Curcumin Intake Affects miRNA Signature in Murine Melanoma with mmu-miR-205-5p Most Significantly Altered

Melanoma is the most aggressive form of skin cancer with estimated 48,000 deaths per year worldwide. The polyphenol curcumin derived from the plant Curcuma longa is well known for its anti-inflammatory and anti-cancerogenic properties. Accordingly, dietary intake of this compound may be suitable for melanoma prevention. However, how this compound affects basic cellular mechanisms in developing melanoma still remains elusive. Therefore, the aim of this study was to investigate for the first time the impact of oral curcumin administration on the miRNA signature of engrafting melanoma. For this purpose, the effects of a 4% curcumin diet were tested on melanoma, which were established by injection of murine B78H1 cells in the flank of C57BL/6 mice. Curcumin diet or standard chow (control) was administered two weeks prior to injection of tumor cells until termination of the experiment. High throughput chip-based array analysis was deployed to detect alterations in the miRNA signature of the tumors. Curcumin treatment significantly reduced the growth of the flank tumors. Furthermore the miRNA expression signature in tumors was substantially altered by curcumin intake with mmu-miR-205-5p over 100 times higher expressed when compared to controls. The expression levels of identified key miRNAs in the tumor samples were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). A comparable expression pattern of these miRNAs was also detected in other curcumin-treated melanoma cell lines under in vitro conditions. Putative targets of curcumin-induced up-regulated miRNAs were enriched in ‘o-glycan biosynthesis’, ‘endoplasmatic reticulum protein processing’ and different cancer-related pathways. Western Blot analyses revealed that of these targets anti-apoptotic B-cell CLL/lymphoma 2 (Bcl-2) and proliferating cell nuclear antigen (PCNA) were significantly down-regulated in curcumin-treated tumors. These findings demonstrate a profound alteration of the miRNA expression signature in engrafting curcumin-treated melanoma with mmu-miR-205-5p being up-regulated most significantly.     

Clonal Differences in Expression of 25-Hydroxyvitamin D3-1α-hydroxylase, of 25-Hydroxyvitamin D3-24-hydroxylase, and of the Vitamin D Receptor in Human Colon Carcinoma Cells: Effects of Epidermal Growth Factor and 1α,25-Dihydroxyvitamin D3

Human colon carcinoma cells express 25-hydroxyvitamin D₃-1α-hydroxylase (CYP27B1) and thus produce the vitamin D receptor (VDR) ligand 1α,25-dihydroxyvitamin D₃ (1,25-D3), which can be metabolized by 25-hydroxyvitamin D₃-24-hydroxylase (CYP24). Expression of VDR, CYP27B1, and CYP24 determines the efficacy of the antimitotic action of 1,25-D3 and is distinctly related to the degree of differentiation of cancerous lesions. In the present study we addressed the question of whether the effects of epidermal growth factor (EGF) and of 1,25-D3 on VDR, CYP27B1, and CYP24 gene expression in human colon carcinoma cell lines also depend on the degree of cellular differentiation. We were able to show that slowly dividing, highly differentiated Caco-2/15 cells responded in a dose-dependent manner to both EGF and 1,25-D3 by up-regulation of VDR and CYP27B1 expression, whereas in highly proliferative, less differentiated cell lines, such as Caco-2/AQ and COGA-1A and -1E, negative regulation was observed. CYP24 mRNA was inducible in all clones by 1,25-D3 but not by EGF. From the observed clonal differences in the regulatory effects of EGF and 1,25-D3 on VDR and CYP27B1 gene expression we suggest that VDR-mediated growth inhibition by 1,25-D3 would be efficient only in highly differentiated carcinomas even when under mitogenic stimulation by EGF.     

In Vivo Epigenomic Profiling of Germ Cells Reveals Germ Cell Molecular Signatures

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Highlights? Modified small-scale ChIP-seq method applicable to small number of cells ? Genome-wide maps of H3K4me3, H3K27me3, H3K27ac, and H2BK20ac of germ cells in vivo ? Identification of active and inactive regulatory elements in germ cells in vivo ? Germ cell H3K27me3 regions are enriched for retrotransposon repeats     

Ghrelin Receptor (GHS-R1A) Antagonism Suppresses Both Alcohol Consumption and the Alcohol Deprivation Effect in Rats following Long-Term Voluntary Alcohol Consumption

Alcohol dependence is a heterogeneous disorder where several signalling systems play important roles. Recent studies implicate that the gut-brain hormone ghrelin, an orexigenic peptide, is a potential mediator of alcohol related behaviours. Ghrelin increases whereas a ghrelin receptor (GHS-R1A) antagonist decreases alcohol consumption as well as operant self-administration of alcohol in rodents that have consumed alcohol for twelve weeks. In the present study we aimed at investigating the effect of acute and repeated treatment with the GHS-R1A antagonist JMV2959 on alcohol intake in a group of rats following voluntarily alcohol consumption for two, five and eight months. After approximately ten months of voluntary alcohol consumption the expression of the GHS-R1A gene (Ghsr) as well as the degree of methylation of a CpG island found in Ghsr was examined in reward related brain areas. In a separate group of rats, we examined the effect of the JMV2959 on alcohol relapse using the alcohol deprivation paradigm. Acute JMV2959 treatment was found to decrease alcohol intake and the effect was more pronounced after five, compared to two months of alcohol exposure. In addition, repeated JMV2959 treatment decreased alcohol intake without inducing tolerance or rebound increase in alcohol intake after the treatment. The GHS-R1A antagonist prevented the alcohol deprivation effect in rats. There was a significant down-regulation of the Ghsr expression in the ventral tegmental area (VTA) in high- compared to low-alcohol consuming rats after approximately ten months of voluntary alcohol consumption. Further analysis revealed a negative correlation between Ghsr expression in the VTA and alcohol intake. No differences in methylation degree were found between high- compared to low-alcohol consuming rats. These findings support previous studies showing that the ghrelin signalling system may constitute a potential target for development of novel treatment strategies for alcohol dependence.     

Trypsin inhibitor activity in mature tobacco and tomato plants is mainly induced locally in response to insect attack,wounding and virus infection

Wounding of plants by insects is often mimicked in the laboratory by mechanical means such as cutting or crushing, and has not been compared directly with other forms of biotic stress such as virus infection. To compare the response of plants to these types of biotic and abiotic stress, trypsin inhibitor (TI) activity induced locally and systemically in mature tobacco (Nicotiana tabacum L.) and tomato (Lycopersicon esculentum L.) plants was followed for 12 days. In tobacco, cutting, crushing and insect feeding all induced comparable levels of TI activity of approx. 5 nmol·(mg leaf protein)^?1 in wounded leaves, while tobacco mosaic virus (TMV) infection of tobacco induced 10-fold lower amounts in the infected leaves. In tomato, feeding by insects also led to the induction of a level of TI activity of 5 nmol·(mg leaf protein)^?1. In contrast, both cutting and crushing of tomato leaves induced 10-fold higher amounts. These data show that biotic stress, in the form of insect feeding and TMV infection, and abiotic stress, in the form of wounding, have different effects on local levels of induced TI activity in mature tobacco and tomato plants. Irrespective of the type of wounding, in neither tobacco nor tomato could systemic induction of TI activity be observed in nearby unwounded leaves, which suggests that systemic induction of TI activity in mature tobacco and tomato plants is different from systemic TI induction in seedlings. Wounding of tobacco leaves, however, did increase the responsiveness to wounding elsewhere in the plant, as measured by an increased induction of TI activity.     

Plant sphingolipids: structural diversity,biosynthesis, first genes and functions

In mammals and Saccharomyces cerevisiae, sphingolipids have been a subject of intensive research triggered by the interest in their structural diversity and in mammalian pathophysiology as well as in the availability of yeast mutants and suppressor strains. More recently, sphingolipids have attracted additional interest, because they are emerging as an important class of messenger molecules linked to many different cellular functions. In plants, sphingolipids show structural features differing from those found in animals and fungi, and much less is known about their biosynthesis and function. This review focuses on the sphingolipid modifications found in plants and on recent advances in the functional characterization of genes gaining new insight into plant sphingolipid biosynthesis. Recent studies indicate that plant sphingolipids may be also involved in signal transduction, membrane stability, host-pathogen interactions and stress responses.     

Testosterone and its effects on courtship in golden-collared manakins (Manacus vitellinus): seasonal, sex, and age differences

Male golden-collared manakins gather on leks and perform an acrobatic display to attract females. In temperate breeding species, testosterone (T) activation of courtship displays has been well studied. Few studies have examined T activation of displays in tropical species; even fewer have explored the activational role of T in elaborate courtship displays such as in the manakin. In some tropical species, including manakins, territorial aggression or song behavior are uncoupled from T. We have previously shown that T activates display behavior in manakin males when endogenous T levels are low in the non-courtship season. To understand how T functions in breeding birds, we examined T levels in a large group of manakins sampled during the courtship and non-courtship season. In addition, during the courtship season, we gave T implants to adult males, juvenile males, and females. We found that T levels were low during the non-courtship season and comparatively higher on average during the courtship season. However, T levels were low in many adult males during the courtship season, especially when compared to temperate breeding species. Regardless of initial endogenous T levels during the courtship season, T implants did not increase the display frequency of adult males. T-treated females and juvenile males did display under similar conditions. Our data suggest that the effects of T on manakin display vary with season, sex, and age and that high T is not necessary for display.     

Mutational analysis of cytochrome b at the ubiquinol oxidation site of yeast complex III

The cytochrome bc1 complex is a dimeric enzyme of the inner mitochondrial membrane that links electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which ubiquinol is oxidized at one center in the enzyme, referred to as center P, and ubiquinone is rereduced at a second center, referred to as center N. To better understand the mechanism of ubiquinol oxidation, we have examined catalytic activities and pre-steady-state reduction kinetics of yeast cytochrome bc1 complexes with mutations in cytochrome b that we expected would affect oxidation of ubiquinol. We mutated two residues thought to be involved in proton conduction linked to ubiquinol oxidation, Tyr132 and Glu272, and two residues proposed to be involved in docking ubiquinol into the center P pocket, Phe129 and Tyr279. Substitution of Phe129 by lysine or arginine yielded a respiration-deficient phenotype and lipid-dependent catalytic activity. Increased bypass reactions were detectable for both variants, with F129K showing the more severe effects. Substitution with lysine leads to a disturbed coordination of a b heme as deduced from changes in the midpoint potential and the EPR signature. Removal of the aromatic side chain in position Tyr279 lowers the catalytic activity accompanied by a low level of bypass reactions. Pre-steady-state kinetics of the enzymes modified at Glu272 and Tyr132 confirmed the importance of their functional groups for electron transfer. Altered center N kinetics and activation of ubiquinol oxidation by binding of cytochrome c in the Y132F and E272D enzymes indicate long range effects of these mutations.