Identification of reference genes for quantitative real-time PCR studies in human cell lines under copper and zinc exposure.

Accurate quantification depends on normalization of the measured gene expression data. In particular, gene expression studies with exposure to metals are challenging due their toxicity and redox-active properties. Here, we assessed the stability of potential reference genes in three cell lines commonly used to study metal cell metabolism: Caco-2 (colon), HepG2 (liver) and THP-1 (peripheral blood) under copper (Cu) or zinc (Zn) exposure. We used combined statistical tools to identify the best reference genes from a set of eleven candidates, which included traditional "housekeeping" genes such as GAPDH and B-ACTIN, in cell lines exposed to high and low, Zn and Cu concentrations. The expression stabilities of ATP5B (ATP synthase) and CYC1 (subunits of the cytochrome) were the highest considering the effect of Zn and Cu treatments whereas SDHA (succinate dehydrogenase) was found to be the most unstable gene. Even though the transcriptional effect of Zn and Cu is very different in term of redox properties, the same best reference genes were identified when Zn or Cu treatments were analyzed together. Our results indicate that ATP5B/CYC1 are the best candidates for reference genes after metal exposure, which can be used as a suitable starting point to evaluate gene expression with other metals or in different cell types in human models.

Quantification and evaluation of Joule heating in on-chip capillary electrophoresis.

We present the use of a novel, picoliter volume interferometer to measure, for the first time, the extent of Joule heating in chip-scale capillary electrophoresis (CE). The simple optical configuration for the on-chip interferometric backscatter detector (OCIBD) consists of an unfocused laser, an unaltered silica chip with a half-cylinder channel and a photodetector. Using OCIBD for millidegree-level noninvasive thermometry, temperature changes associated with Joule heating (2.81 degrees C above ambient) in on-chip CE have been observed in 90 microm wide and 40 microm deep separation channels. The temporal response of Joule heating in isotropically etched channels was exponential, with it taking an excess of 2.7 s to reach equilibrium. Buffer viscosity changes have also been derived from empirical on-chip thermometry data, allowing for the determination of diffusion coefficients for solutes when separated in heated buffers. In addition, OCIBD has allowed the reduction in separation efficiency to be estimated in the absence of laminar flow and due to increased molecular diffusion and lower buffer viscosity. A 7% reduction in separation efficiency was determined for a high current drawing buffer such as Tris-boric acid under an applied field of just 400 V/cm. Results indicate that heating effects in on-chip CE have been underestimated and there is a need to readdress the theoretical model.

A valid quantitative histochemical technique for assaying cytochrome c oxidase in single cells.

A quantitative histochemical method for assaying cytochrome c oxidase (COX) has been validated with two new findings concerning the optimal tissue thickness and a suitable substrate. The kinetics of a COX-catalysed reaction coupled to the oxidation of diaminobenzidine (DAB) were followed at 37 degrees C in single muscle fibres in unfixed sections of mouse gastrocnemius using a real-time image analysis system. The optimum composition of the substrate medium for the reaction was 0.1 mM reduced cytochrome c, 4 mM DAB, 2% dimethylsulphoxide, 2% polyvinyl alcohol and 0.1 mM HEPES buffer, final pH 7.5. The absorbances at 451 nm of the final reaction products, DAB polymer oxides, deposited in the intermyofibrillar mitochondria increased linearly as a function of incubation time for at least 80 s after the start of incubation. The initial velocities (v(i)) of the COX reaction calculated from the gradients of the linear regression best fits for times between 40 and 60 s were reproducible. The v(i) determined in single muscle fibres at a saturated concentration of cytochrome c (0.1 mM) were proportional to section thickness for thicknesses less than 3 microns, but they decreased exponentially when the thickness was greater than 4 microns. Thus, for the quantitative assay, unfixed sections 3 microns thick must be used. The Michaelis constants (Km) determined for commercial cytochrome c in the range of 20-26 microM for COX in three types of skeletal muscle fibres of mouse gastrocnemius were higher than the corresponding in situ Km (12-13 microM) for reduced cytochrome c. However, the Km values for commercial cytochrome c were in good agreement with the value previously determined with homogenates of rat hind limb muscle. Therefore, reduced cytochrome c is a more suitable substrate for the kinetic study and assay of COX in situ.

Immunoquantitation of cytochrome C in cardiac perfusate.

An indirect three step ELISA has been assessed in order to detect the possible release of cytochrome c, a mitochondrial protein, from isolated and perfused guinea-pig heart. The ELISA described in this study is sufficiently sensitive and accurate to measure extracellular cytochrome c.

Redox-linked conformational changes in proteins detected by a combination of infrared spectroscopy and protein electrochemistry. Evaluation of the technique with cytochrome c.

We have developed a new technique for the study of redox-linked conformational changes in proteins, by the combination of two established techniques. Fourier-transform infrared spectroscopy has been used together with direct electrochemistry of the protein at a modified metal electrode surface. The technique has been evaluated with cytochrome c, because of its well-characterized electrochemistry and because the availability of X-ray crystallographic and NMR studies of both redox states of the protein provides a reference against which our data can be compared. In electrochemical control experiments, it was confirmed that the spectroelectrochemical cell design allows fast, accurate and reproducible control of the redox poise of the protein. The resulting reduced-minus-oxidized infrared difference spectra show the changes in the frequencies and intensities of molecular vibrations which arise from the redox-linked conformational change. In contrast to the absolute infrared spectra of proteins, such difference spectra can be sufficiently straightforward to allow interpretation at the level of individual bonds. A complete interpretation of the spectra is beyond the scope of the present paper: however, on the basis of the data presented, we are able to suggest assignments for all except one of the major bands between 1500 cm-1 and 1800 cm-1.