Cross-reactivity of the UC1MT antibody for MT-III was tested by direct ELISA.

<p>Comparison of the standard curves for MT-IIA (blue lines) and MT-III (red lines) demonstrate UC1MT has very little if any cross-reactivity for MT-III. Data are expressed as the mean of triplicate measurements (error bars = SEM).</p

Corticosterone concentrations in plasma after cryolesion injury to the brain were assayed by RIA in wild type and MT-I/II^−/− mice (A).

<p>No significant differences were found between the mouse strains. There was a significant increase in plasma corticosterone after cryolesion injury and sham surgery to a similar extent. Sham surgery does not induce a significant change in hepatic MT-I or MT-II mRNA expression (<b>B</b>). Time points that share letters are not significantly different (n = 5, error bars = SEM).</p

Displacement curves for MT-IIA in MT-I/II^−/− mouse brain homogenate (A) and MT-I/II^−/− mouse liver homogenate (B).

<p>Displacement curves constructed in solutions with protein content of 0.01 mg/ml and 0.1 mg/ml are parallel to the standard curve constructed in PBS. Therefore, no matrix effects were observed at these concentrations, in these tissues (n = 3, error bars = SEM).</p

Liver MT-I/II protein levels after cryolesion injury to the brain were assayed by UC1MT ELISA in wild type mice.

<p>Hepatic MT-I/II protein levels were not increased until 3 DPI and showed a further increase at 7 DPI. Groups that share lower case letters are not significantly different from each other (n = 7, error bars = SEM). signalling mechanism is involved.</p

Standard curve generated by the UC1MT competitive ELISA with 4-parameter logistic curve fitted.

<p>Each point shown is the average of 4 quadruplicate standards.</p

Expression of MT-I and MT-II mRNA in the liver of wild type and MT-I/II^−/− mice after brain injury was quantified by RT-PCR.

<p>(<b>A</b>) MT-I mRNA expression showed its greatest increase at 1 DPI and 3 DPI in wild type mice. (<b>B</b>) MT-II mRNA was increased at 1 DPI in wild type mice but was at peak levels at 3 DPI. MT-I/II^−/− mice were unable to increase MT-I and MT-II mRNA levels to the same extent as wild type mice. Groups that share lower case letters are not significantly different from each other (for both graphs; n = 6–7, error bars = SEM).</p

Soluble Aβ induces prolonged efflux of K⁺ in Tg2576 cortical neurons.

<p>Treatment with 1 µM Aβ_1-40 triggered rapid efflux of K⁺ from Tg2576 neurons (A), which continued over the 120 minutes of recording. Aβ_1-40 treatment caused a rapid influx of H⁺ in Tg2576 neurons (B), which did not stabilise over the 120 minute recording period.</p

Uptake of soluble Aβ by wildtype and Tg2576 cortical neurons <i>in vitro</i>.

<p>Wildtype and Tg2576 cortical neurons were treated with 10 µM of monomeric Aβ_1-40, and immunostained for Aβ after 24 hours. In untreated Tg2576 neurons, Aβ was smoothly distributed throughout the cytoplasm and processes of all neurons (A). When Aβ_1-40 was applied to wildtype neurons, it was internalised and distributed in a punctate manner within the cytoplasm and processes (B). Notably, not all wildtype neurons internalised Aβ_1-40 (B). When Aβ_1-40 was applied to Tg2576 neurons, both smooth and punctately distributed Aβ was detected within neurons (C). scale bar  = 25 µm.</p

Tg2576 cortical neurons are more vulnerable to soluble Aβ-induced neurotoxicity.

<p>Wildtype and Tg2576 cortical neurons were treated daily with 1 µM (A) or 10 µM (B) of monomeric Aβ_1-40 for 6 days, and neuronal viability (intracellular metabolism as assessed by Alamar Blue assay) assessed every 24 hours. At 1 µM concentrations, only Tg2576 neurons were vulnerable to Aβ_1-40, resulting in approximately 30% cell death after 6 days (A). 10 µM Aβ_1-40 was mildly toxic to wildtype neurons over the experimental timecourse, but killed 40% of Tg2576 neurons after 6 days (B). The Alamar Blue neurotoxicity assay produced similar results to direct counting of dying cells via propidium iodide uptake following treatment with 10 µM Aβ_1-40 (C). * - p<0.05, ANOVA. Error bars represent standard error values from at least three replicates per experimental condition. This graph is representative of the results observed from 4 different experiments.</p

Soluble Aβ triggers caspase-3 expression in Tg2576 cortical neurons.

<p>Tg2576 neurons cultured for 14 days <i>in vitro</i> (DIV) showed no signs of caspase-3 activation (A). However, when 7 DIV Tg2576 neurons were treated with 1 µM Aβ_1-40 daily for 6 days, a substantial number of neurons were found to express caspase-3 (B).</p