trans -2-(2,5-Dimethoxy-4-iodophenyl)cyclopropylamine and trans -2-(2,5-dimethoxy-4-bromophenyl)cyclopropylamine as potent agonists for the 5-HT 2 receptor family

A strategy to replace the ethylamine side chain of 2,5-dimethoxy-4-iodoamphetamine (DOI, 1a), and 2,5-dimethoxy-4-bromoamphetamine (DOB, 1b) with a cyclopropylamine moiety was successful in leading to compounds with high affinity at the 5-HT2 family of receptors; and the more potent stereoisomer of the cyclopropane analogues had the expected (−)-(1R,2S)-configuration. Screening for affinity at various serotonin receptor subtypes, however, revealed that the cyclopropane congeners also had increased affinity at several sites in addition to the 5-HT2A and 5-HT2B receptors. Therefore, at appropriate doses – although (−)-4 and (−)-5 may be useful as tools to probe 5-HT2 receptor function – one would need to be mindful that their selectivity for 5-HT2A receptors is somewhat less than for DOI itself

Intracellular Spatial Localization Regulated by the Microtubule Network

The commonly recognized mechanisms for spatial regulation inside the cell are membrane-bounded compartmentalization and biochemical association with subcellular organelles. We use computational modeling to investigate another spatial regulation mechanism mediated by the microtubule network in the cell. Our results demonstrate that the mitotic spindle can impose strong sequestration and concentration effects on molecules with binding affinity for microtubules, especially dynein-directed cargoes. The model can recapitulate the essence of three experimental observations on distinct microtubule network morphologies: the sequestration of germ plasm components by the mitotic spindles in the Drosophila syncytial embryo, the asymmetric cell division initiated by the time delay in centrosome maturation in the Drosophila neuroblast, and the diffusional block between neighboring energids in the Drosophila syncytial embryo. Our model thus suggests that the cell cycle-dependent changes in the microtubule network are critical for achieving different spatial regulation effects. The microtubule network provides a spatially extensive docking platform for molecules and gives rise to a “structured cytoplasm”, in contrast to a free and fluid environment

JHDM1B/FBXL10 is a nucleolar protein that represses transcription of ribosomal RNA genes

JHDM1B is an evolutionarily conserved and ubiquitously expressed member of the JHDM (JmjC-domain-containing his- tone demethylase) family1\u20133. Because it contains an F-box motif, this protein is also known as FBXL10 (ref. 4). With the use of a genome-wide RNAi screen, the JHDM1B worm orthologue (T26A5.5) was identified as a gene that regulates growth5. In the mouse, four independent screens have identified JHDM1B as a putative tumour suppressor by retroviral insertion analysis6\u20139. Here we identify human JHDM1B as a nucleolar protein and show that JHDM1B preferentially binds the transcribed region of ribosomal DNA to repress the transcription of ribosomal RNA genes. We also show that repression of ribosomal RNA genes by JHDM1B is dependent on its JmjC domain, which is necessary for the specific demethylation of trimethylated lysine 4 on histone H3 in the nucleolus. In agreement with the notion that ribosomal RNA synthesis and cell growth are coupled processes, we show a JmjC-domain-dependent negative effect of JHDM1B on cell size and cell proliferation. Because aberrant ribosome biogenesis and the disruption of epigenetic control mechanisms contribute to cellular transformation, these results, together with the low levels of JHDM1B expression found in aggressive brain tumours, suggest a role for JHDM1B in cancer development

A Buoyancy-Based Screen of Drosophila Larvae for Fat-Storage Mutants Reveals a Role for Sir2 in Coupling Fat Storage to Nutrient Availability

Obesity has a strong genetic component, but few of the genes that predispose to obesity are known. Genetic screens in invertebrates have the potential to identify genes and pathways that regulate the levels of stored fat, many of which are likely to be conserved in humans. To facilitate such screens, we have developed a simple buoyancy-based screening method for identifying mutant Drosophila larvae with increased levels of stored fat. Using this approach, we have identified 66 genes that when mutated increase organismal fat levels. Among these was a sirtuin family member, Sir2. Sirtuins regulate the storage and metabolism of carbohydrates and lipids by deacetylating key regulatory proteins. However, since mammalian sirtuins function in many tissues in different ways, it has been difficult to define their role in energy homeostasis accurately under normal feeding conditions. We show that knockdown of Sir2 in the larval fat body results in increased fat levels. Moreover, using genetic mosaics, we demonstrate that Sir2 restricts fat accumulation in individual cells of the fat body in a cell-autonomous manner. Consistent with this function, changes in the expression of metabolic enzymes in Sir2 mutants point to a shift away from catabolism. Surprisingly, although Sir2 is typically upregulated under conditions of starvation, Sir2 mutant larvae survive better than wild type under conditions of amino-acid starvation as long as sugars are provided. Our findings point to a Sir2-mediated pathway that activates a catabolic response to amino-acid starvation irrespective of the sugar content of the diet

The Tumor Suppressive Role of eIF3f and Its Function in Translation Inhibition and rRNA Degradation

Deregulated translation plays an important role in human cancer. We previously reported decreased eukaryotic initiation factor 3 subunit f (eIF3f) expression in pancreatic cancer. Whether decreased eIF3f expression can transform normal epithelial cells is not known. In our current study, we found evidence that stable knockdown of eIF3f in normal human pancreatic ductal epithelial cells increased cell size, nuclear pleomorphism, cytokinesis defects, cell proliferation, clonogenicity, apoptotic resistance, migration, and formation of 3-dimensional irregular masses. Our findings support the tumor suppressive role of eIF3f in pancreatic cancer. Mechanistically, we found that eIF3f inhibited both cap-dependent and cap-independent translation. An increase in the ribosomal RNA (rRNA) level was suggested to promote the generation of cancer. The regulatory mechanism of rRNA degradation in mammals is not well understood. We demonstrated here that eIF3f promotes rRNA degradation through direct interaction with heterogeneous nuclear ribonucleoprotein (hnRNP) K. We showed that hnRNP K is required for maintaining rRNA stability: under stress conditions, eIF3f dissociates hnRNP K from rRNA, thereby preventing it from protecting rRNA from degradation. We also demonstrated that rRNA degradation occurred in non-P body, non-stress granule cytoplasmic foci that contain eIF3f. Our findings established a new mechanism of rRNA decay regulation mediated by hnRNP K/eIF3f and suggest that the tumor suppressive function of eIF3f may link to impaired rRNA degradation and translation

NOF1 Encodes an Arabidopsis Protein Involved in the Control of rRNA Expression

The control of ribosomal RNA biogenesis is essential for the regulation of protein synthesis in eukaryotic cells. Here, we report the characterization of NOF1 that encodes a putative nucleolar protein involved in the control of rRNA expression in Arabidopsis. The gene has been isolated by T-DNA tagging and its function verified by the characterization of a second allele and genetic complementation of the mutants. The nof1 mutants are affected in female gametogenesis and embryo development. This result is consistent with the detection of NOF1 mRNA in all tissues throughout plant life's cycle, and preferentially in differentiating cells. Interestingly, the closely related proteins from zebra fish and yeast are also necessary for cell division and differentiation. We showed that the nof1-1 mutant displays higher rRNA expression and hypomethylation of rRNA promoter. Taken together, the results presented here demonstrated that NOF1 is an Arabidopsis gene involved in the control of rRNA expression, and suggested that it encodes a putative nucleolar protein, the function of which may be conserved in eukaryotes

Cks1 Is Required for Tumor Cell Proliferation but Not Sufficient to Induce Hematopoietic Malignancies

The Cks1 component of the SCFSkp2 complex is necessary for p27Kip1 ubiquitylation and degradation. Cks1 expression is elevated in various B cell malignancies including Burkitt lymphoma and multiple myeloma. We have previously shown that loss of Cks1 results in elevated p27Kip1 levels and delayed tumor development in a mouse model of Myc-induced B cell lymphoma. Surprisingly, loss of Skp2 in the same mouse model also resulted in elevated p27Kip1 levels but exhibited no impact on tumor onset. This raises the possibility that Cks1 could have other oncogenic activities than suppressing p27Kip1. To challenge this notion we have targeted overexpression of Cks1 to B cells using a conditional retroviral bone marrow transduction-transplantation system. Despite potent ectopic overexpression, Cks1 was unable to promote B cell hyperproliferation or B cell malignancies, indicating that Cks1 is not oncogenic when overexpressed in B cells. Since Skp2 overexpression can drive T-cell tumorigenesis or other cancers we also widened the quest for oncogenic activity of Cks1 by ubiquitously expressing Cks1 in hematopoetic progenitors. At variance with c-Myc overexpression, which caused acute myeloid leukemia, Cks1 overexpression did not induce myeloproliferation or leukemia. Therefore, despite being associated with a poor prognosis in various malignancies, sole Cks1 expression is insufficient to induce lymphoma or a myeloproliferative disease in vivo

Exploring the molecular mechanisms underlying the potentiation of exogenous growth hormone on alcohol-induced fatty liver diseases in mice

<p>Abstract</p> <p>Background</p> <p>Growth hormone (GH) is an essential regulator of intrahepatic lipid metabolism by activating multiple complex hepatic signaling cascades. Here, we examined whether chronic exogenous GH administration (via gene therapy) could ameliorate liver steatosis in animal models of alcoholic fatty liver disease (AFLD) and explored the underlying molecular mechanisms.</p> <p>Methods</p> <p>Male C57BL/6J mice were fed either an alcohol or a control liquid diet with or without GH therapy for 6 weeks. Biochemical parameters, liver histology, oxidative stress markers, and serum high molecular weight (HMW) adiponectin were measured. Quantitative real-time PCR and western blotting were also conducted to determine the underlying molecular mechanism.</p> <p>Results</p> <p>Serum HMW adiponectin levels were significantly higher in the GH1-treated control group than in the control group (3.98 ± 0.71 μg/mL vs. 3.07 ± 0.55 μg/mL; <it>P </it>< 0.001). GH1 therapy reversed HMW adiponectin levels to the normal levels in the alcohol-fed group. Alcohol feeding significantly reduced hepatic adipoR2 mRNA expression compared with that in the control group (0.71 ± 0.17 vs. 1.03 ± 0.19; <it>P </it>< 0.001), which was reversed by GH therapy. GH1 therapy also significantly increased the relative mRNA (1.98 ± 0.15 vs. 0.98 ± 0.15) and protein levels of SIRT1 (2.18 ± 0.37 vs. 0.99 ± 0.17) in the alcohol-fed group compared with those in the control group (both, <it>P </it>< 0.001). The alcohol diet decreased the phosphorylated and total protein levels of hepatic AMP-activated kinase-α (AMPKα) (phosphorylated protein: 0.40 ± 0.14 vs. 1.00 ± 0.12; total protein: 0.32 ± 0.12 vs. 1.00 ± 0.14; both, <it>P </it>< 0.001) and peroxisome proliferator activated receptor-α (PPARα) (phosphorylated protein: 0.30 ± 0.09 vs. 1.00 ± 0.09; total protein: 0.27 ± 0.10 vs. 1.00 ± 0.13; both, <it>P </it>< 0.001), which were restored by GH1 therapy. GH therapy also decreased the severity of fatty liver in alcohol-fed mice.</p> <p>Conclusions</p> <p>GH therapy had positive effects on AFLD and may offer a promising approach to prevent or treat AFLD. These beneficial effects of GH on AFLD were achieved through the activation of the hepatic adiponectin-SIRT1-AMPK and PPARα-AMPK signaling systems.</p

In Vivo Monitoring of mRNA Movement in Drosophila Body Wall Muscle Cells Reveals the Presence of Myofiber Domains

Background: In skeletal muscle each muscle cell, commonly called myofiber, is actually a large syncytium containing numerous nuclei. Experiments in fixed myofibers show that mRNAs remain localized around the nuclei in which they are produced. Methodology/Principal Findings: In this study we generated transgenic flies that allowed us to investigate the movement of mRNAs in body wall myofibers of living Drosophila embryos. We determined the dynamic properties of GFP-tagged mRNAs using in vivo confocal imaging and photobleaching techniques and found that the GFP-tagged mRNAs are not free to move throughout myofibers. The restricted movement indicated that body wall myofibers consist of three domains. The exchange of mRNAs between the domains is relatively slow, but the GFP-tagged mRNAs move rapidly within these domains. One domain is located at the centre of the cell and is surrounded by nuclei while the other two domains are located at either end of the fiber. To move between these domains mRNAs have to travel past centrally located nuclei. Conclusions/Significance: These data suggest that the domains made visible in our experiments result from prolonged interactions with as yet undefined structures close to the nuclei that prevent GFP-tagged mRNAs from rapidly moving between the domains. This could be of significant importance for the treatment of myopathies using regenerative cellbase