Implications of using the fluorescent probes, dihydrorhodamine 123 and 2',7'-dichlorodihydrofluorescein diacetate, for the detection of UVA-induced reactive oxygen species.

During investigation of UVA-induced oxidative stress in HaCaT keratinocytes with dihydrorhodamine 123 (DHR123) and 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA), exaggerated baseline values were observed within control samples, suggesting a mechanism of probe oxidation and subsequent change in fluorescence intensity (FI) independent of cellular ROS generation. The effects of diluent, UVA pre-treatment and loading protocols upon the FI of the probes have therefore been investigated. The study confirmed the capacity of Dulbecco's Modified Eagle's Medium (DMEM) to confer fluorescence intensity changes in both probes, most notably DCF-DA. In addition, UVA pre-treatment compromises the effectiveness of DHR123 and DCF-DA to detect ROS generated in a cell-free system. In vitro data shows a greater UVA-induced FI increase in HaCaT cells loaded with probe before rather than after UVA treatment. This study has important implications for future research, the understanding of previous studies and associated confounding effects using DHR123 and DCF-DA as ROS sensitive probes.

Direct, real-time monitoring of superoxide generation in isolated mitochondria.

Mitochondria are one of the major sources of reactive oxygen species (ROS) in mammalian cells. The generation of ROS underlies many physiological and pathophysiological processes that occur within cellular systems. Superoxide ([image omitted] ) is the proximal ROS generated during electron 'leakage' from the mitochondrial electron transport chain (mETC) and is known to be released at mitochondrial complex I and complex III. Monitoring mitochondrial [image omitted] production directly and in real-time offers the potential to improve understanding of the complex mechanisms involved during mitochondrial [image omitted] generation. This study reports the novel application of a cytochrome c functionalized amperometric sensor for monitoring [image omitted] generation in isolated mitochondrial fractions. The non-invasive sensor system described allowed a comparison of [image omitted] production following specific inhibition of complex I and complex III of the mETC to be made directly and in real-time.

The development and in vitro characterisation of an intracellular nanosensor responsive to reactive oxygen species.

Advances in sensor technologies have enhanced our understanding of the roles played by reactive oxygen species (ROS) in a number of physiological and pathological processes. However, high inter-reactivity and short life spans has made real-time monitoring of ROS in cellular systems challenging. Fluorescent dyes capable of intracellular ROS measurements have been reported. However, these dyes are known to be intrinsically cytotoxic and thus can potentially significantly alter cellular metabolism and adversely influence in vitro data. Reported here is the development and in vitro application of a novel ROS responsive nanosensor, based on PEBBLE (Probes Encapsulated By Biologically Localised Embedding) technology. The ROS sensitive fluorescent probe dihydrorhodamine 123 (DHR 123) was employed as the sensing element of the PEBBLE through entrapment within a porous, bio-inert polyacrylamide nanostructure enabling passive monitoring of free radical flux within the intracellular environment. Successful delivery of the nanosensors into NR8383 rat alveolar macrophage cells via phagocytosis was achieved. Stimulation of PEBBLE loaded NR8383 cells with phorbol-12-myristate-13-acetate (PMA) enabled real time monitoring of ROS generation within the cell without affecting cellular viability. These data suggest that PEBBLE nanosensors could offer significant advantages over existing technologies used in monitoring the intracellular environment.

An electrochemical sensor array system for the direct, simultaneous in vitro monitoring of nitric oxide and superoxide production by cultured cells.

A new approach for an amperometric array sensor platform employing arrays of sensors in a 24-well cell culture plate format has been developed for simultaneous in vitro determination of nitric oxide (NO) and superoxide free radicals (O(2)(-)) produced by stimulated cells. The work reported focuses on the direct, real-time monitoring of extracellular production of these two analytes, as well as the effects of their interaction. The sensor platform was manufactured by a combination of sputtering gold electrodes and screen-printing carbon electrodes. The O(2)(-) sensor uses covalent immobilization of cytochrome c via a binder, DTSSP (3,3'-dithio-bis(sulphosuccinimidylpropionate) onto the surface of the Au electrodes, whereas the NO sensor system involves an NiTSPc (nickel tetrasulfonated phthalocyanine) film electrodeposited onto the surface of the carbon electrodes and subsequently covered with an external layer of Nafion. For in vitro demonstration of the platforms as a potential drug-screening system, A172 glioblastoma cells were cultured and transferred into the 24-well arrays. Simultaneous and direct monitoring of NO and O(2)(-) production as a response to chemicals of biomedical relevance was carried out. The results obtained demonstrated that it would be possible to envisage a drug screening platform for compounds designed to be inhibitors of nitric oxide synthase or to have an inhibitory effect on superoxide free radical production. By suitable modification of the electrodes employed it would also be possible to extend the platform to measure alternative species.

Combined system for the simultaneous optical and electrochemical monitoring of intra- and extracellular NO produced by glioblastoma cells.

A combined, optospectroscopic and electrochemical assay system for the simultaneous monitoring of intra- and extracellular production of biologically important species has been developed and assessed. The present model system evaluates intra- and extracellular nitric oxide produced by stimulated glioblastoma multiform cell line (A172). The production of endogenous NO was induced by phorbol-12-myristate-13-acetate and inhibited by N(omega)-nitro-l-arginine methyl ester. Intracellular production of NO was monitored via fluorescence image analysis using a 4,5-diaminofluorescein probe, while extracellular NO release was monitored via a chemically modified electrode, which was incorporated into an optically transparent cell chip. The results indicated that there was no mutual interference between the optical and electrochemical measurement systems. The response time of the combined optical/electrochemical system was found to be in the range of a few tens of seconds.

Simultaneous intra- and extracellular superoxide monitoring using an integrated optical and electrochemical sensor system.

A new integrated optical and electrochemical sensor system for simultaneous monitoring of intra- and extracellular superoxide (O(2)(-)) was developed using an array-based cell chip. For in vitro assays, A172 human glioblastoma cells were transferred into the cell chip and stimulated by phorbol 12-myristate 13-acetate (PMA). Intracellular O(2)(-) generation was detected via fluorescence image analysis with a dye probe, dihydrorhodamine 123 (DHR 123). Extracellular O(2)(-) was detected using an amperometric sensor constructed by immobilisation of cytochrome c using a binder, 3,3'-dithiobis(sulphosuccinimidylpropionate), to attach the redox protein onto the surface of electrodeposited Au electrodes incorporated into the optically transparent cell chip. The simultaneous intra- and extracellular production of O(2)(-) was successfully observed from PMA-stimulated A172 cells and inhibited by superoxide dismutase (SOD). The quantification of O(2)(-) concentration based on a mathematical model study and possible applications using the sensor system developed were discussed. The results confirm that there was no detectable interference or crosstalk between the optical and electrochemical assays. Feasibility of the integration of the two methods, optical and electrochemical, and the neutralisation of the intra- and extracellular O(2)(-) levels by SOD have been demonstrated.

ALP and MLP distribution during myofibrillogenesis in cultured cardiomyocytes.

The Z-line is a multifunctional macromolecular complex that anchors sarcomeric actin filaments, mediates interactions with intermediate filaments and costameres, and recruits signaling molecules. Antiparallel alpha-actinin homodimers, present at Z-lines, cross-link overlapping actin filaments and also bind other cytoskeletal and signaling elements. Two LIM domain containing proteins, alpha-actinin associated LIM protein (ALP) and muscle LIM protein (MLP), interact with alpha-actinin, distribute in vivo to Z-lines or costameres, respectively, and, when absent, are associated with heart disease. Here we describe the behavior of ALP and MLP during myofibrillogenesis in cultured embryonic chick cardiomyocytes. As myofibrils develop, ALP and MLP are observed in distinct distribution patterns in the cell. ALP is coincident with alpha-actinin from the first stage of myofibrillogenesis and co-distributes with alpha-actinin to Z-lines and intercalated discs in mature myofibrils. Interestingly, we also demonstrate using ALP-GFP transfection experiments and an in vitro binding assay that the ALP-alpha-actinin binding interaction is not required to target ALP to the Z-line. In contrast, MLP localization is not co-incident with that of alpha-actinin until late stages of myofibrillogenesis; however, it is present in premyofibrils and nascent myofibrils prior to the incorporation of other costameric components such as vinculin, vimentin, or desmin. Our observations support the view that ALP function is required specifically at actin anchorage sites. The subcellular distribution pattern of MLP during myofibrillogenesis suggests that it functions during differentiation prior to the establishment of costameres.

Expression of the gene encoding the LIM protein CRP2: a developmental profile.

Members of the cysteine-rich protein (CRP) family are evolutionarily conserved Lin-11, Isl-1, Mac-3 (LIM) domain proteins that have been implicated in cell differentiation. Here we describe the expression pattern of the CRP family member CRP2 in mouse. Unlike other CRP family members, which are expressed primarily in muscle, CRP2 is more broadly expressed. In addition to expression in vascular smooth muscle cells, we also detect CRP2-specific transcripts in mesenchymal derivatives and several epithelia.