Ca2+-activated Cl- current in retinal arteriolar smooth muscle.

PURPOSE: To characterize the biophysical, pharmacologic, and functional properties of the Ca(2+)-activated Cl(-) current in retinal arteriolar myocytes. METHODS: Whole-cell perforated patch-clamp recordings were made from myocytes within intact isolated arteriolar segments. Arteriolar tone was assessed using pressure myography. RESULTS: Depolarizing of voltage steps to -40 mV and greater activated an L-type Ca(2+) current (I(Ca(L))) that was followed by a sustained current. Large tail currents (I(tail)) were observed on stepping back to -80 mV. The sustained current and I(tail) reversed close to 0 mV in symmetrical Cl(-) concentrations. The ion selectivity sequence for I(tail) was I(-)> Cl(-)> glucuronate. Outward I(tail) was sensitive to the Cl(-) channel blockers 9-anthracene-carboxylic acid (9-AC; 1 mM), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS; 1 mM), and disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS; 1 mM), but only DIDS produced a substantial (78%) block of inward tail currents at -100 mV. I(tail) was decreased in magnitude when the normal bathing medium was substituted with Ca(2+)-free solution or if I(Ca(L)) was inhibited by 1 microM nimodipine. Caffeine (10 mM) produced large transient currents that reversed close to the Cl(-) equilibrium potential and were blocked by 1 mM DIDS or 100 microM tetracaine. DIDS had no effect on basal vascular tone in pressurized arterioles but dramatically reduced the level of vasoconstriction observed in the presence of 10 nM endothelin-1. CONCLUSIONS: Retinal arteriolar myocytes have I(Cl(Ca)), which may be activated by Ca(2+) entry through L-type Ca(2+) channels or Ca(2+) release from intracellular stores. This current appears to contribute to agonist-induced retinal vasoconstriction.

Photosensitiser delivery for photodynamic therapy. Part 2: systemic carrier platforms

Background: The treatment of solid tumours and angiogenic ocular diseases by photodynamic therapy (PDT) requires the injection of a photosensitiser (PS) to destroy target cells through a combination of visible light irradiation and molecular oxygen. There is currently great interest in the development of efficient and specific carrier delivery platforms for systemic PDT. Objective: This article aims to review recent developments in systemic carrier delivery platforms for PDT, with an emphasis on target specificity. Methods: Recent publications, spanning the last five years, concerning delivery carrier platforms for systemic PDT were reviewed, including PS conjugates, dendrimers, micelles, liposomes and nanoparticles. Results/conclusion: PS conjugates and supramolecular delivery platforms can improve PDT selectivity by exploiting cellular and physiological specificities of the targeted tissue. Overexpression of receptors in cancer and angiogenic endothelial cells allows their targeting by affinity-based moieties for the selective uptake of PS conjugates and encapsulating delivery carriers, while the abnormal tumour neovascularisation induces a specific accumulation of heavy weighted PS carriers by enhanced permeability and retention (EPR) effect. in addition, polymeric prodrug delivery platforms triggered by the acidic nature of the tumour environment or the expression of proteases can be designed. Promising results obtained with recent systemic carrier platforms will, in due course, be translated into the clinic for highly efficient and selective PDT protocols.

Multiply reconfigurable ‘plug and play’ molecular logic via self-assembly

A substantial set of ion-driven molecular logic gates are implemented in turn by arranging the association between easily available lumophores and receptors in detergent micelles.

Development and validation of a dried blood spot-LC-APCI-MS assay for estimation of canrenone in paediatric samples

A selective and sensitive liquid chromatography (LC)-atmospheric pressure chemical ionisation (APCI)-mass spectroscopic (MS) assay of canrenone has been developed and validated employing Dried Blood Spots (DBS) as the sample collection medium. DBS samples were prepared by applying 30 mu l of spiked whole blood onto Guthrie cards. A 6 mm disc was punched from the each DBS and extracted with 2 ml of methanolic solution of 17 alpha-methyltestosterone (Internal Standard). The methanolic extract was evaporated to dryness and reconstituted in acetonitrile:water (1:9, v/v). The reconstituted solution was further subjected to solid phase extraction using HLB cartridges. Chromatographic separation was achieved using Waters Sunfire C18 reversed-phase column using isocratic elution, followed by a high organic wash to clear late eluting/highly retained components. The mobile phase consisted of methanol:water (60:40, v/v) pumped at a flow rate of 0.3 ml/min. LC-APCI-MS detection was performed in the selected-ion monitoring (SIM) mode using target ions at m/z 341.1 and 303.3 for canrenone and internal standard respectively. The selectivity of the method was established by analysing DBS samples from 6 different sources (individuals). The calibration curve for canrenone was found to be linear over 25-1000 ng/ml (r >0.994). Accuracy (% RE) and precision (% CV) values for within and between day were

Rapid confirmatory method for the determination of sixteen synthetic growth promoters and bisphenol A in bovine milk using dispersive solid-phase extraction and liquid chromatography-tandem mass spectrometry

A rapid liquid chromatographic-tandem mass spectrometric (LC-MS/MS) multi-residue method for the simultaneous quantitation and identification of sixteen synthetic growth promoters and bisphenol A in bovine milk has been developed and validated. Sample preparation was straightforward, efficient and economically advantageous. Milk was extracted with acetonitrile followed by phase separation with NaCl. After centrifugation, the extract was purified by dispersive solid-phase extraction with C18 sorbent material. The compounds were analysed by reversed-phase LC-MS/MS using both positive and negative ionization and operated in multiple reaction monitoring (MRM) mode, acquiring two diagnostic product ions from each of the chosen precursor ions for unambiguous confirmation. Total chromatographic run time was less than 10 min for each sample. The method was validated at a level of 1 mu g L-1. A wide variety of deuterated internal standards were used to improve method performance. The accuracy and precision of the method were satisfactory for all analytes. The confirmative quantitative liquid chromatographic tandem mass spectrometric (LC-MS/MS) method was validated according to Commission Decision 2002/657/EC. The decision limit (CC alpha) and the detection capability (CC beta) were found to be below the chosen validation level of 1 mu g L-1 for all compounds. (C) 2010 Elsevier B.V. All rights reserved.

Existence and stability of multisite breathers in honeycomb and hexagonal lattices

We study the existence and stability of multisite discrete breathers in two prototypical non-square Klein-Gordon lattices, namely a honeycomb and a hexagonal one. In the honeycomb case we consider six-site configurations and find that for soft potential and positive coupling the out-of-phase breather configuration and the charge-two vortex breather are linearly stable, while the in-phase and charge-one vortex states are unstable. In the hexagonal lattice, we first consider three-site configurations. In the case of soft potential and positive coupling, the in-phase configuration is unstable and the charge-one vortex is linearly stable. The out-of-phase configuration here is found to always be linearly unstable. We then turn to six-site configurations in the hexagonal lattice. The stability results in this case are the same as in the six-site configurations in the honeycomb lattice. For all configurations in both lattices, the stability results are reversed in the setting of either hard potential or negative coupling. The study is complemented by numerical simulations which are in very good agreement with the theoretical predictions. Since neither the form of the on-site potential nor the sign of the coupling parameter involved have been prescribed, this description can accommodate inverse-dispersive systems (e. g. supporting backward waves) such as transverse dust-lattice oscillations in dusty plasma (Debye) crystals or analogous modes in molecular chains.

C5a-mediated neutrophil phagocytic dysfunction is RhoA-dependent and predicts nosocomial infection in critically ill patients.

Critically ill patients are at heightened risk for nosocomial infections. The anaphylatoxin C5a impairs phagocytosis by neutrophils. However, the mechanisms by which this occurs and the relevance for acquisition of nosocomial infection remain undetermined. We aimed to characterize mechanisms by which C5a inhibits phagocytosis in vitro and in critically ill patients, and to define the relationship between C5a-mediated dysfunction and acquisition of nosocomial infection. In healthy human neutrophils, C5a significantly inhibited RhoA activation, preventing actin polymerization and phagocytosis. RhoA inhibition was mediated by PI3Kd. The effects on RhoA, actin, and phagocytosis were fully reversed by GM-CSF. Parallel observations were made in neutrophils from critically ill patients, that is, impaired phagocytosis was associated with inhibition of RhoA and actin polymerization, and reversed by GM-CSF. Among a cohort of 60 critically ill patients, C5a-mediated neutrophil dysfunction (as determined by reduced CD88 expression) was a strong predictor for subsequent acquisition of nosocomial infection (relative risk, 5.8; 95% confidence interval, 1.5-22; P = .0007), and remained independent of time effects as assessed by survival analysis (hazard ratio, 5.0; 95% confidence interval, 1.3-8.3; P = .01). In conclusion, this study provides new insight into the mechanisms underlying immunocompromise in critical illness and suggests novel avenues for therapy and prevention of nosocomial infection.

Vascular stem cells and ischaemic retinopathies

Retinal ischaemic disorders such as diabetic retinopathy and retinal vein occlusion are common. The hypoxia-related stimuli from oxygen-deprived neural and glial networks can drive expression of growth factors and cytokines which induce leakage from the surviving vasculature and/or pre-retinal and papillary neovascularisation. If left untreated, retinal vascular stasis, hypoxia or ischaemia can lead to macular oedema or fibro-vascular scar formation which are associated with severe visual impairment, and even blindness. Current therapies for ischaemic retinopathies include laser photocoagulation, injection of corticosteroids or VEGF-antibodies and vitreoretinal surgery, however they carry significant side effects. As an alternative approach, we propose that if reparative intra-retinal angiogenesis can be harnessed at the appropriate stage, ischaemia could be contained or reversed. This review provides evidence that reperfusion of ischaemic retina and suppression of sight-threatening sequelae is possible in both experimental and clinical settings. In particular, there is emphasis on the clinical potential for endothelial progenitor cells (EPCs) to promote vascular repair and reversal of ischaemic injury in various tissues including retina. Gathering evidence from an extensive published literature, we outline the molecular and phenotypic nature of EPCs, how they are altered in disease and provide a rationale for harnessing the vascular reparative properties of various cell sub-types. When some of the remaining questions surrounding the clinical use of EPCs are addressed, they may provide an exciting new therapeutic option for treating ischaemic retinopathies. (C) 2011 Elsevier Ltd. All rights reserved.

CTGF/CCN2 activates canonical Wnt signalling in mesangial cells through LRP6: Implications for the pathogenesis of diabetic nephropathy

We describe the activation of Wnt signalling in mesangial cells by CCN2. CCN2 stimulates phosphorylation of LRP6 and GSK-3 beta resulting in accumulation and nuclear localisation of beta-catenin, TCF/LEF activity and expression of Wnt targets. This is coincident with decreased phosphorylation of beta-catenin on Ser 33/37 and increased phosphorylation on Tyr142. DKK-1 and LRP6 siRNA reversed CCN2's effects. Microarray analyses of diabetic patients identified differentially expressed Wnt components. beta-Catenin is increased in type 1 diabetic and UUO mice and in in vitro models of hyperglycaemia and hypertension. These findings suggest that Wnt/CCN2 signalling plays a role in the pathogenesis of diabetic nephropathy. (C) 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Regulation of OPA1-mediated mitochondrial fusion by leucine zipper/EF-hand-containing transmembrane protein-1 plays a role in apoptosis

Carboxyl-terminal modulator protein (CTMP) is a tumor suppressor-like binding partner of Protein kinase B (PKB/Akt) that negative regulates this kinase. In the course of our recent work, we identified that CTMP is consistently associated with leucine zipper/EF-hand-containing transmembrane-1 (LETM1). Here, we report that adenovirus-LETM1 increased the sensitivity of HeLa cells to apoptosis, induced by either staurosporine or actinomycin D. As shown previously, LETM1 localized to the inner mitochondrial membrane. Electron-microscopy analysis of adenovirus-LETM1 transduced cells revealed that mitochondrial cristae were swollen in these cells, a phenotype similar to that observed in optic atrophy type-1 (OPA1)-ablated cells. OPA1 cleavage was increased in LETM1-overexpressing cells, and this phenotype was reversed by overexpression of OPA1 variant-7, a cleavage resistant form of OPA1. Taken together, these data suggest that LETM1 is a novel binding partner for CTMP that may play an important role in mitochondrial fragmentation via OPA1-cleavage. (C) 2009 Elsevier Inc. All rights reserved

MicroRNA-155 Promotes Resolution of Hypoxia-Inducible Factor 1{alpha} Activity

The hypoxia-inducible factor (HIF) is a key regulator of the transcriptional response to hypoxia. While the mechanism underpinning HIF activation is well understood, little is known about its resolution. Both the protein and the mRNA levels of HIF-1a (but not HIF-2a) were decreased in intestinal epithelial cells exposed to prolonged hypoxia. Coincident with this, microRNA (miRNA) array analysis revealed multiple hypoxia-inducible miRNAs. Among these was miRNA-155 (miR-155), which is predicted to target HIF-1a mRNA. We confirmed the hypoxic upregulation of miR-155 in cultured cells and intestinal tissue from mice exposed to hypoxia. Furthermore, a role for HIF-1a in the induction of miR-155 in hypoxia was suggested by the identification of hypoxia response elements in the miR-155 promoter and confirmed experimentally. Application of miR-155 decreased the HIF-1a mRNA, protein, and transcriptional activity in hypoxia, and neutralization of endogenous miR-155 reversed the resolution of HIF-1a stabilization and activity. Based on these data and a mathematical model of HIF-1a suppression by miR-155, we propose that miR-155 induction contributes to an isoform-specific negative-feedback loop for the resolution of HIF-1a activity in cells exposed to prolonged hypoxia, leading to oscillatory behavior of HIF-1a-dependent transcription.

Rapid synthesis of nucleotide pyrophosphate linkages in a ball mill

Using a ball mill, rapid, atom-economic coupling between adenosine-5'-phosphoromorpholidate and phosphorylated ribose derivatives as their sodium or barium salts was achieved. Facile purification by reversed-phase HPLC enabled product isolation within hours.

Theoretical and experimental correlations of gas dissolution, diffusion, and thermodynamic properties in determination of gas permeability and selectivity in supported ionic liquid membranes

Supported ionic liquid membranes (SILMs) has the potential to be a new technological platform for gas/organic vapour separation because of the unique non-volatile nature and discriminating gas dissolution properties of room temperature ionic liquids (ILs). This work starts with an examination of gas dissolution and transport properties in bulk imidazulium cation based ionic liquids [C_nmim][NTf2] (n = 2.4, 6, 8.10) from simple gas H₂, N₂, to polar CO₂, and C₂H₆, leading to a further analysis of how gas dissolution and diffusion are influenced by molecular specific gas-SILMs interactions, reflected by differences in gas dissolution enthalpy and entropy. These effects were elucidated again during gas permeation studies by examining how changes in these properties and molecular specific interactions work together to cause deviations from conventional solution–diffusion theory and their impact on some remarkably contrasting gas perm-selectivity performance. The experimental perm-selectivity for all tested gases showed varied and contrasting deviation from the solution–diffusion, depending on specific gas-IL combinations. It transpires permeation for simpler non-polar gases (H₂, N₂) is diffusion controlled, but strong molecular specific gas-ILs interactions led to a different permeation and selectivity performance for C₂H₆ and CO₂. With exothermic dissolution enthalpy and large order disruptive entropy, C₂H₆ displayed the fastest permeation rate at increased gas phase pressure in spite of its smallest diffusivity among the tested gases. The C₂H₆ gas molecules “peg” on the side alkyl chain on the imidazulium cation at low concentration, and are well dispersed in the ionic liquids phase at high concentration. On the other hand strong CO₂-ILs affinity resulted in a more prolonged “residence time” for the gas molecule, typified by reversed CO₂/N₂ selectivity and slowest CO₂ transport despite CO₂ possess the highest solubility and comparable diffusivity in the ionic liquids. The unique transport and dissolution behaviour of CO₂ are further exploited by examining the residing state of CO₂ molecules in the ionic liquid phase, which leads to a hypothesis of a condensing and holding capacity of ILs towards CO₂, which provide an explanation to slower CO₂ transport through the SILMs. The pressure related exponential increase in permeations rate is also analysed which suggests a typical concentration dependent diffusion rate at high gas concentration under increased gas feed pressure. Finally the strong influence of discriminating and molecular specific gas-ILs interactions on gas perm-selectivity performance points to future specific design of ionic liquids for targeted gas separations.

SURFACE-ENHANCED RAMAN-SCATTERING STUDIES OF METALLOPORPHYRINS ON SILVER SOLS, MELLFS, AND ELECTRODES - EVIDENCE FOR REVERSIBLE PHOTOINDUCED DEMETALATION OF A SILVER(II) PORPHYRIN

A series of metalloporphyrins of the type M(TMPyP) (where M = Ag(II), Zn(II), Cu(II) and TMPyP = meso-tetrakis(4-N-methylpyridyl)porphyrin) have been investigated in solution and on the surface of silver sols, electrodes, and MELLFs (metal liquidlike films). Similar spectra were recorded on all three surfaces but significant differences in detailed behavior were found. In particular, a novel, reversible, and rapid photoinduced demetalation reaction has been observed for the AgII(TMPyP)/MELLF system. An apparently similar demetalation reaction for the same metalloporphyrin was observed on Ag electrodes but this reversed at a very much slower rate. No demetalation of Ag(II)(TMPyP) was observed with Ag sols nor with any of the other metalloporphyrins at any of the surfaces investigated. The implications of the findings in relation to the nature of the MELLF environment are briefly considered.

Platelet Redox Balance in Diabetic Patients with Hypertension improved by n-3 fatty acids

OBJECTIVE:
Patients with type 2 diabetes mellitus (T2DM) are at increased risk of developing cardiovascular disease, largely as a result of defective production of cardioprotective nitric oxide and a concomitant rise in oxidative stress. Dietary interventions that could reverse this trend would be extremely beneficial. Here we investigated whether dietary n-3 polyunsaturated fatty acid (n-3 PUFA) supplementation positively affected platelet nitroso-redox imbalance.
RESEARCH DESIGN AND METHODS:
We randomized hypertensive T2DM patients (T2DM HT; n = 22) and age-and-sex matched hypertensive study participants without diabetes (HT alone; n = 23) in a double-blind, crossover fashion to receive 8 weeks of n-3 PUFAs (1.8 g eicosapentaenoic acid and 1.5 g docosahexaenoic acid) or identical olive oil capsules (placebo), with an intervening 8-week washout period. Platelet nitrite and superoxide were measured and compared before and after treatment; 8-isoprostane was determined by ELISA and subcellular compartmentalization of the NAD(P)H oxidase subunit p47-phox examined by Western blotting.
RESULTS:
The n-3 PUFA supplementation reduced 8-isoprostane and superoxide levels in platelets from T2DM HT, but not HT alone, participants, without effect on nitrite production. This coincided with a significant decrease in p47-phox membrane localization and a similar reduction in superoxide to that achieved with apocynin. At baseline, a subcohort of T2DM HT and HT alone participants showed evidence of nitric oxide synthase (NOS)-derived superoxide production, indicating defective enzymatic activity. This was reversed significantly in T2DM HT participants after treatment, demonstrating improved NOS function.
CONCLUSIONS:
Our finding that n-3 PUFAs diminish platelet superoxide production in T2DM HT patients in vivo suggests a therapeutic role for these agents in reducing the vascular-derived oxidative stress associated with diabetes.