbon dications were taken up by energized mitochondria, whereas the 2- and 4-carbon dications were not. The accumulation of the dication was greater than that of the monocation methyltriphenylphosphonium. However, the uptake of dications was only described by the Nernst equation at low levels of accumulation, and beyond a threshold membrane potential of 90-100 mV there was negligible increase in dication uptake. Interestingly, the 5- and 6-carbon dications were not accumulated by cells, due to lack of permeation through the plasma membrane. These findings indicate that conjugating compounds to dications offers only a minor increase over monocations in delivery to mitochondria. Instead, this suggests that it may be possible to form dications within mitochondria that then remain within the cell.     

Pest problems: The view of Nga Whenua Rahui

Abstract

Nga Whenua Rahui began in the early 1980s, when Maori owners of commercially unproductive lands in Waikare (Northland) were being pressured into felling their native forests or selling property in order to meet their management costs, such as rates, fencing, and control of weeds and pests. The original concept recommended by the then Director of Maori Affairs, Tom Parore, was that a “rental” be paid by the Crown to Maori land owners as an incentive to keep their last remnants of indigenous forest alive and available for public enjoyment, and to cover the associated management costs.     

Bronchial epithelial cells are rendered insensitive to glucocorticoid transactivation by transforming growth factor-β1

Background

We have previously shown that transforming growth factor-beta (TGF-beta) impairs glucocorticoid (GC) function in pulmonary epithelial cell-lines. However, the signalling cascade leading to this impairment is unknown. In the present study, we provide the first evidence that TGF-beta impairs GC action in differentiated primary air-liquid interface (ALI) human bronchial epithelial cells (HBECs). Using the BEAS-2B bronchial epithelial cell line, we also present a systematic examination of the known pathways activated by TGF-beta, in order to ascertain the molecular mechanism through which TGF-beta impairs epithelial GC action.

Methods

GC transactivation was measured using a Glucocorticoid Response Element (GRE)–Secreted embryonic alkaline phosphatase (SEAP) reporter and measuring GC-inducible gene expression by qRT-PCR. GC transrepression was measured by examining GC regulation of pro-inflammatory mediators. TGF-beta signalling pathways were investigated using siRNA and small molecule kinase inhibitors. GRα level, phosphorylation and sub-cellular localisation were determined by western blotting, immunocytochemistry and localisation of GRα–Yellow Fluorescent Protein (YFP). Data are presented as the mean ± SEM for n independent experiments in cell lines, or for experiments on primary HBEC cells from n individual donors. All data were statistically analysed using GraphPad Prism 5.0 (Graphpad, San Diego, CA). In most cases, two-way analyses of variance (ANOVA) with Bonferroni post-hoc tests were used to analyse the data. In all cases, P <0.05 was considered to be statistically significant.

Results

TGF-beta impaired Glucocorticoid Response Element (GRE) activation and the GC induction of several anti-inflammatory genes, but did not broadly impair the regulation of pro-inflammatory gene expression in A549 and BEAS-2B cell lines. TGF-beta-impairment of GC transactivation was also observed in differentiated primary HBECs. The TGF-beta receptor (ALK5) inhibitor SB431541 fully prevented the GC transactivation impairment in the BEAS-2B cell line. However, neither inhibitors of the known downstream non-canonical signalling pathways, nor knocking down Smad4 by siRNA prevented the TGF-beta impairment of GC activity.

Conclusions

Our results indicate that TGF-beta profoundly impairs GC transactivation in bronchial epithelial cells through activating ALK5, but not through known non-canonical pathways, nor through Smad4-dependent signalling, suggesting that TGF-beta may impair GC action through a novel non-canonical signalling mechanism.     

Localisation and characterisation of cell wall mannan polysaccharides in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

Polysaccharides containing -1,4-mannosyl residues (mannans) are abundant in the lignified secondary cell walls of gymnosperms, and are also found as major seed storage polysaccharides in some plants, such as legume species. Although they have been found in a variety of angiosperm tissues, little is known about their presence and tissue localisation in the model angiosperm, Arabidopsis thaliana (L.) Heynh. In this study, antibodies that specifically recognised mannans in competitive ELISA experiments were raised in rabbits. Using these antibodies, we showed that Golgi-rich vesicles derived from Arabidopsis callus were able to synthesise mannan polysaccharides in vitro. Immunofluorescence light microscopy and immunogold electron microscopy of Arabidopsis inflorescence stem sections revealed that the mannan polysaccharide epitopes were localised in the thickened secondary cell walls of xylem elements, xylem parenchyma and interfascicular fibres. Similarly, mannan epitopes were present in the xylem of the leaf vascular bundles. Surprisingly, the thickened epidermal cell walls of both leaves and stems also contained abundant mannan epitopes. Low levels were observed in most other cell types examined. Thus, mannans are widespread in Arabidopsis tissues, and may be of particular significance in both lignified and non-lignified thickened cell walls. Polysaccharide analysis using carbohydrate gel electrophoresis (PACE) of cell wall preparations digested with a specific mannanase showed that there is glucomannan in inflorescence stems. The findings show that Arabidopsis can be used as a model plant in studies of the synthesis and functions of mannans.Abbreviations BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - PACE polysaccharide analysis by carbohydrate gel electrophoresis     

Sources and patterns of referrals of oral cancer: role of general practitioners.

Referrals of patients with oral squamous cell carcinomas to an oral medicine clinic were assessed with regard to the sources, delays, and pattern of referrals from general medical practitioners and general dental practitioners. Slightly more patients were referred by dental practitioners than by medical practitioners, but general medical practitioners were far more likely to see advanced tumours and to request an urgent second opinion or suggest a diagnosis of malignant disease. The greatest delay overall was caused by the patients in seeking advice from their practitioner, particularly those who attended a general medical practitioner. Both groups of practitioners requested a hospital opinion within roughly a month--a reasonable interval. Subsequent delays were minimal. Delays occur mainly because the patients are slow in seeking professional advice and, in general, do not appear to have been reduced over the decade since a previous British study on referral patterns was carried out. This study emphasises the importance of educating patients about oral cancer since it is they who appear to be mainly responsible for the delays in diagnosis. The results also help to dispel the myth that general medical practitioners might be less competent at diagnosis and referral of patients with oral cancer than are dental practitioners, though we are aware of misdiagnoses from both groups.     

The mitochondria-targeted anti-oxidant MitoQ reduces aspects of mitochondrial fission in the 6-OHDA cell model of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder for which available treatments provide symptom relief but do not stop disease progression. Mitochondria, and in particular mitochondrial dynamics, have been postulated as plausible pharmacological targets. Mitochondria-targeted antioxidants have been developed to prevent mitochondrial oxidative damage, and to alter the involvement of reactive oxygen species (ROS) in signaling pathways. In this study, we have dissected the effect of MitoQ, which is produced by covalent attachment of ubiquinone to a triphenylphosphonium lipophilic cation by a ten carbon alkyl chain. MitoQ was tested in an in vitro PD model which involves addition of 6-hydroxydopamine (6-OHDA) to SH-SY5Y cell cultures. At sublethal concentrations of 50 μM, 6-OHDA did not induce increases in protein carbonyl, mitochondrial lipid peroxidation or mitochondrial DNA damage. However, after 3 h of treatment, 6-OHDA disrupts the mitochondrial morphology and activates the machinery of mitochondrial fission, but not fusion. Addition of 6-OHDA did not increase the levels of fission 1, mitofusins 1 and 2 or optic atrophy 1 proteins, but does lead to the translocation of dynamin related protein 1 from the cytosol to the mitochondria. Pre-treatment with MitoQ (50 nM, 30 min) results in the inhibition of the mitochondrial translocation of Drp1. Furthermore, MitoQ also inhibited the translocation of the pro-apoptotic protein Bax to the mitochondria. These findings provide mechanistic evidence for a role for redox events contributing to mitochondrial fission and suggest the potential of mitochondria-targeted therapeutics in diseases that involve mitochondrial fragmentation due to oxidative stress.     

Cortical mechanisms for trans-saccadic memory and integration of multiple object features

Constructing an internal representation of the world from successive visual fixations, i.e. separated by saccadic eye movements, is known as trans-saccadic perception. Research on trans-saccadic perception (TSP) has been traditionally aimed at resolving the problems of memory capacity and visual integration across saccades. In this paper, we review this literature on TSP with a focus on research showing that egocentric measures of the saccadic eye movement can be used to integrate simple object features across saccades, and that the memory capacity for items retained across saccades, like visual working memory, is restricted to about three to four items. We also review recent transcranial magnetic stimulation experiments which suggest that the right parietal eye field and frontal eye fields play a key functional role in spatial updating of objects in TSP. We conclude by speculating on possible cortical mechanisms for governing egocentric spatial updating of multiple objects in TSP.     

A ratiometric fluorescent probe for assessing mitochondrial phospholipid peroxidation within living cells

Mitochondrial oxidative damage contributes to a wide range of pathologies, and lipid peroxidation of the mitochondrial inner membrane is a major component of this disruption. However, despite its importance, there are no methods to assess mitochondrial lipid peroxidation within cells specifically. To address this unmet need we have developed a ratiometric, fluorescent, mitochondria-targeted lipid peroxidation probe, MitoPerOx. This compound is derived from the C11-BODIPY(581/591) probe, which contains a boron dipyromethane difluoride (BODIPY) fluorophore conjugated via a dienyl link to a phenyl group. In response to lipid peroxidation the fluorescence emission maximum shifts from ～590 to ～520nm. To target this probe to the matrix-facing surface of the mitochondrial inner membrane we attached a triphenylphosphonium lipophilic cation, which leads to its selective uptake into mitochondria in cells, driven by the mitochondrial membrane potential. Here we report on the development and characterization of MitoPerOx. We found that MitoPerOx was taken up very rapidly into mitochondria within cells, where it responded to changes in mitochondrial lipid peroxidation that could be measured by fluorimetry, confocal microscopy, and epifluorescence live cell imaging. Importantly, the peroxidation-sensitive change in fluorescence at 520nm relative to that at 590nm enabled the use of the probe as a ratiometric fluorescent probe, greatly facilitating assessment of mitochondrial lipid peroxidation in cells.