Targeting MDMX and PKCδ to improve current uveal melanoma therapeutic strategies.

Uveal melanoma (UM) is the most frequent ocular cancer in adults, accounting for ~5% of the total melanoma incidence. Although the primary tumor is well treatable, patients frequently develop metastases for which no curative therapy exists. Highly activated protein kinase C (PKC) is a common feature of UM and has shown potential as therapeutic intervention for UM patients. Unfortunately, PKC inhibition as single treatment appears to have only limited clinical benefit. Combining PKC inhibition with activation of p53, which is rarely mutated in UM, by MDM2 inhibitors has shown promising results in vitro and in vivo. However, clinical studies have shown strong adverse effects of MDM2 inhibition. Therefore, we investigated alternative approaches to achieve similar anticancer effects, but with potentially less adverse effects. We studied the potential of targeting MDMX, an essential p53 inhibitor during embryonal development but less universally expressed in adult tissues compared with MDM2. Therefore, targeting MDMX is predicted to have less adverse effects in patients. Depletion of MDMX, like the pharmacological activation of p53, inhibits the survival of UM cells, which is enhanced in combination with PKC inhibition. Also pan-PKC inhibitors elicit adverse effects in patients. As the PKC family consists of 10 different isoforms, it could be hypothesized that targeting a single PKC isoform would have less adverse effects compared with a pan-PKC inhibitor. Here we show that specifically depleting PKCδ inhibits UM cell growth, which can be further enhanced by p53 reactivation. In conclusion, our data show that the synergistic effects of p53 activation by MDM2 inhibition and broad spectrum PKC inhibition on survival of UM cells can also largely be achieved by the presumably less toxic combination of depletion of MDMX and targeting a specific PKC isoform, PKCδ.

Multiple strategies of Lake Victoria cichlids to cope with lifelong hypoxia include hemoglobin switching.

Many fish species adapt to hypoxia by reducing their metabolic rate and increasing hemoglobin-oxygen (Hb-O(2)) affinity. Pilot studies with young broods of cichlids showed that the young could survive severe hypoxia in contrast with the adults. It was therefore hypothesized that early exposure results in improved oxygen transport. This hypothesis was tested using split brood experiments. Broods of Astatoreochromis alluaudi, Haplochromis ishmaeli, and a tilapia hybrid (Oreochromis) were raised either under normoxia (NR; 80-90% air saturation) or hypoxia (HR; 10% air saturation). The activity of the mitochondrial citrate synthase was not different between NR and HR tilapia, but was significantly decreased in HR A. alluaudi and H. ishmaeli, indicating lowered maximum aerobic capacities. On the other hand, hemoglobin and hematocrit levels were significantly higher in all HR fish of the three species, reflecting a physiological adaptation to safeguard oxygen transport capacity. In HR tilapia, intraerythrocytic GTP levels were decreased, suggesting an adaptive increase of blood-O(2) affinity. Similar changes were not found in HR H. ishmaeli. In this species, however, all HR specimens exhibited a distinctly different iso-Hb pattern compared with their NR siblings, which correlated with a higher intrinsic Hb-O(2) affinity in the former. All HR cichlids thus reveal left-shifted Hb-O(2) equilibrium curves, mediated by either decreased allosteric interaction or, in H. ishmaeli, by the production of new hemoglobins. It is concluded that the adaptation to lifelong hypoxia is mainly due to improved oxygen transport.

Differential role of adrenoceptors in control of plasma glucose and fatty acids in carp, Cyprinus carpio (L.).

Carp were cannulated in the dorsal aorta, and after 2 days of recovery they were infused with 1) norepinephrine, 2) yohimbine (alpha(2)-antagonist) plus norepinephrine, 3) clonidine (alpha(2)-agonist), and 4) isoproterenol (nonselective beta-agonist). Norepinephrine lowered the plasma free fatty acid (FFA) level and raised the plasma glucose level for several hours. Norepinephrine in combination with the alpha(2)-antagonist yohimbine resulted in retardation of the FFA decrease, indicating the involvement of alpha(2)-adrenoceptors. Infusion with the partial alpha(2)-agonist clonidine had a smaller effect. Infusion with isoproterenol caused a marked increase of glucose levels, and unexpectedly a decline of plasma FFA levels, indicating a direct involvement of beta-adrenoceptors. Combination of isoproterenol with either atenolol (beta(1)-antagonist) or ICI-118,551 (beta(2)-antagonist) showed that both beta(1)- and beta(2)-adrenoceptors were involved in the glucose release by isoproterenol. Remarkably, the decline of FFA levels was augmented in the presence of ICI-118,551, whereas with atenolol present plasma FFA levels were increased by isoproterenol. Thus it is concluded that in carp both beta(1)- and beta(2)-adrenoceptors mediate glucose release, whereas lipolysis is controlled by inhibitory beta(1)-adrenoceptors and stimulatory beta(2)-adrenoceptors, as well as by inhibitory alpha(2)-adrenoceptors.

Rapid and simple isolation of pure photosystem II core and reaction center particles from spinach.

Pure and active oxygen-evolving PS II core particles containing 35 Chl per reaction center were isolated with 75% yield from spinach PS II membrane fragments by incubation with n-dodecyl-ß-D-maltoside and a rapid one step anion-exchange separation. By Triton X-100 treatment on the column these particles could be converted with 55% yield to pure and active PS II reaction center particles, which contained 6 Chl per reaction center.