Targeted inhibition of metastatic melanoma through interference with Pin1-FOXM1 signaling.

Melanoma is the most lethal form of skin cancer and successful treatment of metastatic melanoma remains challenging. BRAF/MEK inhibitors only show a temporary benefit due to rapid occurrence of resistance, whereas immunotherapy is mainly effective in selected subsets of patients. Thus, there is a need to identify new targets to improve treatment of metastatic melanoma. To this extent, we searched for markers that are elevated in melanoma and are under regulation of potentially druggable enzymes. Here, we show that the pro-proliferative transcription factor FOXM1 is elevated and activated in malignant melanoma. FOXM1 activity correlated with expression of the enzyme Pin1, which we found to be indicative of a poor prognosis. In functional experiments, Pin1 proved to be a main regulator of FOXM1 activity through MEK-dependent physical regulation during the cell cycle. The Pin1-FOXM1 interaction was enhanced by BRAF(V600E), the driver oncogene in the majority of melanomas, and in extrapolation of the correlation data, interference with\ Pin1 in BRAF(V600E)-driven metastatic melanoma cells impaired both FOXM1 activity and cell survival. Importantly, cell-permeable Pin1-FOXM1-blocking peptides repressed the proliferation of melanoma cells in freshly isolated human metastatic melanoma ex vivo and in three-dimensional-cultured patient-derived melanoids. When combined with the BRAF(V600E)-inhibitor PLX4032 a robust repression in melanoid viability was obtained, establishing preclinical value of patient-derived melanoids for prognostic use of drug sensitivity and further underscoring the beneficial effect of Pin1-FOXM1 inhibitory peptides as anti-melanoma drugs. These proof-of-concept results provide a starting point for development of therapeutic Pin1-FOXM1 inhibitors to target metastatic melanoma.

Rat islet isolation yield and function are donor strain dependent.

Effective rat islet isolation is pertinent for successful islet transplantation and islet studies in vitro. To determine which rat strain yields the highest number of pure and functional islets, four commonly used rat strains were compared with regard to islet yield, islet purity and islet function. Secretory responses were assessed by stimulation with glucose, and by stimulation with glucose plus 3-isobutyl-1-methylxanthine (IBMX). We show that rat islet function and isolation yield are donor strain dependent. Albino Oxford (AO) rats donated twice as many islets than Wistar, Lewis and Sprague Dawley (SD) rats. Stimulation with glucose plus IBMX resulted in an average five-fold increase of the stimulation index of AO, Lewis, Wistar and SD rats compared to stimulation with glucose only. AO islets had improved secretory responses after a one-week culture period, but required the addition of IBMX to glucose to elicit a distinguished stimulated insulin secretion after 2 days of culture. Islets from SD rats showed inferior results with regard to purity immediately after isolation and with regard to function after short- and after long-time culture. Because Lewis islets possessed the highest secretory response to glucose (without IBMX) immediately after isolation, Lewis rats may be preferred as islet donors for immediate use. The addition of IBMX to glucose for in vitro functional testing is recommended because it elicits high insulin secretory responses of islets regardless of the rat strain. AO rats are preferred for culture experiments since the number of experimental animals is reduced two-fold compared to Lewis, Wistar and SD rats.

Response of Encapsulated Rat Pancreatic Islets to Hypoxia.

Hypoxia contributes to encapsulated pancreatic islet graft failure. To gain insight into the mechanisms that lead to hypoxia-induced graft failure, encapsulated islet function, vitality, and cell replication were assessed after 2 and 5 days of hypoxic (1% O2) and normoxic (20% O2) culture. The mRNA expression levels of Bcl-2, Bax, inducible nitric oxide synthase (iNOS), and monocyte chemoattractant protein 1 (MCP-1) were assessed, as well as the amount of nitrite and MCP-1 in the culture medium. Hypoxia was associated with loss of encapsulated islet function and vitality, but not with an increase in islet cell replication. Loss of vitality was due to necrosis, and only modestly due to apoptosis. Hypoxia was not associated with changes in the Bcl-2/Bax mRNA ratio, but it did increase the expression of iNOS and MCP-1 mRNA. The increased mRNA levels were, however, not associated with elevated concentrations of nitrite nor with elevated levels of MCP-1 protein. The increased iNOS mRNA levels imply a role for NO in the completion of cell death by hypoxia. The increased MCP-1 mRNA levels suggest that encapsulated islets in vivo contribute to their own graft failure by attracting cytokine-producing macrophages. The discrepancy between iNOS mRNA and nitrite is explained by the longer half-life of NO during hypoxia. MCP-1 protein levels are underestimated as a consequence of the lower number of vital cells in combination with a higher proteolytic activity due to necrosis. Thus, strategies to eliminate hypoxia may not only improve islet function and vitality, but may also reduce the attraction of macrophages by encapsulated islets.

Response of encapsulated rat pancreatic islets to hypoxia.

Hypoxia contributes to encapsulated pancreatic islet graft failure. To gain insight into the mechanisms that lead to hypoxia-induced graft failure, encapsulated islet function, vitality, and cell replication were assessed after 2 and 5 days of hypoxic (1% O2) and normoxic (20% O2) culture. The mRNA expression levels of Bcl-2, Bax, inducible nitric oxide synthase (iNOS), and monocyte chemoattractant protein 1 (MCP-1) were assessed, as well as the amount of nitrite and MCP-1 in the culture medium. Hypoxia was associated with loss of encapsulated islet function and vitality, but not with an increase in islet cell replication. Loss of vitality was due to necrosis, and only modestly due to apoptosis. Hypoxia was not associated with changes in the Bcl-2/Bax mRNA ratio, but it did increase the expression of iNOS and MCP-1 mRNA. The increased mRNA levels were, however, not associated with elevated concentrations of nitrite nor with elevated levels of MCP-1 protein. The increased iNOS mRNA levels imply a role for NO in the completion of cell death by hypoxia. The increased MCP-1 mRNA levels suggest that encapsulated islets in vivo contribute to their own graft failure by attracting cytokine-producing macrophages. The discrepancy between iNOS mRNA and nitrite is explained by the longer half-life of NO during hypoxia. MCP-1 protein levels are underestimated as a consequence of the lower number of vital cells in combination with a higher proteolytic activity due to necrosis. Thus, strategies to eliminate hypoxia may not only improve islet function and vitality, but may also reduce the attraction of macrophages by encapsulated islets.

Reduced Position Effect in Mature Transgenic Plants Conferred by the Chicken Lysozyme Matrix-Associated Region.

Matrix-associated regions may be useful for studying the role of chromatin architecture in transgene activity of transformed plants. The chicken lysozyme A element was shown to have specific affinity for tobacco nuclear matrices, and its influence on the variability of transgene expression in tobacco plants was studied. T-DNA constructs in which this element flanked either the [beta]-glucuronidase (GUS) reporter gene or both reporter and selection gene were introduced in tobacco. The variation in GUS gene activity was reduced significantly among mature first-generation transgenic plants carrying the A element. Average GUS activity became somewhat higher, but the maximum attainable level of gene expression was similar for all three constructs. Transient gene expression assays showed that the A element did not contain general enhancer functions; therefore, its presence seemed to prevent the lower levels of transgene expression. The strongest reduction in variability was found in plants transformed with the construct carrying the A elements at the borders of the T-DNA. In this population, expression levels became copy number dependent. The presence of two A elements in the T-DNA did not interfere with meiosis.

Methane oxidation by methanotrophs and its effects on the phosphate flux over the sediment-water interface in a eutrophic lake.

The effect of methane oxidation in aerobic sediment on oxygen consumption and phosphate flux was investigated in diffusion chambers. The diffusion chambers consisted of two compartments separated by a Teflon membrane. In the upper chamber a thin sediment layer was present and the lower chamber was continuously flushed with gas. The hydrophobic membrane allowed for diffusion of gases from the lower chamber through the sediment layer toward the headspace of the upper chamber. In experiments with a methane oxidation rate of 9.8 mmol m(-2) day(-1), the oxygen consumption rate increased by a factor of two compared with controls without methane oxidation (8.6 vs 17.7 mmol m(-2) day(-1)). Methane oxidation significantly decreased oxygen penetration depth (2.5-4.0 vs 1.0-2.0 mm). However, despite the shrinkage of the oxidized microlayer, no differences were found in phosphate flux across the sediment water interface. Batch experiments with standard additions of methane revealed that the growth of methanotrophic bacteria contributes to the phosphate uptake of aerobic sediment. From the batch experiments a molar ratio of carbon to phosphate of 45 mol:mol was calculated for the growth of methanotrophs. Results suggest that a decrease in chemical phosphate adsorption caused by a decrease in the oxygen penetration depth could be compensated for entirely by the growth of methanotrophic bacteria.

Radical reactions of c60.

Photochemically generated benzyl radicals react with C(60) producing radical and nonradical adducts Rn C(60) (R = C(6)H(5)CH(2)) with n = 1 to at least 15. The radical adducts with n = 3 and 5 are stable above 50 degrees C and have been identified by electron spin resonance (ESR) spectroscopy as the allylic R(3)C(60)(.) (3) and cyclopentadienyl R(5)C(60)(.) (5) radicals. The unpaired electrons are highly localized on the C(60) surface. The extraordinary stability of these radicals can be attributed to the steric protection of the surface radical sites by the surrounding benzyl substituents. Photochemically generated methyl radicals also add readily to C(60). Mass spectrometric analyses show the formation of (CH(3))nC(60) with n = 1 to at least 34.

Lever attacking and pressing as a function of conditioning and extinguishing a lever-press avoidance response in rats.

Six experimental rats were conditioned to press one of two available levers to avoid shock. The levers registered bites as well as presses. For four of these rats, shock was contingent on lever bites when a specified time period had elapsed after the previous shock. An extinction period, in which only periodic noncontingent shocks were presented, followed avoidance training. Six yoked-control rats received the same sequence of shocks as did the corresponding experimental rats in both the conditioning and extinction phases. All six experimental rats repeatedly bit the avoidance lever. Four bit it more than the nonavoidance lever during conditioning, and five bit it more during extinction. Five of the six experimental rats consistently bit the levers many more times during each session than did their respective control rats, suggesting that avoidance conditioning facilitated lever biting. Rates of lever biting and pressing by all of the experimental rats and by some of the control rats were highest immediately following shock throughout both phases. During later portions of the intervals following shock, characteristic effects of conditioning and extinction were observed. This finding suggests that extinction of avoidance behavior by unavoidable shock presentations can be demonstrated more readily when shock-elicited responding is extricated from the data.