Transfer of exosomal microRNA-203-3p from dendritic cells to bone marrow-derived macrophages reduces development of atherosclerosis by downregulating Ctss in mice.

Dendritic cell-derived exosomes have been proven to be efficient adjuvant options for anti-tumor vaccines in cancer immunotherapy. However, their potency in atherosclerosis remains unclear. Here we summarize the association of microRNA-203-3p (miR-203-3p) with dendritic cell-derived exosomes and atherosclerosis. Firstly, dendritic cell-derived exosomes and bone marrow-derived macrophages were isolated, after which expression of miR-203-3p and cathepsin S was determined. After the establishment of atherosclerosis mouse models, gain- and loss-of-function experiments were conducted for the analysis of effects of miR-203-3p and cathepsin S on foam-cell formation, lipid accumulation, collagen deposition and serum total cholesterol. The results found high expression of cathepsin S in atherosclerosis mice and downregulation of miR-203-3p in the serum of atherosclerosis patients and ox-LDL-simulated bone marrow-derived macrophages. Cathepsin S was the target gene of miR-203-3p. miR-203-3p transporting from exosomes to bone marrow-derived macrophages resulted in inhibition of cathepsin S expression and atherosclerosis-related phenotypes in bone marrow-derived macrophages, thus alleviating atherosclerosis in mice, and this process was found to involve the p38/MAPK signaling pathway. These findings provided evidence that the transfer of miR-203-3p by dendritic cell-derived exosomes targeted cathepsin S in bone marrow-derived macrophages to attenuate atherosclerosis progression in mice, serving as a promising clinical target for atherosclerosis.

A choreography of centrosomal mRNAs reveals a conserved localization mechanism involving active polysome transport.

Local translation allows for a spatial control of gene expression. Here, we use high-throughput smFISH to screen centrosomal protein-coding genes, and we describe 8 human mRNAs accumulating at centrosomes. These mRNAs localize at different stages during cell cycle with a remarkable choreography, indicating a finely regulated translational program at centrosomes. Interestingly, drug treatments and reporter analyses reveal a common translation-dependent localization mechanism requiring the nascent protein. Using ASPM and NUMA1 as models, single mRNA and polysome imaging reveals active movements of endogenous polysomes towards the centrosome at the onset of mitosis, when these mRNAs start localizing. ASPM polysomes associate with microtubules and localize by either motor-driven transport or microtubule pulling. Remarkably, the Drosophila orthologs of the human centrosomal mRNAs also localize to centrosomes and also require translation. These data identify a conserved family of centrosomal mRNAs that localize by active polysome transport mediated by nascent proteins.

Endocytosis Controls siRNA Efficiency: Implications for siRNA Delivery Vehicle Design and Cell-Specific Targeting.

While small interfering RNAs (siRNAs) are commonly used for laboratory studies, development of siRNA therapeutics has been slower than expected, due, in part, to a still limited understanding of the endocytosis and intracellular trafficking of siRNA-containing complexes. With the recent characterization of multiple clathrin-/caveolin-independent endocytic pathways, that is, those mediated by Graf1, Arf6, and flotillin, it has become clear that the endocytic mechanism influences subsequent intracellular processing of the internalized cargo. To explore siRNA delivery in light of these findings, we developed a novel assay that differentiates uptake by each of the endocytic pathways and can be used to determine whether endocytosis by a pathway leads to the initiation of RNA interference (RNAi). Using Lipofectamine 2000 (LF2K), we determined the endocytosis pathway leading to active silencing (whether by clathrin, caveolin, Arf6, Graf1, flotillin, or macropinocytosis) across multiple cell types (HeLa, H1299, HEK293, and HepG2). We showed that LF2K is internalized by Graf1-, Arf6-, or flotillin-mediated endocytosis for the initiation of RNAi, depending on cell type. In addition, we found that a portion of siRNA-containing complexes is internalized by pathways that do not lead to initiation of silencing. Inhibition of these pathways enhanced intracellular levels of siRNAs with concomitant enhancement of silencing.

Selective Export into Extracellular Vesicles and Function of tRNA Fragments during T Cell Activation.

The discovery of microRNA (miRNA) sorting into extracellular vesicles (EVs) revealed a novel mode of intercellular communication and uncovered a link between cellular endomembrane compartments and small RNAs in EV-secreting cells. Using a two-step ultracentrifugation procedure to isolate EVs released by T cells, we found that 45% of tRNA fragments (tRFs), but fewer than 1% of miRNAs, were significantly enriched in EVs compared with the corresponding cellular RNA. T cell activation induced the EV-mediated release of a specific set of tRFs derived from the 5' end and 3'-internal region of tRNAs without variable loops. Inhibition of EV biogenesis pathways specifically led to the accumulation of these activation-induced EV-enriched tRFs within multivesicular bodies (MVBs). Introducing antisense oligonucleotides to inhibit these tRFs enhanced T cell activation. Taken together, these results demonstrate that T cells selectively release tRFs into EVs via MVBs and suggest that this process may remove tRFs that repress immune activation.

Expression of tomato prosystemin gene in Arabidopsis reveals systemic translocation of its mRNA and confers necrotrophic fungal resistance.

Systemin (SYS), an octadecapeptide hormone processed from a 200-amino-acid precursor (prosystemin, PS), plays a central role in the systemic activation of defense genes in tomato in response to herbivore and pathogen attacks. However, whether PS mRNA is transferable and its role in systemic defense responses remain unknown. We created the transgenic tomato PS gene tagged with the green fluorescent protein (PS-GFP) using a shoot- or root-specific promoter, and the constitutive 35S promoter in Arabidopsis. Subcellular localization of PS-/SYS-GFP was observed using confocal laser scanning microscopy and gene transcripts were determined using quantitative real-time PCR. In Arabidopsis, PS protein can be processed and SYS is secreted. Shoot-/root-specific expression of PS-GFP in Arabidopsis, and grafting experiments, revealed that the PS mRNA moves in a bi-directional manner. We also found that ectopic expression of PS improves Arabidopsis resistance to the necrotrophic fungus Botrytis cinerea, consistent with substantial upregulation of the transcript levels of specific pathogen-responsive genes. Our results provide novel insights into the multifaceted mechanism of SYS signaling transport and its potential application in genetic engineering for increasing pathogen resistance across diverse plant families.

Nuclear retention of the lncRNA SNHG1 by doxorubicin attenuates hnRNPC-p53 protein interactions.

The protein p53 plays a crucial role in the regulation of cellular responses to diverse stresses. Thus, a major priority in cell biology is to define the mechanisms that regulate p53 activity in response to stresses or maintain it at basal levels under normal conditions. Moreover, further investigation is required to establish whether RNA participates in regulating p53's interaction with other proteins. Here, by conducting systematic experiments, we discovered a p53 interactor-hnRNPC-that directly binds to p53, destabilizes it, and prevents its activation under normal conditions. Upon doxorubicin treatment, the lncRNA SNHG1 is retained in the nucleus through its binding with nucleolin and it competes with p53 for hnRNPC binding, which upregulates p53 levels and promotes p53-dependent apoptosis by impairing hnRNPC regulation of p53 activity. Our results indicate that a balance between lncRNA SNHG1 and hnRNPC regulates p53 activity and p53-dependent apoptosis upon doxorubicin treatment, and further indicate that a change in lncRNA subcellular localization under specific circumstances is biologically significant.

Daclatasvir Prevents Hepatitis C Virus Infectivity by Blocking Transfer of the Viral Genome to Assembly Sites.

BACKGROUND & AIMS: Daclatasvir is a direct-acting antiviral agent and potent inhibitor of NS5A, which is involved in replication of the hepatitis C virus (HCV) genome, presumably via membranous web shaping, and assembly of new virions, likely via transfer of the HCV RNA genome to viral particle assembly sites. Daclatasvir inhibits the formation of new membranous web structures and, ultimately, of replication complex vesicles, but also inhibits an early assembly step. We investigated the relationship between daclatasvir-induced clustering of HCV proteins, intracellular localization of viral RNAs, and inhibition of viral particle assembly. METHODS: Cell-culture-derived HCV particles were produced from Huh7.5 hepatocarcinoma cells in presence of daclatasvir for short time periods. Infectivity and production of physical particles were quantified and producer cells were subjected to subcellular fractionation. Intracellular colocalization between core, E2, NS5A, NS4B proteins, and viral RNAs was quantitatively analyzed by confocal microscopy and by structured illumination microscopy. RESULTS: Short exposure of HCV-infected cells to daclatasvir reduced viral assembly and induced clustering of structural proteins with non-structural HCV proteins, including core, E2, NS4B, and NS5A. These clustered structures appeared to be inactive assembly platforms, likely owing to loss of functional connection with replication complexes. Daclatasvir greatly reduced delivery of viral genomes to these core clusters without altering HCV RNA colocalization with NS5A. In contrast, daclatasvir neither induced clustered structures nor inhibited HCV assembly in cells infected with a daclatasvir-resistant mutant (NS5A-Y93H), indicating that daclatasvir targets a mutual, specific function of NS5A inhibiting both processes. CONCLUSIONS: In addition to inhibiting replication complex biogenesis, daclatasvir prevents viral assembly by blocking transfer of the viral genome to assembly sites. This leads to clustering of HCV proteins because viral particles and replication complex vesicles cannot form or egress. This dual mode of action of daclatasvir could explain its efficacy in blocking HCV replication in cultured cells and in treatment of patients with HCV infection.

Gap junction mediated miRNA intercellular transfer and gene regulation: A novel mechanism for intercellular genetic communication.

Intercellular genetic communication is an essential requirement for coordination of cell proliferation and differentiation and has an important role in many cellular processes. Gap junction channels possess large pore allowing passage of ions and small molecules between cells. MicroRNAs (miRNAs) are small regulatory RNAs that can regulate gene expression broadly. Here, we report that miRNAs can pass through gap junction channels in a connexin-dependent manner. Connexin43 (Cx43) had higher permeability, whereas Cx30 showed little permeability to miRNAs. In the tested connexin cell lines, the permeability to miRNAs demonstrated: Cx43 > Cx26/30 > Cx26 > Cx31 > Cx30 = Cx-null. However, consistent with a uniform structure of miRNAs, there was no significant difference in permeability to different miRNAs. The passage is efficient; the miRNA level in the recipient cells could be up to 30% of the donor level. Moreover, the transferred miRNA is functional and could regulate gene expression in neighboring cells. Connexin mutation and gap junctional blockers could eliminate this miRNA intercellular transfer and gene regulation. These data reveal a novel mechanism for intercellular genetic communication. Given that connexin expression is cell-specific, this connexin-dependent, miRNA intercellular genetic communication may play an important role in synchronizing and coordinating proliferation and differentiation of specific cell types during multicellular organ development.

MicroRNA Cargo of Extracellular Vesicles from Alcohol-exposed Monocytes Signals Naive Monocytes to Differentiate into M2 Macrophages.

Membrane-coated extracellular vesicles (EVs) released by cells can serve as vehicles for delivery of biological materials and signals. Recently, we demonstrated that alcohol-treated hepatocytes cross-talk with immune cells via exosomes containing microRNA (miRNAs). Here, we hypothesized that alcohol-exposed monocytes can communicate with naive monocytes via EVs. We observed increased numbers of EVs, mostly exosomes, secreted by primary human monocytes and THP-1 monocytic cells in the presence of alcohol in a concentration- and time-dependent manner. EVs derived from alcohol-treated monocytes stimulated naive monocytes to polarize into M2 macrophages as indicated by increased surface expression of CD68 (macrophage marker), M2 markers (CD206 (mannose receptor) and CD163 (scavenger receptor)), secretion of IL-10, and TGFß and increased phagocytic activity. miRNA profiling of the EVs derived from alcohol-treated THP-1 monocytes revealed high expression of the M2-polarizing miRNA, miR-27a. Treatment of naive monocytes with control EVs overexpressing miR-27a reproduced the effect of EVs from alcohol-treated monocytes on naive monocytes and induced M2 polarization, suggesting that the effect of alcohol EVs was mediated by miR-27a. We found that miR-27a modulated the process of phagocytosis by targeting CD206 expression on monocytes. Importantly, analysis of circulating EVs from plasma of alcoholic hepatitis patients revealed increased numbers of EVs that contained high levels of miR-27a as compared with healthy controls. Our results demonstrate the following: first, alcohol increases EV production in monocytes; second, alcohol-exposed monocytes communicate with naive monocytes via EVs; and third, miR-27a cargo in monocyte-derived EVs can program naive monocytes to polarize into M2 macrophages.

Yeast Integral Membrane Proteins Apq12, Brl1, and Brr6 Form a Complex Important for Regulation of Membrane Homeostasis and Nuclear Pore Complex Biogenesis.

Proper functioning of intracellular membranes is critical for many cellular processes. A key feature of membranes is their ability to adapt to changes in environmental conditions by adjusting their composition so as to maintain constant biophysical properties, including fluidity and flexibility. Similar changes in the biophysical properties of membranes likely occur when intracellular processes, such as vesicle formation and fusion, require dramatic changes in membrane curvature. Similar modifications must also be made when nuclear pore complexes (NPCs) are constructed within the existing nuclear membrane, as occurs during interphase in all eukaryotes. Here we report on the role of the essential nuclear envelope/endoplasmic reticulum (NE/ER) protein Brl1 in regulating the membrane composition of the NE/ER. We show that Brl1 and two other proteins characterized previously-Brr6, which is closely related to Brl1, and Apq12-function together and are required for lipid homeostasis. All three transmembrane proteins are localized to the NE and can be coprecipitated. As has been shown for mutations affecting Brr6 and Apq12, mutations in Brl1 lead to defects in lipid metabolism, increased sensitivity to drugs that inhibit enzymes involved in lipid synthesis, and strong genetic interactions with mutations affecting lipid metabolism. Mutations affecting Brl1 or Brr6 or the absence of Apq12 leads to hyperfluid membranes, because mutant cells are hypersensitive to agents that increase membrane fluidity. We suggest that the defects in nuclear pore complex biogenesis and mRNA export seen in these mutants are consequences of defects in maintaining the biophysical properties of the NE.

Neuron-wide RNA transport combines with netrin-mediated local translation to spatially regulate the synaptic proteome.

The persistence of experience-dependent changes in brain connectivity requires RNA localization and protein synthesis. Previous studies have demonstrated a role for local translation in altering the structure and function of synapses during synapse formation and experience-dependent synaptic plasticity. In this study, we ask whether in addition to promoting local translation, local stimulation also triggers directed trafficking of RNAs from nucleus to stimulated synapses. Imaging of RNA localization and translation in cultured Aplysia sensory-motor neurons revealed that RNAs were delivered throughout the arbor of the sensory neuron, but that translation was enriched only at sites of synaptic contact and/or synaptic stimulation. Investigation of the mechanisms that trigger local translation revealed a role for calcium-dependent retrograde netrin-1/DCC receptor signaling. Spatially restricting gene expression by regulating local translation rather than by directing the delivery of mRNAs from nucleus to stimulated synapses maximizes the readiness of the entire neuronal arbor to respond to local cues.

Long-distance transport of Gibberellic Acid Insensitive mRNA in Nicotiana benthamiana.

BACKGROUND: The Gibberellic Acid (GA) signal is governed by the GAI (Gibberellic Acid Insensitive) repressor, which is characterized by a highly conserved N-terminal DELLA domain. Deletion of the DELLA domain results in constitutive suppression of GA signaling. As the GAI transcript is transportable in phloem elements, a Δ-DELLA GAI (gai) transgenic stock plant can reduce the stature of a scion through transport of gai mRNA from the stock. However, little is known about the characteristics of a scion on a gai stock. RESULTS: Arabidopsis Δ-DELLA GAI (gai) was fused with a T7 epitope tag and expressed under the control of a companion cell-specific expression promoter, Commelina yellow mottle virus promoter (CoYMVp), to enhance transport in the phloem. The CoYMVp:Atgai-T7 (CgT) transgenic Nicotiana benthamiana exhibited a dwarf phenotype and lower sensitivity to GA enhancement of shoot stature. A wild-type (WT) scion on a CgT stock contained both Atgai-T7 mRNA and the translated product. Microarray analysis to clarify the effect of the CgT stock on the gene expression pattern in the scion clearly revealed that the WT scions on CgT stocks had fewer genes whose expression was altered in response to GA treatment. An apple rootstock variety, Malus prunifolia, integrating CoYMVp:Atgai moderately reduced the tree height of the apple cultivar scion. CONCLUSIONS: Our results demonstrate that Atgai mRNA can move from companion cells to sieve tubes and that the translated product remains at the sites to which it is transported, resulting in attenuation of GA responses by reducing the expression of many genes. The induction of semi-dwarfism in an apple cultivar on root stock harbouring Atgai suggests that long-distance transport of mRNA from grafts would be applicable to horticulture crops.

Transcriptional regulation of immediate-early gene response by THOC5, a member of mRNA export complex, contributes to the M-CSF-induced macrophage differentiation.

Hematopoiesis and commitment to a restricted lineage are guided by a timely expressed set of cytokine receptors and their downstream transcription factors. A member of the mRNA export complex, THOC5 (suppressors of the transcriptional defects of hpr1 delta by overexpression complex 5) is a substrate for several tyrosine kinases such as macrophage colony-stimulating factor (M-CSF) receptor and various leukemogenic tyrosine kinases, such as Bcr-Abl, or NPM-ALK. THOC5 tyrosine phosphorylation is elevated in stem cells from patients with chronic myeloid leukemia, suggesting that THOC5 may be involved in leukemia development. THOC5 is also an essential element in the maintenance of hematopoiesis in adult mice. In this report, we show that THOC5 is located in the nuclear speckles, and that it is translocated from the nucleus to cytoplasm during M-CSF-induced bone marrow-derived macrophage differentiation. Furthermore, we have identified THOC5 target genes by trancriptome analysis, using tamoxifen-inducible THOC5 knockout macrophages. Although only 99 genes were downregulated in THOC5-depleted macrophages, half of the genes are involved in differentiation and/or migration. These include well-known regulators of myeloid differentiation inhibitor of DNA binding (Id)1, Id3, Smad family member 6 (Smad6) and Homeobox (Hox)A1. In addition, a subset of M-CSF-inducible genes, such as Ets family mRNAs are THOC5 target mRNAs. Upon depletion of THOC5, unspliced v-ets erythroblastosis virus E26 oncogene homolog (Ets1) mRNA was accumulated in the nucleus. Furthermore, THOC5 was recruited to chromatin where Ets1 was transcribed and bound to unspliced and spliced Ets1 transcripts, indicating that THOC5 has a role in processing/export of M-CSF-inducible genes. In conclusion, regulation of immediate-early gene response by THOC5, a member of mRNA export complex contributes to the M-CSF-induced macrophage differentiation.

A novel function for CUGBP2 in controlling the pro-inflammatory stimulus in H9c2 cells: subcellular trafficking of messenger molecules.

Accumulating evidence demonstrates that chronic inflammation plays an important role in heart hypertrophy and cardiac diseases. However, the fine-tuning of cellular and molecular mechanisms that connect inflammatory process and cardiac diseases is still under investigation. Many reports have demonstrated that the overexpression of the cyclooxygenase-2 (COX-2), a key enzyme in the conversion of arachidonic acid to prostaglandins and other prostanoids, is correlated with inflammatory processes. Increased level of prostaglandin E2 was also found in animal model of left ventricle of hypertrophy. Based on previous observations that demonstrated a regulatory loop between COX-2 and the RNA-binding protein CUGBP2, we studied cellular and molecular mechanisms of a pro-inflammatory stimulus in a cardiac cell to verify if the above two molecules could be correlated with the inflammatory process in the heart. A cellular model of investigation was established and H9c2 was used. We also demonstrated a regulatory connection between COX-2 and CUGBP2 in the cardiac cells. Based on a set of different assays including gene silencing and fluorescence microscopy, we describe a novel function for the RNA-binding protein CUGBP2 in controlling the pro-inflammatory stimulus: subcellular trafficking of messenger molecules to specific cytoplasmic stress granules to maintain homeostasis.

Cotton AnnGh3 encoding an annexin protein is preferentially expressed in fibers and promotes initiation and elongation of leaf trichomes in transgenic Arabidopsis.

The annexins are a multifamily of calcium-regulated phospholipid-binding proteins. To investigate the roles of annexins in fiber development, four genes encoding putative annexin proteins were isolated from cotton (Gossypium hirsutum) and designated AnnGh3, AnnGh4, AnnGh5, and AnnGh6. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) results indicated that AnnGh3, AnnGh4, and AnnGh5 were preferentially expressed in fibers, while the transcripts of AnnGh6 were predominantly accumulated in roots. During fiber development, the transcripts of AnnGh3/4/5 genes were mainly accumulated in rapidly elongating fibers. With fiber cells further developed, their expression activity was dramatically declined to a relatively low level. In situ hybridization results indicated that AnnGh3 and AnnGh5 were expressed in initiating fiber cells (0-2 DPA). Additionally, their expression in fibers was also regulated by phytohormones and [Ca(2+)]. Subcellular localization analysis discovered that AnnGh3 protein was localized in the cytoplasm. Overexpression of AnnGh3 in Arabidopsis resulted in a significant increase in trichome density and length on leaves of the transgenic plants, suggesting that AnnGh3 may be involved in fiber cell initiation and elongation of cotton.

Molecular basis for the action of a dietary flavonoid revealed by the comprehensive identification of apigenin human targets.

Flavonoids constitute the largest class of dietary phytochemicals, adding essential health value to our diet, and are emerging as key nutraceuticals. Cellular targets for dietary phytochemicals remain largely unknown, posing significant challenges for the regulation of dietary supplements and the understanding of how nutraceuticals provide health value. Here, we describe the identification of human cellular targets of apigenin, a flavonoid abundantly present in fruits and vegetables, using an innovative high-throughput approach that combines phage display with second generation sequencing. The 160 identified high-confidence candidate apigenin targets are significantly enriched in three main functional categories: GTPase activation, membrane transport, and mRNA metabolism/alternative splicing. This last category includes the heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2), a factor involved in splicing regulation, mRNA stability, and mRNA transport. Apigenin binds to the C-terminal glycine-rich domain of hnRNPA2, preventing hnRNPA2 from forming homodimers, and therefore, it perturbs the alternative splicing of several human hnRNPA2 targets. Our results provide a framework to understand how dietary phytochemicals exert their actions by binding to many functionally diverse cellular targets. In turn, some of them may modulate the activity of a large number of downstream genes, which is exemplified here by the effects of apigenin on the alternative splicing activity of hnRNPA2. Hence, in contrast to small-molecule pharmaceuticals designed for defined target specificity, dietary phytochemicals affect a large number of cellular targets with varied affinities that, combined, result in their recognized health benefits.

Macrophage microvesicles induce macrophage differentiation and miR-223 transfer.

Microvesicles are small membrane-bound particles comprised of exosomes and various-sized extracellular vesicles. These are released by several cell types. Microvesicles have a variety of cellular functions from communication to mediating growth and differentiation. Microvesicles contain proteins and nucleic acids. Previously, we showed that plasma microvesicles contain microRNAs (miRNAs). Based on our previous report, the majority of peripheral blood microvesicles are derived from platelets, while mononuclear phagocytes, including macrophages, are the second most abundant population. Here, we characterized macrophage-derived microvesicles and explored their role in the differentiation of naive monocytes. We also identified the miRNA content of the macrophage-derived microvesicles. We found that RNA molecules contained in the macrophage-derived microvesicles were transported to target cells, including mono cytes, endothelial cells, epithelial cells, and fibroblasts. Furthermore, we found that miR-223 was transported to target cells and was functionally active. Based on our observations, we hypothesize that microvesicles bind to and activate target cells. Furthermore, we find that microvesicles induce the differentiation of macrophages. Thus, defining key components of this response may identify novel targets to regulate host defense and inflammation.

Localization of Notch signaling molecules and their effect on cellular proliferation in adult rat pituitary.

Notch signaling is a cell-to-cell signaling system involved in the maintenance of precursor cells in many tissues. Although Notch signaling has been reported in the pituitary gland, the histological characteristics of Notch receptors and ligands in the gland are unknown. Here, we report the histological gene expression pattern of Notch receptors and ligands and the role of Notch signaling in cellular proliferation in adult rat pituitary gland. In situ hybridization detected transcripts of Notch1 and 2 and Jagged1 and 2. Double-staining with a combination of in situ hybridization and immunohistochemistry revealed that their mRNAs were localized in almost half of the S100-protein-positive cells, which are generally regarded as marginal layer cells and folliculo-stellate cells. In primary culture of anterior pituitary cells, proliferation of S100-protein-positive cells was modulated by Notch signaling inhibitor and solubilized Notch ligand. Furthermore, quantitative analysis revealed that the inhibition of Notch signaling led to the down-regulation of mRNA for the Notch target gene Hes1 and the up-regulation of p57 gene expression. These findings suggest that Notch signaling is involved in the proliferation of S100-protein-positive cells, presumably precursor cells, in adult rat pituitary gland.

Expression and regulation of beta-defensin 11 in the oviduct in response to estrogen and in ovarian tumors of chickens.

Avian beta-defensins (AvBDs), also known as gallinacins, are small cationic peptides having three cysteine disulfide bonds between their cysteine residues. They play essential roles in the innate immune system as well as stimulate proliferation of epithelial cells and fibroblasts. Although we found the avian homolog of human beta-defensin 11 to be highly expressed in chicks treated with the diethylstilbestrol (DES, a synthetic estrogen agonist), little is known about the hormonal and transcriptional regulation of AvBD-11 in the chicken oviduct and its expression in cancerous ovaries of chickens. Results of this study of young chicks revealed that DES induced AvBD-11 mRNA and protein in the oviduct, specifically luminal and glandular epithelial cells. In addition, microRNA-1615 was discovered to influence AvBD-11 expression via its 3'-UTR which suggests post-transcriptional regulation of AvBD-11 expression in chickens. Furthermore, we compared the expression patterns of the AvBD-11 gene in normal and cancerous ovaries from laying hens which are models for human epithelial ovarian cancer. Our results demonstrated that AvBD-11 is most abundant in the glandular epithelium of endometrioid-type ovarian tumors, but not normal ovaries of laying hens. Collectively, these results suggest that AvBD-11 is an estrogen-induced gene during oviduct development and that it may be used as a biomarker for diagnosis of ovarian cancer and for monitoring effects of therapeutics on progression of ovarian carcinogenesis.

Antibiotics that bind to the A site of the large ribosomal subunit can induce mRNA translocation.

In the absence of elongation factor EF-G, ribosomes undergo spontaneous, thermally driven fluctuation between the pre-translocation (classical) and intermediate (hybrid) states of translocation. These fluctuations do not result in productive mRNA translocation. Extending previous findings that the antibiotic sparsomycin induces translocation, we identify additional peptidyl transferase inhibitors that trigger productive mRNA translocation. We find that antibiotics that bind the peptidyl transferase A site induce mRNA translocation, whereas those that do not occupy the A site fail to induce translocation. Using single-molecule FRET, we show that translocation-inducing antibiotics do not accelerate intersubunit rotation, but act solely by converting the intrinsic, thermally driven dynamics of the ribosome into translocation. Our results support the idea that the ribosome is a Brownian ratchet machine, whose intrinsic dynamics can be rectified into unidirectional translocation by ligand binding.