This latter finding is of particular importance as previous studies have demonstrated that LKB1 is nuclear localized and sequestered from its cytosolic targets such as AMPK. In contrast, coexpression of STRAD with LKB1 in culture cell systems results in nuclear export of the LKB1/STRAD/MO25 ternary complex, suggesting that the assembly state of the LKB1 subunit complex is responsible for nuclear export. However, whether this interaction has any physiological BAY 43-9006 meanings is still controversial since we were unable to show any significant difference in STRAD or MO25 expression or association with LKB1 in physiologic conditions. On the other hand, we have shown that inhibition of Fyn kinase efficiently upregulates AMPK activity through LKB1 redistribution. Thus, the regulation of Fyn binding/phosphorylation of LKB1 can account for the normal physiologic regulation of nuclear LKB1 export. The role of Fyn-LKB1 interaction in the control of AMPK and downstream signalling in cultured C2C12 myotubes and skeletal muscle in vivo was clearly demonstrated by using a cell permeable synthetic peptide of the LKB1 proline-rich domain. This peptide was able to dissociate Fyn from LKB1 and importantly, it increased the phosphorylation levels of the activating site of the AMPK a subunit resulting in increased AMPK activity, as detected by phosphorylation of the specific AMPK substrate ACC. This latter finding also suggests that developing novel therapeutic approaches that disrupt the Fyn-LKB1 interaction will have the biological effect of activating AMPK, a pathway that in principal will increase fatty acid utilization and improve insulin sensitivity in peripheral tissues. In summary, this study has identified the structural elements responsible for the direct binding interaction between the tyrosine kinase Fyn and the LKB1 catalytic subunit that is primarily responsible for the activation of the energy-sensing AMPK. Disruption of this interaction using LKB1-proline rich domain mimicking peptides recapitulates the pharmacological effects of Fyn kinase inhibition/deletion in vitro and in vivo. MicroRNAs are abundant small RNA molecules that act as post-transcriptional regulators of gene expression in higher organisms. Two properties of miRNAs make them especially promising as extracellular disease biomarkers. Firstly, miRNAs act as master regulators of cellular pathways. Therefore, by measuring miRNA expression, inferences can be made about pathophysiological processes in the tissue of origin. Secondly, miRNAs exhibit remarkable stability in extracellular biofluids such as serum on account of their association with proteins/lipoproteins or by encapsulation within extracellular vesicles. Serum miRNAs have attracted much interest as biomarkers for a wide range of disease conditions, especially in the case of human malignancy.

In this work we have analyzed the contribution of factors others than T3 itself, that might be involved in the regulation of gene expression and that may play diverse roles in vivo. This experimental set up allowed us to confirm that genes which expression in the mouse cerebral cortex is strongly dependent on thyroid hormones are indeed cellular targets of T3. The altered expression previously observed in vivo after induction of hypothyroidism was not due to a distal effect of the hypothyroid condition, but reflected a cellular effect of the hormone. The cellular system employed in this work is based on the culture of primary cells isolated from the embryonic mouse brain. These cultures are enriched in neurons but also contain astrocytes and unidentified cells. Therefore any effect observed by hormonal manipulations of these cells could have its origin in neurons or in astrocytes, depending among other factors on the relative expression of the target gene in each of these cell types. First we analyzed the specific contribution of the TRa1 and TRb receptor subtypes by measuring the response to T3 of primary cells derived from the cerebral cortex of TRa1 and TRb KO mice. The results confirm that at the cellular level both TRa1 and TRb mediate the effects of T3, with two exceptions, Dio3 and Aldh1a1. Dio3 was already shown to be regulated specifically by TRa1, and we confirm this fact in the cultured neurons. Also Aldh1a1 appears to be regulated specifically by TRa1, since no induction by T3 was observed in cells derived from TRa1 KO mice. Klf9 and Aldh1a3 show no preference for each of the TR subtypes. A recent global analysis of TR specificity in HeLa cells expressing exogenous TRa1 or TRb1 concluded that there are no complete TR subtype specificity, although TRa1 or TRb1 showed some gene preferences, depending on the time of exposure and the dose of T3. In established neural cell lines expression TRa1 or TRb1 lead to substantial differences in the gene network regulated by T3, without correlation with differential chromatin occupancy. Direct transcriptional regulation by T3, has previously been shown for Hr, Klf9 Dio3, and Shh. Here we confirm that also in Y-27632 cerebrocortical primary cells Hr, Klf9, and Shh are direct transcriptional targets of T3, given that the effect of the hormone was not blocked by previous treatment with CHX to inhibit protein synthesis. We could not determine whether Dio3 was also transcriptionally regulated by T3 because it was not stimulated by T3 at 6 hours of incubation, preventing to test the effect of CHX. However, a specific TRa1 binding site has been demonstrated in the upstream region of the gene. The fact that we could not determine whether the effect of T3 was direct, suggest the possibility that the full effect of T3 requires interaction with other intermediate proteins. Dio3 is a transcriptional target of Shh which, as mentioned above is a direct T3 target. This raises the possibility that rapid and full induction of Dio3 by T3 is the result of a direct transcriptional effect of TRa1 potentiated by the T3- dependent accumulation of Shh protein.

However, our expression data did not support co-regulation of SREBF2 and miR-33 expression across 10 types of chicken tissues examined. Predicting targets is an important first step to determine the function of a miRNA. Many algorithms and databases for miRNA target predictions have been established, and among them, miRanda, TargetScan, and PicTar, appear to be the most widely used miRNA target prediction methods. In this study, 378 genes were predicted as the target genes of miR-33 among the total 11,891 chicken genes within the 39UTR database using “miRanda”. The “TargetScan” principle was also applied in the prediction procedures: the target site should match to the seed region of miRNA, the 8th nucleotide of miRNA should also be a match or the target nucleotide corresponding to the first nucleotide of miRNA should be an A. One of the predicted target genes of miR-33 named FTO is a member of the non-heme dioxygenase superfamily, and has been recently implicated in regulation of lipid and energy metabolism. Dual-luciferase reporter assays and site mutation analyses validated that chicken FTO was a target gene of miR-33. Because in chickens de novo fatty acid synthesis occurs primarily in the liver, we further studied the possibility that miR-33 targets FTO in the chicken liver. One of the most powerful and straightforward ways to determine the relationship between a miRNA and a mRNA in tissues or cells is to determine the effect of knockdown of the miRNA on the expression of the mRNA of interest. Using LNAanti-miR-33, we successfully reduced the expression of endogenous miR-33 in primary chicken hepatocytes, and this reduction was associated with an up-regulated expression of FTO mRNA. This association supports that the FTO gene is targeted by miR-33 in chicken hepatocytes. We also observed that miR-33 and FTO mRNA expression were inversely correlated in chicken liver at most of the developmental ages examined. At day 35 and day 42 of age, the expressions of miR-33 and FTO mRNA were not inversely correlated. This suggests that the expression of FTO at these two stages may be regulated LDK378 predominantly by mechanisms other than miR-33. In the chicken, FTO is widely expressed. Expression of FTO in the hypothalamic nuclei involved in energy balance regulation has been shown to respond to nutritional manipulations such as feeding and fasting. Fasting has been shown to also increase FTO gene expression in the cerebrum, liver, breast muscle and subcutaneous fat. Alterations in feeding status resulted in significant changes in FTO expression in the liver, but not in other tissues of broiler chickens. In addition to this, hepatic FTO expression changes in response to metabolic states, and glucose reduces hepatic FTO mRNA expression independently of body weight. Since miR-33 inhibits the expression of FTO, it might play a role in mediating the nutritional regulation of FTO expression in chicken liver.

This investigation establishes the first record of Bd and Ranavirus presence in amphibian trade in Hong Kong and demonstrates an opportunity for exposure to native amphibians. In addition, this is the first report to the authors’ knowledge of ranavirus detection in the three species testing positive in this study. Unlike previous amphibian trade investigations in Southeast Asia, a relatively high proportion of Hong Kong’s traded animals tested positive for both of these significant pathogens. Risk of pathogen spillover and potential establishment is elevated by the regions’ high volume of domestic trade in species with known pathogen susceptibility and the likelihood to persist in the wild in a wide range of habitats if released or escaped, including the Chinese bullfrog and African clawed frog. The previous lack of Bd detection in the wild and in Hong Kong trade by Rowley et al. is surprising, given the findings of this study and the long-term presence of international amphibian trade in the region. Indications of amphibian escape or release from the exotic pet trade into the wild date back as far as 1977, with Japanese red-bellied newts recorded from Sha Tau Kok in the New Territories. Due to the narrow diversity of native amphibian species previously evaluated for Bd infection and sampling bias towards post-metamorphic specimens, it remains possible that amphibian pathogens have evaded detection. Previous surveys throughout Asia have generally demonstrated widespread Bd distribution at low prevalence, and have provided some evidence for the presence of an endemic Asian lineage of Bd. Therefore, although the possibility of an historic introduction of Bd and long-term presence in Hong Kong cannot be fully disregarded, the absence of both prior detection and disease-suspected population declines suggests this phenomenon would be of low conservation concern relative to the contemporary importation of exotic disease strains that typically express higher virulence than strains considered endemic to the region. The risk of establishment of highly virulent trade-associated strains of Bd and ranavirus in Hong Kong depends largely on the continued importation and domestic sale of diseased amphibians. An analysis of trade activity from 2005–2006 showed the importation of nearly 4.3 million live amphibians into Hong Kong, comprised of at least 45 species originating from 11 countries, nine of which have reported presence of Bd and/or ranavirus in wild or traded herptiles. The majority of this trade volume involved bullfrogs intended for human consumption in Hong Kong and originated in either Thailand or China, where these pathogens have been detected in farmed and free-ranging amphibians. The risk of pathogen spillover from trade into the wild in Hong Kong is heightened by several factors additional to those mentioned above. First, each of these pathogens may cause mortality in the host with the absence of other clinical signs or lesions prior to death, which makes visual identification of illness HhAntag691 side effects difficult for traders.

Mammalian cells have a high capacity system for oxidative disposal of branched-chain amino acids. In contrast to other essential amino acids, which are primarily oxidized in the liver, the most active system for the oxidation of BCAA is located in skeletal muscle cells. The degradation of BCAA mainly occurs in the mitochondria via reversible transamination by branched-chain aminotransferase to produce the corresponding branched-chain a-keto acids, which in turn are subjected to oxidative decarboxylation by branched-chain a-keto acid dehydrogenase to produce CoA esters. The enzymes that catalyze these two reactions are common to the three BCAA. The second step enzyme, BCKDH, catalyzes an irreversible reaction that commits individual BCKA to their respective degradation pathways and is considered to be the most important regulatory enzyme in the catabolism of the three BCAA. BCKDH activity is Axitinib regulated by BCKDH kinase ; BCKDH phosphorylation attenuates its enzyme activity. In this study, microarray analysis revealed that the BCAA catabolic pathway was coordinately activated in skeletal muscle of transgenic mice overexpressing PGC-1a. Thus, we investigated whether PGC-1a stimulates BCAA metabolism with an increase in the expression of enzymes involved in BCAA metabolism, such as BCAT, BCKDH and BCKDK, using cultured cells and murine skeletal muscle overexpressing PGC-1a. The high volume of global trade in potentially diseased amphibians has sparked a series of investigations into its role as a primary driver of the emergence and spread of amphibian chytrid fungus and ranaviruses, threatening global amphibian biodiversity. With respect to Bd, particular concern has been expressed regarding the transport of American bullfrogs, due to the species’ propensity to carry infection asymptomatically and serve as a reservoir of disease. Millions of L. catesbeianus are traded globally for consumption annually. High prevalence of Bd infection has been detected among this species sold in markets in the USA, imported primarily from Southeast Asia and South America. Furthermore, Bd-positive water often accompanying commercial amphibian shipments likewise represent a potential source of spread. Similarly, ranaviruses are emerging pathogens capable of causing mass mortality and localized population decline in amphibians, as well as reptiles and fish, and their spread shares many nuances with the global dispersal of Bd. Transmission of viral particles occurs through direct contact with infected individuals and exposure to contaminated water or soil. Its ability to infect three classes of ectotherms and the lack of an effective therapeutic treatment warrants serious consideration. The geographic spread of ranavirus also demonstrates strong association with the trade in live amphibians, most notably the trade in tiger salamanders and American bullfrogs. Investigations for the presence of these pathogens in both traded and free-ranging amphibians in Asian countries have produced mixed results, ranging from lack of detection to widespread low prevalence.