Functional expression of IL-12 receptor by human eosinophils: IL-12 promotes eosinophil apoptosis.

PubMed

Nutku, E; Zhuang, Q; Soussi-Gounni, A; Aris, F; Mazer, B D; Hamid, Q

2001-07-15

In murine models of allergic inflammation, IL-12 has been shown to decrease tissue eosinophilia, but the underlying mechanisms are not known. We evaluated the expression of IL-12R and the effect of IL-12 on eosinophil survival. In situ hybridization demonstrated the presence of mRNA and immunoreactivity for IL-12Rbeta1 and -beta2 subunits in human peripheral blood eosinophils. Surface expression of IL-12Rbeta1 and -beta2 subunits on freshly isolated human eosinophils was optimally expressed after incubation with PMA. To determine the functional significance of IL-12R studies, we studied cell viability and apoptosis. Morphological analysis and propidium iodide staining for cell cycle demonstrated that recombinant human IL-12 increased in vitro human eosinophil apoptosis in a dose-dependent manner. Addition of IL-5 together with IL-12 abrogated eosinophil apoptosis, suggesting that IL-12 and IL-5 have antagonistic effects. Our findings provide evidence for a novel role for IL-12 in regulating eosinophil function by increasing eosinophil apoptosis.

Exendin-4 Upregulates Adiponectin Level in Adipocytes via Sirt1/Foxo-1 Signaling Pathway

PubMed Central

Wang, Anping; Li, Ting; An, Ping; Yan, Wenhua; Zheng, Hua; Wang, Baoan; Mu, Yiming

2017-01-01

Glucagon-like peptide-1 (GLP-1) receptor plays an essential role in regulating glucose metabolism. GLP-1 receptor agonists have been widely used for treating diabetes and other insulin resistance-related diseases. However, mechanisms underlying the anti-diabetic effects of GLP-1 receptor agonists remain largely unknown. In this study, we investigated the effects of GLP-1 agonist exendin-4 on the expression of adiponectin, an insulin sensitizing hormone. We found that exendin-4 increased the expression and secretion of adiponectin both in vitro and in vivo. Our data showed that exendin-4 upregulated adiponectin expression at both mRNA and protein levels in adipocytes and adipose tissues. The effects of exendin-4 on adiponectin expression were dependent on the GLP-1 receptor. We further demonstrated important roles of Sirt1 and transcriptional factor Foxo-1 in mediating the function of exendin-4 in regulating adiponectin expression. Suppression of Sirt1 or Foxo-1 expression significantly impaired exendin-4-induced adiponectin expression. Consistently, exendin-4 up-regulated Sirt1 and Foxo-1 expression in vivo. Our work is the first study demonstrating the role of Sirt1/Foxo-1 in regulating the regulatory function of a GLP-1 receptor agonist in adiponectin expression both in vitro and in vivo. The results provide important information for the mechanism underlying the function of GLP-1R on improving insulin resistance and related diseases. PMID:28122026

AKT inhibition mitigates GRP78 (glucose-regulated protein) expression and contribution to chemoresistance in endometrial cancers.

PubMed

Gray, Michael J; Mhawech-Fauceglia, Paulette; Yoo, Eunjeong; Yang, Wangrong; Wu, Eijean; Lee, Amy S; Lin, Yvonne G

2013-07-01

Overexpression of the unfolded protein response master regulator GRP78 is associated with poor prognosis and therapeutic resistance in numerous human cancers, yet its role in endometrial cancers (EC) is undefined. To better understand the contribution of GRP78 to EC, we examined its expression levels in EC patient samples and EC cell lines. We demonstrate that GRP78 overexpression occurs more frequently in EC tissues compared with that found in normal endometrium, and that GRP78 expression occurs in most EC cell lines examined. Functional analysis demonstrated that GRP78 is inducible by cisplatin in EC cells, and siRNA knockdown of GRP78 augments chemotherapy-mediated cell death. Examination of AKT and GRP78 expression demonstrated that inhibition of AKT activity by MK2206 blocks GRP78 expression in EC cells. SiRNA studies also revealed that knockdown of GRP78 reduces but does not abrogate AKT activity, demonstrating that GRP78 is required for optimal AKT activity. In the presence of MK2206, siRNA knockdown of GRP78 does not augment AKT mediated survival in response to cisplatin treatment, suggesting that GRP78's antiapoptosis functions are part of the AKT survival pathway. Targeted therapies that reduce GRP78 expression or activity in cancers may serve to increase the effectiveness of current therapies for EC patients. Copyright © 2012 UICC.

Hand2 loss-of-function in Hand1-expressing Cells Reveals Distinct Roles In Epicardial And Coronary Vessel Development

PubMed Central

Barnes, Ralston M.; Firulli, Beth A.; VanDusen, Nathan J.; Morikawa, Yuka; Conway, Simon J.; Cserjesi, Peter; Vincentz, Joshua W.; Firulli, Anthony B.

2011-01-01

Rationale The bHLH transcription factors Hand1 and Hand2 are essential for embryonic development. Given their requirement for cardiogenesis, it is imperative to determine their impact on cardiovascular function. Objective Deduce the role of Hand2 within the epicardium. Method & Results We engineered a Hand1 allele expressing Cre recombinase. Cardiac Hand1 expression is largely limited to cells of the primary heart field, overlapping little with Hand2 expression. Hand1 is expressed within the septum transversum (ST) and the Hand1-lineage marks the proepicardial organ and epicardium. To examine Hand factor functional overlap, we conditionally deleted Hand2 from Hand1-expressing cells. Hand2 mutants display defective epicardialization and fail to form coronary arteries, coincident with altered ECM deposition and Pdgfr expression. Conclusion These data demonstrate a hierarchal relationship whereby transient Hand1 ST expression defines epicardial precursors that are subsequently dependent upon Hand2 function. PMID:21350214

Differential expression pattern of UBX family genes in Caenorhabditis elegans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamauchi, Seiji; Sasagawa, Yohei; Ogura, Teru

2007-06-29

UBX (ubiquitin regulatory X)-containing proteins belong to an evolutionary conserved protein family and determine the specificity of p97/VCP/Cdc48p function by binding as its adaptors. Caenorhabditis elegans was found to possess six UBX-containing proteins, named UBXN-1 to -6. However, no general or specific function of them has been revealed. During the course of understanding not only their function but also specified function of p97, we investigated spatial and temporal expression patterns of six ubxn genes in this study. Transcript analyses showed that the expression pattern of each ubxn gene was different throughout worm's development and may show potential developmental dynamics inmore » their function, especially ubxn-5 was expressed specifically in the spermatogenic germline, suggesting a crucial role in spermatogenesis. In addition, as ubxn-4 expression was induced by ER stress, it would function as an ERAD factor in C. elegans. In vivo expression analysis by using GFP translational fusion constructs revealed that six ubxn genes show distinct expression patterns. These results altogether demonstrate that the expression of all six ubxn genes of C. elegans is differently regulated.« less

Expression, purification and functional characterization of human equilibrative nucleoside transporter subtype-1 (hENT1) protein from Sf9 insect cells.

PubMed

Rehan, Shahid; Jaakola, Veli-Pekka

2015-10-01

Human equilibrative nucleoside transporter-1 (hENT1) is the major plasma membrane transporter involved in transportation of natural nucleosides as well as nucleoside analog drugs, used in anti-cancer and anti-viral therapies. Despite extensive biochemical and pharmacological studies, little is known about the structure-function relationship of this protein. The major obstacles to purification include a low endogenous expression level, the lack of an efficient expression and purification protocol, and the hydrophobic nature of the protein. Here, we report protein expression, purification and functional characterization of hENT1 from Sf9 insect cells. hENT1 expressed by Sf9 cells is functionally active as demonstrated by saturation binding with a Kd of 1.2±0.2nM and Bmax of 110±5pmol/mg for [(3)H]nitrobenzylmercaptopurine ribonucleoside ([(3)H]NBMPR). We also demonstrate purification of hENT1 using FLAG antibody affinity resin in lauryl maltose neopentyl glycol detergent with a Kd of 4.3±0.7nM. The yield of hENT1 from Sf9 cells was ∼0.5mg active transporter per liter of culture. The purified protein is functionally active, stable, homogenous and appropriate for further biophysical and structural studies. Copyright © 2015 Elsevier Inc. All rights reserved.

Dental Enamel: Genes Define Biomechanics

PubMed Central

Rauth, Rick J.; Potter, Karen S.; Ngan, Amanda Y.-W.; Saad, Deema M.; Mehr, Rana; Luong, Vivian Q.; Schuetter, Verna L.; Miklus, Vetea G.; Chang, PeiPei; Paine, Michael L.; Lacruz, Rodrigo S.; Snead, Malcolm L.; White, Shane N.

2010-01-01

Regulated gene expression assembles an extracellular proteinaceous matrix to control biomineralization and the resultant biomechanical function of tooth enamel. The importance of the dominant enamel matrix protein, amelogenin (Amel); a minor transiently expressed protein, dentin sialoprotein (Dsp); an electrogenic sodium bicarbonate cotransporter (NBCe1); the timely removal of the proteinaceous matrix by a serine protease, Kallikrein-4 (Klk4); and the late-stage expression of Amelotin (Amtn) on enamel biomechanical function were demonstrated and measured using mouse models. PMID:20066874

An Unexpected Function of the Prader-Willi Syndrome Imprinting Center in Maternal Imprinting in Mice

PubMed Central

Wu, Mei-Yi; Jiang, Ming; Zhai, Xiaodong; Beaudet, Arthur L.; Wu, Ray-Chang

2012-01-01

Genomic imprinting is a phenomenon that some genes are expressed differentially according to the parent of origin. Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurobehavioral disorders caused by deficiency of imprinted gene expression from paternal and maternal chromosome 15q11–q13, respectively. Imprinted genes at the PWS/AS domain are regulated through a bipartite imprinting center, the PWS-IC and AS-IC. The PWS-IC activates paternal-specific gene expression and is responsible for the paternal imprint, whereas the AS-IC functions in the maternal imprint by allele-specific repression of the PWS-IC to prevent the paternal imprinting program. Although mouse chromosome 7C has a conserved PWS/AS imprinted domain, the mouse equivalent of the human AS-IC element has not yet been identified. Here, we suggest another dimension that the PWS-IC also functions in maternal imprinting by negatively regulating the paternally expressed imprinted genes in mice, in contrast to its known function as a positive regulator for paternal-specific gene expression. Using a mouse model carrying a 4.8-kb deletion at the PWS-IC, we demonstrated that maternal transmission of the PWS-IC deletion resulted in a maternal imprinting defect with activation of the paternally expressed imprinted genes and decreased expression of the maternally expressed imprinted gene on the maternal chromosome, accompanied by alteration of the maternal epigenotype toward a paternal state spread over the PWS/AS domain. The functional significance of this acquired paternal pattern of gene expression was demonstrated by the ability to complement PWS phenotypes by maternal inheritance of the PWS-IC deletion, which is in stark contrast to paternal inheritance of the PWS-IC deletion that resulted in the PWS phenotypes. Importantly, low levels of expression of the paternally expressed imprinted genes are sufficient to rescue postnatal lethality and growth retardation in two PWS mouse models. These findings open the opportunity for a novel approach to the treatment of PWS. PMID:22496793

An unexpected function of the Prader-Willi syndrome imprinting center in maternal imprinting in mice.

PubMed

Wu, Mei-Yi; Jiang, Ming; Zhai, Xiaodong; Beaudet, Arthur L; Wu, Ray-Chang

2012-01-01

Genomic imprinting is a phenomenon that some genes are expressed differentially according to the parent of origin. Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurobehavioral disorders caused by deficiency of imprinted gene expression from paternal and maternal chromosome 15q11-q13, respectively. Imprinted genes at the PWS/AS domain are regulated through a bipartite imprinting center, the PWS-IC and AS-IC. The PWS-IC activates paternal-specific gene expression and is responsible for the paternal imprint, whereas the AS-IC functions in the maternal imprint by allele-specific repression of the PWS-IC to prevent the paternal imprinting program. Although mouse chromosome 7C has a conserved PWS/AS imprinted domain, the mouse equivalent of the human AS-IC element has not yet been identified. Here, we suggest another dimension that the PWS-IC also functions in maternal imprinting by negatively regulating the paternally expressed imprinted genes in mice, in contrast to its known function as a positive regulator for paternal-specific gene expression. Using a mouse model carrying a 4.8-kb deletion at the PWS-IC, we demonstrated that maternal transmission of the PWS-IC deletion resulted in a maternal imprinting defect with activation of the paternally expressed imprinted genes and decreased expression of the maternally expressed imprinted gene on the maternal chromosome, accompanied by alteration of the maternal epigenotype toward a paternal state spread over the PWS/AS domain. The functional significance of this acquired paternal pattern of gene expression was demonstrated by the ability to complement PWS phenotypes by maternal inheritance of the PWS-IC deletion, which is in stark contrast to paternal inheritance of the PWS-IC deletion that resulted in the PWS phenotypes. Importantly, low levels of expression of the paternally expressed imprinted genes are sufficient to rescue postnatal lethality and growth retardation in two PWS mouse models. These findings open the opportunity for a novel approach to the treatment of PWS.

Synthetic estrogen derivatives demonstrate the functionality of intracellular GPR30.

PubMed

Revankar, Chetana M; Mitchell, Hugh D; Field, Angela S; Burai, Ritwik; Corona, Cesear; Ramesh, Chinnasamy; Sklar, Larry A; Arterburn, Jeffrey B; Prossnitz, Eric R

2007-08-17

Estrogen mediates its effects through multiple cellular receptors. In addition to the classical nuclear estrogen receptors (ERalpha and ERbeta), estrogen also signals through the seven-transmembrane G-protein-coupled receptor (GPCR) GPR30. Although estrogen is a cell-permeable ligand, it is often assumed that all GPCRs function solely as cell surface receptors. Our previous results showed that GPR30 appeared to be expressed predominantly in the endoplasmic reticulum. A critical question that arises is whether this localization represents the site of functional receptor. To address this question, we synthesized a collection of cell-permeable and cell-impermeable estrogen derivatives. We hypothesized that if functional GPR30 were expressed at the cell surface, both permeable and impermeable derivatives would show activity. However, if functional GPR30 were predominantly intracellular, like ERalpha, only the permeable ligands should show activity. Cell permeability was assessed using cells expressing ERalpha as a model intracellular estrogen-binding receptor. Our results reveal that despite exhibiting similar binding affinities for GPR30, only the cell-permeable ligands are capable of stimulating rapid calcium mobilization and phosphoinositide 3-kinase (PI3K) activation. We conclude that GPR30 expressed intracellularly is capable of initiating cellular signaling and that there is insufficient GPR30 expressed on the cell surface to initiate signaling in response to impermeable ligands in the cell lines examined. To our knowledge, this is the first definitive demonstration of a functional intracellular transmembrane estrogen receptor.

Galectin-9-CD44 interaction enhances stability and function of adaptive regulatory T cells | Center for Cancer Research

Cancer.gov

The β-galactoside-binding protein galectin-9 is critical in regulating the immune response, but the mechanism by which it functions remains unclear. We have demonstrated that galectin-9 is highly expressed by induced regulatory T cells (iTreg) and was crucial for the generation and function of iTreg cells, but not natural regulatory T (nTreg) cells. Galectin-9 expression

IL-6 mediates differentiation disorder during spermatogenesis in obesity-associated inflammation by affecting the expression of Zfp637 through the SOCS3/STAT3 pathway.

PubMed

Huang, Guizhen; Yuan, Miao; Zhang, Jie; Li, Jun; Gong, Di; Li, Yanyan; Zhang, Jie; Lin, Ping; Huang, Lugang

2016-06-22

Zfp637 is a recently identified zinc finger protein, and its functions remain largely unknown. Here, we innovatively demonstrate the effects of Zfp637 on the differentiation of mouse spermatogonia and on its downstream target gene SOX2 in vitro. Obesity has been recognized as a chronic inflammatory disease that leads to decreased sexual function and sexual development disorders. We observed higher levels of IL-6 in serum and testis homogenates from obese mice compared with control mice. We also demonstrated that high levels of IL-6 inhibited Zfp637 expression, and we elucidated the underlying mechanisms. SOCS3 overexpression and STAT3 phosphorylation inhibitor (AG490) were used to investigate the function of the SOCS3/STAT3 pathway during this process. Our results showed that exposure of mouse spermatogonial cells to high levels of IL-6 inhibited Zfp637 expression by increasing SOCS3 expression and inhibiting the phosphorylation of STAT3, further reducing cellular differentiation. Consistent with the in vitro results, we observed increasing expression levels of SOCS3 and SOX2, but a reduction of Zfp637 expression, in obese mouse testes. In conclusion, Zfp637 plays a crucial role in spermatogenesis by downregulating SOX2 expression, and IL-6 can decrease the expression of Zfp637 through the SOCS3/STAT3 signaling pathway.

Asialoglycoprotein receptor 1 is a specific cell-surface marker for isolating hepatocytes derived from human pluripotent stem cells.

PubMed

Peters, Derek T; Henderson, Christopher A; Warren, Curtis R; Friesen, Max; Xia, Fang; Becker, Caroline E; Musunuru, Kiran; Cowan, Chad A

2016-05-01

Hepatocyte-like cells (HLCs) are derived from human pluripotent stem cells (hPSCs) in vitro, but differentiation protocols commonly give rise to a heterogeneous mixture of cells. This variability confounds the evaluation of in vitro functional assays performed using HLCs. Increased differentiation efficiency and more accurate approximation of the in vivo hepatocyte gene expression profile would improve the utility of hPSCs. Towards this goal, we demonstrate the purification of a subpopulation of functional HLCs using the hepatocyte surface marker asialoglycoprotein receptor 1 (ASGR1). We analyzed the expression profile of ASGR1-positive cells by microarray, and tested their ability to perform mature hepatocyte functions (albumin and urea secretion, cytochrome activity). By these measures, ASGR1-positive HLCs are enriched for the gene expression profile and functional characteristics of primary hepatocytes compared with unsorted HLCs. We have demonstrated that ASGR1-positive sorting isolates a functional subpopulation of HLCs from among the heterogeneous cellular population produced by directed differentiation. © 2016. Published by The Company of Biologists Ltd.

Rat Models of Cardiovascular Disease Demonstrate Distinctive Pulmonary Gene Expressions for Vascular Response Genes: Impact of Ozone Exposure

EPA Science Inventory

Comparative gene expression profiling of multiple tissues from rat strains with genetic predisposition to diverse cardiovascular diseases (CVD) can help decode the transcriptional program that governs organ-specific functions. We examined expressions of CVD genes in the lungs of ...

Testosterone Regulates Erectile Function and Vcsa1 Expression in the Corpora of Rats

PubMed Central

Chua, Rowena G.; Calenda, Giulia; Zhang, Xinhua; Siragusa, Joseph; Tong, Yuehong; Tar, Moses; Aydin, Memduh; DiSanto, Michael E.; Melman, Arnold; Davies, Kelvin P.

2009-01-01

Summary Vcsa1 plays an important role in the erectile physiology of the rat. We conducted experiments to determine if erectile function, testosterone levels and Vcsa1 expression were correlated. In orchiectomized rats, total testosterone in blood fell from an average of 4ng/ml to <0.04ng/ml. Erectile function was significantly lower compared to controls and Vcsa1 expression was significantly (>6-fold) decreased. Injection of orchiectomized animals with testosterone (2mg in 100ml sesame oil every 4 days for two weeks) restored average levels of testosterone to 2ng/ml, increased erectile function and significantly increased Vcsa1 expression. In isolated corporal cells there was testosterone dependent Vcsa1 expression. However, intracorporal injection of orchiectomized animals with a plasmid expressing Vcsa1 or its gene product Sialorphin (previously demonstrated to improve erectile function in old animals) gave no significant improvement in erectile function. Also, the ability of Sialorphin to reduce tension in corporal smooth muscle strips isolated from orchiectomized animals was impaired compared to controls. PMID:19428993

Na/K-ATPase/src complex mediates regulation of CD40 in renal parenchyma.

PubMed

Xie, Jeffrey X; Zhang, Shungang; Cui, Xiaoyu; Zhang, Jue; Yu, Hui; Khalaf, Fatimah K; Malhotra, Deepak; Kennedy, David J; Shapiro, Joseph I; Tian, Jiang; Haller, Steven T

2017-12-22

Recent studies have highlighted a critical role for CD40 in the pathogenesis of renal injury and fibrosis. However, little is currently understood about the regulation of CD40 in this setting. We use novel Na/K-ATPase cell lines and inhibitors in order to demonstrate the regulatory function of Na/K-ATPase with regards to CD40 expression and function. We utilize 5/6 partial nephrectomy as well as direct infusion of a Na/K-ATPase ligand to demonstrate this mechanism exists in vivo. We demonstrate that knockdown of the α1 isoform of Na/K-ATPase causes a reduction in CD40 while rescue of the α1 but not the α2 isoform restores CD40 expression in renal epithelial cells. Second, because the major functional difference between α1 and α2 is the ability of α1 to form a functional signaling complex with Src, we examined whether the Na/K-ATPase/Src complex is important for CD40 expression. We show that a gain-of-Src binding α2 mutant restores CD40 expression while loss-of-Src binding α1 reduces CD40 expression. Furthermore, loss of a functional Na/K-ATPase/Src complex also disrupts CD40 signaling. Importantly, we show that use of a specific Na/K-ATPase/Src complex antagonist, pNaKtide, can attenuate cardiotonic steroid (CTS)-induced induction of CD40 expression in vitro. Because the Na/K-ATPase/Src complex is also a key player in the pathogenesis of renal injury and fibrosis, our new findings suggest that Na/K-ATPase and CD40 may comprise a pro-fibrotic feed-forward loop in the kidney and that pharmacological inhibition of this loop may be useful in the treatment of renal fibrosis. © The Author 2017. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

Prolonged, brain-wide expression of nuclear-localized GCaMP3 for functional circuit mapping

PubMed Central

Kim, Christina K.; Miri, Andrew; Leung, Louis C.; Berndt, Andre; Mourrain, Philippe; Tank, David W.; Burdine, Rebecca D.

2014-01-01

Larval zebrafish offer the potential for large-scale optical imaging of neural activity throughout the central nervous system; however, several barriers challenge their utility. First, ~panneuronal probe expression has to date only been demonstrated at early larval stages up to 7 days post-fertilization (dpf), precluding imaging at later time points when circuits are more mature. Second, nuclear exclusion of genetically-encoded calcium indicators (GECIs) limits the resolution of functional fluorescence signals collected during imaging. Here, we report the creation of transgenic zebrafish strains exhibiting robust, nuclearly targeted expression of GCaMP3 across the brain up to at least 14 dpf utilizing a previously described optimized Gal4-UAS system. We confirmed both nuclear targeting and functionality of the modified probe in vitro and measured its kinetics in response to action potentials (APs). We then demonstrated in vivo functionality of nuclear-localized GCaMP3 in transgenic zebrafish strains by identifying eye position-sensitive fluorescence fluctuations in caudal hindbrain neurons during spontaneous eye movements. Our methodological approach will facilitate studies of larval zebrafish circuitry by both improving resolution of functional Ca2+ signals and by allowing brain-wide expression of improved GECIs, or potentially any probe, further into development. PMID:25505384

TMV-Gate vectors: Gateway compatible tobacco mosaic virus based expression vectors for functional analysis of proteins

PubMed Central

Kagale, Sateesh; Uzuhashi, Shihomi; Wigness, Merek; Bender, Tricia; Yang, Wen; Borhan, M. Hossein; Rozwadowski, Kevin

2012-01-01

Plant viral expression vectors are advantageous for high-throughput functional characterization studies of genes due to their capability for rapid, high-level transient expression of proteins. We have constructed a series of tobacco mosaic virus (TMV) based vectors that are compatible with Gateway technology to enable rapid assembly of expression constructs and exploitation of ORFeome collections. In addition to the potential of producing recombinant protein at grams per kilogram FW of leaf tissue, these vectors facilitate either N- or C-terminal fusions to a broad series of epitope tag(s) and fluorescent proteins. We demonstrate the utility of these vectors in affinity purification, immunodetection and subcellular localisation studies. We also apply the vectors to characterize protein-protein interactions and demonstrate their utility in screening plant pathogen effectors. Given its broad utility in defining protein properties, this vector series will serve as a useful resource to expedite gene characterization efforts. PMID:23166857

Molecular anatomy of the developing limb in the coquí frog, Eleutherodactylus coqui.

PubMed

Gross, Joshua B; Kerney, Ryan; Hanken, James; Tabin, Clifford J

2011-01-01

The vertebrate limb demonstrates remarkable similarity in basic organization across phylogenetically disparate groups. To gain further insight into how this morphological similarity is maintained in different developmental contexts, we explored the molecular anatomy of size-reduced embryos of the Puerto Rican coquí frog, Eleutherodactylus coqui. This animal demonstrates direct development, a life-history strategy marked by rapid progression from egg to adult and absence of a free-living, aquatic larva. Nonetheless, coquí exhibits a basal anuran limb structure, with four toes on the forelimb and five toes on the hind limb. We investigated the extent to which coquí limb bud development conforms to the model of limb development derived from amniote studies. Toward this end, we characterized dynamic patterns of expression for 13 critical patterning genes across three principle stages of limb development. As expected, most genes demonstrate expression patterns that are essentially unchanged compared to amniote species. For example, we identified an EcFgf8-expression domain within the apical ectodermal ridge (AER). This expression pattern defines a putatively functional AER signaling domain, despite the absence of a morphological ridge in coquí embryos. However, two genes, EcMeis2 and EcAlx4, demonstrate altered domains of expression, which imply a potential shift in gene function between coquí frogs and amniote model systems. Unexpectedly, several genes thought to be critical for limb patterning in other systems, including EcFgf4, EcWnt3a, EcWnt7a, and EcGremlin, demonstrated no evident expression pattern in the limb at the three stages we analyzed. The absence of EcFgf4 and EcWnt3a expression during limb patterning is perhaps not surprising, given that neither gene is critical for proper limb development in the mouse, based on knockout and expression analyses. In contrast, absence of EcWnt7a and EcGremlin is surprising, given that expression of these molecules appears to be absolutely essential in all other model systems so far examined. Although this analysis substantiates the existence of a core set of ancient limb-patterning molecules, which likely mediate identical functions across highly diverse vertebrate forms, it also reveals remarkable evolutionary flexibility in the genetic control of a conserved morphological pattern across evolutionary time. © 2011 Wiley Periodicals, Inc.

Functional conservation of atonal and Math1 in the CNS and PNS

NASA Technical Reports Server (NTRS)

Ben-Arie, N.; Hassan, B. A.; Bermingham, N. A.; Malicki, D. M.; Armstrong, D.; Matzuk, M.; Bellen, H. J.; Zoghbi, H. Y.

2000-01-01

To determine the extent to which atonal and its mouse homolog Math1 exhibit functional conservation, we inserted (beta)-galactosidase (lacZ) into the Math1 locus and analyzed its expression, evaluated consequences of loss of Math1 function, and expressed Math1 in atonal mutant flies. lacZ under the control of Math1 regulatory elements duplicated the previously known expression pattern of Math1 in the CNS (i.e., the neural tube, dorsal spinal cord, brainstem, and cerebellar external granule neurons) but also revealed new sites of expression: PNS mechanoreceptors (inner ear hair cells and Merkel cells) and articular chondrocytes. Expressing Math1 induced ectopic chordotonal organs (CHOs) in wild-type flies and partially rescued CHO loss in atonal mutant embryos. These data demonstrate that both the mouse and fly homologs encode lineage identity information and, more interestingly, that some of the cells dependent on this information serve similar mechanoreceptor functions.

Sonic Hedgehog Expression in Corticofugal Projection Neurons Directs Cortical Microcircuit Formation

PubMed Central

Harwell, Corey C.; Parker, Philip R.L.; Gee, Steven M.; Okada, Ami; McConnell, Susan K.; Kreitzer, Anatol C.; Kriegstein, Arnold R.

2012-01-01

SUMMARY The precise connectivity of inputs and outputs is critical for cerebral cortex function; however, the cellular mechanisms that establish these connections are poorly understood. Here, we show that the secreted molecule Sonic Hedgehog (Shh) is involved in synapse formation of a specific cortical circuit. Shh is expressed in layer V corticofugal projection neurons and the Shh receptor, Brother of CDO (Boc), is expressed in local and callosal projection neurons of layer II/III that synapse onto the subcortical projection neurons. Layer V neurons of mice lacking functional Shh exhibit decreased synapses. Conversely, the loss of functional Boc leads to a reduction in the strength of synaptic connections onto layer Vb, but not layer II/III, pyramidal neurons. These results demonstrate that Shh is expressed in postsynaptic target cells while Boc is expressed in a complementary population of presynaptic input neurons, and they function to guide the formation of cortical microcircuitry. PMID:22445340

Cyclophilin B as a co-regulator of prolactin-induced gene expression and function in breast cancer cells

PubMed Central

Fang, Feng; Zheng, Jiamao; Galbaugh, Traci L; Fiorillo, Alyson A; Hjort, Elizabeth E; Zeng, Xianke; Clevenger, Charles V

2010-01-01

The effects of prolactin (PRL) during the pathogenesis of breast cancer are mediated in part though Stat5 activity enhanced by its interaction with its transcriptional inducer, the prolyl isomerase cyclophilin B (CypB). We have demonstrated that knockdown of CypB decreases cell growth, proliferation, and migration, and CypB expression is associated with malignant progression of breast cancer. In this study, we examined the effect of CypB knockdown on PRL signaling in breast cancer cells. CypB knockdown with two independent siRNAs was shown to impair PRL-induced reporter expression in breast cancer cell line. cDNA microarray analysis was performed on these cells to assess the effect of CypB reduction, and revealed a significant decrease in PRL-induced endogenous gene expression in two breast cancer cell lines. Parallel functional assays revealed corresponding alterations of both anchorage-independent cell growth and cell motility of breast cancer cells. Our results demonstrate that CypB expression levels significantly modulate PRL-induced function in breast cancer cells ultimately resulting in enhanced levels of PRL-responsive gene expression, cell growth, and migration. Given the increasingly appreciated role of PRL in the pathogenesis of breast cancer, the actions of CypB detailed here are of biological significance. PMID:20237142

Cyclophilin B as a co-regulator of prolactin-induced gene expression and function in breast cancer cells.

PubMed

Fang, Feng; Zheng, Jiamao; Galbaugh, Traci L; Fiorillo, Alyson A; Hjort, Elizabeth E; Zeng, Xianke; Clevenger, Charles V

2010-06-01

The effects of prolactin (PRL) during the pathogenesis of breast cancer are mediated in part though Stat5 activity enhanced by its interaction with its transcriptional inducer, the prolyl isomerase cyclophilin B (CypB). We have demonstrated that knockdown of CypB decreases cell growth, proliferation, and migration, and CypB expression is associated with malignant progression of breast cancer. In this study, we examined the effect of CypB knockdown on PRL signaling in breast cancer cells. CypB knockdown with two independent siRNAs was shown to impair PRL-induced reporter expression in breast cancer cell line. cDNA microarray analysis was performed on these cells to assess the effect of CypB reduction, and revealed a significant decrease in PRL-induced endogenous gene expression in two breast cancer cell lines. Parallel functional assays revealed corresponding alterations of both anchorage-independent cell growth and cell motility of breast cancer cells. Our results demonstrate that CypB expression levels significantly modulate PRL-induced function in breast cancer cells ultimately resulting in enhanced levels of PRL-responsive gene expression, cell growth, and migration. Given the increasingly appreciated role of PRL in the pathogenesis of breast cancer, the actions of CypB detailed here are of biological significance.

Coagulation factor VII is regulated by androgen receptor in breast cancer.

PubMed

Naderi, Ali

2015-02-01

Androgen receptor (AR) is widely expressed in breast cancer; however, there is limited information on the key molecular functions and gene targets of AR in this disease. In this study, gene expression data from a cohort of 52 breast cancer cell lines was analyzed to identify a network of AR co-expressed genes. A total of 300 genes, which were significantly enriched for cell cycle and metabolic functions, showed absolute correlation coefficients (|CC|) of more than 0.5 with AR expression across the dataset. In this network, a subset of 35 "AR-signature" genes were highly co-expressed with AR (|CC|>0.6) that included transcriptional regulators PATZ1, NFATC4, and SPDEF. Furthermore, gene encoding coagulation factor VII (F7) demonstrated the closest expression pattern with AR (CC=0.716) in the dataset and factor VII protein expression was significantly associated to that of AR in a cohort of 209 breast tumors. Moreover, functional studies demonstrated that AR activation results in the induction of factor VII expression at both transcript and protein levels and AR directly binds to a proximal region of F7 promoter in breast cancer cells. Importantly, AR activation in breast cancer cells induced endogenous factor VII activity to convert factor X to Xa in conjunction with tissue factor. In summary, F7 is a novel AR target gene and AR activation regulates the ectopic expression and activity of factor VII in breast cancer cells. These findings have functional implications in the pathobiology of thromboembolic events and regulation of factor VII/tissue factor signaling in breast cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

PRL-3 promotes breast cancer progression by downregulating p14ARF-mediated p53 expression.

PubMed

Xie, Hua; Wang, Hao

2018-03-01

Prior studies have demonstrated that phosphatase of regenerating liver-3 (PRL-3) serves avital function in cell proliferation and metastasis in breast cancer. However, the molecular mechanisms underlying the function of PRL-3 in breast cancer remain unknown. PRL-3 expression was analyzed in 24 pairs of breast cancer and normal tissues using the reverse transcription-quantitative polymerase chain reaction assay. The results of the present study identified that the expression of PLR-3 in breast cancer tissues was increased 4.2-fold, compared with normal tissues. Notably, overexpression of PRL-3 significantly promoted the proliferation of cancer cells and inhibited endogenous p53 expression by downregulating the expression level of p14 alternate reading frame (p14 ARF ). In addition, decreased expression levels of PRL-3 resulted in decreased breast cancer cell proliferation and increased expression level of p14 ARF . These results suggested that PRL-3 enhances cell proliferation by downregulating p14 ARF expression, which results in decreased levels ofp53. The results of the present study demonstrated that PRL-3 promotes tumor proliferation by affecting the p14 ARF -p53 axis, and that it may serve as a prognostic marker for patients with breast cancer.

Ageing causes cytoplasmic retention of MaxiK channels in rat corporal smooth muscle cells

PubMed Central

Davies, KP; Stanevsky, Y; Moses, T; Chang, JS; Chance, MR; Melman, A

2007-01-01

The MaxiK channel plays a critical role in the regulation of corporal smooth muscle tone and thereby erectile function. Given that ageing results in a decline in erectile function, we determined changes in the expression of MaxiK, which might impact erectile function. Quantitative-polymerase chain reaction demonstrated that although there is no significant change in transcription of the α- and β-subunits that comprise the MaxiK channel, there are significant changes in the expression of transcripts encoding different splice variants. One transcript, SV1, is 13-fold increased in expression in the ageing rat corpora. SV1 has previously been reported to trap other isoforms of the MaxiK channel in the cytoplasm. Correlating with increased expression of SV1, we observed in older rats there is approximately a 13-fold decrease in MaxiK protein in the corpora cell membrane and a greater proportion is retained in the cytoplasm (approximately threefold). These experiments demonstrate that ageing of the corpora is accompanied by changes in alternative splicing and cellular localization of the MaxiK channel. PMID:17287835

Ageing causes cytoplasmic retention of MaxiK channels in rat corporal smooth muscle cells.

PubMed

Davies, K P; Stanevsky, Y; Tar, M T; Moses, T; Chang, J S; Chance, M R; Melman, A

2007-01-01

The MaxiK channel plays a critical role in the regulation of corporal smooth muscle tone and thereby erectile function. Given that ageing results in a decline in erectile function, we determined changes in the expression of MaxiK, which might impact erectile function. Quantitative-polymerase chain reaction demonstrated that although there is no significant change in transcription of the alpha- and beta-subunits that comprise the MaxiK channel, there are significant changes in the expression of transcripts encoding different splice variants. One transcript, SV1, is 13-fold increased in expression in the ageing rat corpora. SV1 has previously been reported to trap other isoforms of the MaxiK channel in the cytoplasm. Correlating with increased expression of SV1, we observed in older rats there is approximately a 13-fold decrease in MaxiK protein in the corpora cell membrane and a greater proportion is retained in the cytoplasm (approximately threefold). These experiments demonstrate that ageing of the corpora is accompanied by changes in alternative splicing and cellular localization of the MaxiK channel.

Analytical time-domain Green’s functions for power-law media

PubMed Central

Kelly, James F.; McGough, Robert J.; Meerschaert, Mark M.

2008-01-01

Frequency-dependent loss and dispersion are typically modeled with a power-law attenuation coefficient, where the power-law exponent ranges from 0 to 2. To facilitate analytical solution, a fractional partial differential equation is derived that exactly describes power-law attenuation and the Szabo wave equation [“Time domain wave-equations for lossy media obeying a frequency power-law,” J. Acoust. Soc. Am. 96, 491–500 (1994)] is an approximation to this equation. This paper derives analytical time-domain Green’s functions in power-law media for exponents in this range. To construct solutions, stable law probability distributions are utilized. For exponents equal to 0, 1∕3, 1∕2, 2∕3, 3∕2, and 2, the Green’s function is expressed in terms of Dirac delta, exponential, Airy, hypergeometric, and Gaussian functions. For exponents strictly less than 1, the Green’s functions are expressed as Fox functions and are causal. For exponents greater than or equal than 1, the Green’s functions are expressed as Fox and Wright functions and are noncausal. However, numerical computations demonstrate that for observation points only one wavelength from the radiating source, the Green’s function is effectively causal for power-law exponents greater than or equal to 1. The analytical time-domain Green’s function is numerically verified against the material impulse response function, and the results demonstrate excellent agreement. PMID:19045774

Unconventional myosin ID is expressed in myelinating oligodendrocytes.

PubMed

Yamazaki, Reiji; Ishibashi, Tomoko; Baba, Hiroko; Yamaguchi, Yoshihide

2014-10-01

Myelin is a dynamic multilamellar structure that ensheathes axons and is crucial for normal neuronal function. In the central nervous system (CNS), myelin is produced by oligodendrocytes that wrap many layers of plasma membrane around axons. The dynamic membrane trafficking system, which relies on motor proteins, is required for myelin formation and maintenance. Previously, we found that myosin ID (Myo1d), a class I myosin, is enriched in the rat CNS myelin fraction. Myo1d is an unconventional myosin and has been shown to be involved in membrane trafficking in the recycling pathway in an epithelial cell line. Western blotting revealed that Myo1d expression begins early in myelinogenesis and continues to increase into adulthood. The localization of Myo1d in CNS myelin has not been reported, and the function of Myo1d in vivo remains unknown. To demonstrate the expression of Myo1d in CNS myelin and to begin to explore the function of Myo1d in myelination, we produced a new antibody against Myo1d that has a high titer and specificity for rat Myo1d. By using this antibody, we demonstrated that Myo1d is expressed in rat CNS myelin and is especially abundant in abaxonal and adaxonal regions (the outer and inner cytoplasm-containing loops, respectively), but that expression is low in peripheral nervous system myelin. In culture, Myo1d was expressed in mature rat oligodendrocytes. Furthermore, an increase in expression of Myo1d during maturation of CNS white matter (cerebellum and corpus callosum) was demonstrated by histological analysis. These results suggest that Myo1d may be involved in the formation and/or maintenance of CNS myelin. © 2014 Wiley Periodicals, Inc.

Cardiomyocyte-expressed farnesoid-X-receptor is a novel apoptosis mediator and contributes to myocardial ischaemia/reperfusion injury

PubMed Central

Pu, Jun; Yuan, Ancai; Shan, Peiren; Gao, Erhe; Wang, Xiaoliang; Wang, Yajing; Lau, Wayne Bond; Koch, Walter; Ma, Xin-Liang; He, Ben

2013-01-01

Aims Emerging evidence indicates that nuclear receptors play a critical regulatory role in cardiovascular physiology/pathology. Recently, farnesoid-X-receptor (FXR), a member of the metabolic nuclear receptor superfamily, has been demonstrated to be expressed in vascular cells, with important roles in vascular physiology/pathology. However, the potential cardiac function of FXR remains unclear. We investigated the cardiac expression and biological function of FXR. Methods and results Farnesoid-X-receptor was detected in both isolated neonatal rat cardiac myocytes and fibroblasts. Natural and synthetic FXR agonists upregulated cardiac FXR expression, stimulated myocyte apoptosis, and reduced myocyte viability dose- and time-dependently. Mechanistic studies demonstrated that FXR agonists disrupted mitochondria, characterized by mitochondrial permeability transition pores activation, mitochondrial potential dissipation, cytochrome c release, and both caspase-9 and -3 activation. Such mitochondrial apoptotic responses were abolished by siRNA-mediated silencing of endogenous FXR or pharmacological inhibition of mitochondrial death signalling. Furthermore, low levels of FXR were detected in the adult mouse heart, with significant (∼2.0-fold) upregulation after myocardial ischaemia/reperfusion (MI/R). Pharmacological inhibition or genetic ablation of FXR significantly reduced myocardial apoptosis by 29.0–53.4%, decreased infarct size by 23.4–49.7%, and improved cardiac function in ischaemic/reperfused myocardium. Conclusion These results demonstrate that nuclear receptor FXR acts as a novel functional receptor in cardiac tissue, regulates apoptosis in cardiomyocytes, and contributes to MI/R injury. PMID:22307460

Promoting Expressive Language in Young Children with or At-Risk for Autism Spectrum Disorder in a Preschool Classroom

ERIC Educational Resources Information Center

Lane, Justin D.; Shepley, Collin; Lieberman-Betz, Rebecca

2016-01-01

Young children with autism spectrum disorder (ASD) often demonstrate delays in expressive communication, impacting their ability to independently function in typical environments. Individuals with ASD who develop expressive language during early childhood experience better outcomes later in life; therefore, examination of naturalistic language…

Qigesan inhibits migration and invasion of esophageal cancer cells via inducing connexin expression and enhancing gap junction function.

PubMed

Shi, Huijuan; Shi, Dongxuan; Wu, Yansong; Shen, Qiang; Li, Jing

2016-09-28

Qigesan (QGS), a well-known traditional Chinese medicinal formula, has long been used to treat patients with esophageal cancer. However, the anticancer mechanisms of action of QGS remain unknown. This study aims to determine whether QGS regulates gap junction (GJ) function and affects the invasiveness of esophageal cancer cells. Our results demonstrate that QGS markedly inhibits the migration and invasion of esophageal cancer cells in vitro. We further show that QGS enhances the function of GJ in esophageal cancer cells. We therefore hypothesized that enhanced connexin expression leads to enhanced GJ function and inhibition of metastasis. We found that QGS enhances expression of connexin 26 and connexin 43 in esophageal cancer cells. This study suggests that QGS increases GJ function via enhancing the expression of connexins, resulting in reduced esophageal cancer cell migration and invasion. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Anti-leukemic activity and tolerability of anti-human CD47 monoclonal antibodies

PubMed Central

Pietsch, E C; Dong, J; Cardoso, R; Zhang, X; Chin, D; Hawkins, R; Dinh, T; Zhou, M; Strake, B; Feng, P-H; Rocca, M; Santos, C Dos; Shan, X; Danet-Desnoyers, G; Shi, F; Kaiser, E; Millar, H J; Fenton, S; Swanson, R; Nemeth, J A; Attar, R M

2017-01-01

CD47, a broadly expressed cell surface protein, inhibits cell phagocytosis via interaction with phagocyte-expressed SIRPα. A variety of hematological malignancies demonstrate elevated CD47 expression, suggesting that CD47 may mediate immune escape. We discovered three unique CD47-SIRPα blocking anti-CD47 monoclonal antibodies (mAbs) with low nano-molar affinity to human and cynomolgus monkey CD47, and no hemagglutination and platelet aggregation activity. To characterize the anti-cancer activity elicited by blocking CD47, the mAbs were cloned into effector function silent and competent Fc backbones. Effector function competent mAbs demonstrated potent activity in vitro and in vivo, while effector function silent mAbs demonstrated minimal activity, indicating that blocking CD47 only leads to a therapeutic effect in the presence of Fc effector function. A non-human primate study revealed that the effector function competent mAb IgG1 C47B222-(CHO) decreased red blood cells (RBC), hematocrit and hemoglobin by >40% at 1 mg/kg, whereas the effector function silent mAb IgG2σ C47B222-(CHO) had minimal impact on RBC indices at 1 and 10 mg/kg. Taken together, our findings suggest that targeting CD47 is an attractive therapeutic anti-cancer approach. However, the anti-cancer activity observed with anti-CD47 mAbs is Fc effector dependent as are the side effects observed on RBC indices. PMID:28234345

Transcriptional repression of epithelial cell adhesion molecule (EpCAM) contributes to p53 control of breast cancer invasion

PubMed Central

Sankpal, NV; Willman, MW; Fleming, TP; Mayfield, J; Gillanders, WE

2014-01-01

p53 is a tumor suppressor gene with well-characterized roles in cell cycle regulation, apoptosis and the maintenance of genome stability. Recent evidence suggests that p53 may also contribute to the regulation of migration and invasion. Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that is overexpressed in the majority of human epithelial carcinomas, including breast and colorectal carcinomas. We demonstrate by chromatin immunoprecipitation assays that p53 interacts with a candidate p53 binding site within the EpCAM gene. p53-mediated transcriptional repression of EpCAM was confirmed in gain-of-function, and loss-of-function experimental systems. Induction of wildtype p53 was associated with a significant dose-dependent decrease in EpCAM expression; conversely, specific ablation of p53 was associated with a significant increase in EpCAM expression. At the functional level, specific ablation of p53 expression is associated with increased breast cancer invasion, and this effect is abrogated by concomitant specific ablation of EpCAM expression. Taken together, these biochemical and functional data are the first demonstration that (1) wildtype p53 protein binds to a response element within the EpCAM gene and negatively regulates EpCAM expression, and (2) transcriptional repression of EpCAM contributes to p53 control of breast cancer invasion. PMID:19141643

The guinea-pig expresses functional CYP2C and P-glycoprotein: further validation of its usefulness in drug biotransformation/transport studies.

PubMed

Hasibu, Ibrahim; Patoine, Dany; Pilote, Sylvie; Drolet, Benoit; Simard, Chantale

2015-04-01

The guinea-pig is an excellent animal model for studying cardiopulmonary physiology/pharmacology. Interestingly, it also possesses a number of drug-metabolizing enzymes found in humans, such as CYP1A, CYP2D and CYP3A. To evaluate the hypothesis that the guinea-pig also expresses a functional CYP2C drug-metabolizing enzyme and the P-glycoprotein (P-gp) drug transporter in various tissues. cDNAs encoding CYP2C and P-gp were obtained from guinea-pig liver or small intestine and sequenced. Western blotting was performed to confirm the expression of CYP2C and P-gp. The functional enzymatic activity of guinea-pig CYP2C was evaluated with microsomal preparations using diclofenac and tolbutamide as specific drug substrates in HPLC analyses. To further study both P-gp and CYP2C functional activities, the guinea-pig ABCB1/MDR1 and CYP2C genes were cloned. The recombinant plasmids were then transfected in HEK293 (human embryonic kidney) cells and either calcein-acetoxymethyl ester (AM) accumulation assays or 14,15-EET/DHET formation experiments were performed to evaluate either P-gp transport activity or CYP2C epoxygenase activity, respectively. The guinea-pig tissue distribution of P-gp was studied by Western blotting. Functional expression of CYP2C was demonstrated in guinea-pig liver microsomal preparations. CYP2C-mediated biotransformation of diclofenac and tolbutamide were shown. Expression of P-gp protein was detected in guinea-pig liver and small intestine. Functional activity of guinea-pig P-gp was demonstrated in ABCB1/MDR1-transfected cells. GP-CYP2C-transfected cells also showed functional epoxygenase activity. The guinea-pig expresses functional CYP2C and P-gp, thus suggesting its usefulness for further validating data obtained with other animal models in drug biotransformation/transport studies. Copyright © 2015 John Wiley & Sons, Ltd.

When your face describes your memories: facial expressions during retrieval of autobiographical memories.

PubMed

El Haj, Mohamad; Daoudi, Mohamed; Gallouj, Karim; Moustafa, Ahmed A; Nandrino, Jean-Louis

2018-05-11

Thanks to the current advances in the software analysis of facial expressions, there is a burgeoning interest in understanding emotional facial expressions observed during the retrieval of autobiographical memories. This review describes the research on facial expressions during autobiographical retrieval showing distinct emotional facial expressions according to the characteristics of retrieved memoires. More specifically, this research demonstrates that the retrieval of emotional memories can trigger corresponding emotional facial expressions (e.g. positive memories may trigger positive facial expressions). Also, this study demonstrates the variations of facial expressions according to specificity, self-relevance, or past versus future direction of memory construction. Besides linking research on facial expressions during autobiographical retrieval to cognitive and affective characteristics of autobiographical memory in general, this review positions this research within the broader context research on the physiologic characteristics of autobiographical retrieval. We also provide several perspectives for clinical studies to investigate facial expressions in populations with deficits in autobiographical memory (e.g. whether autobiographical overgenerality in neurologic and psychiatric populations may trigger few emotional facial expressions). In sum, this review paper demonstrates how the evaluation of facial expressions during autobiographical retrieval may help understand the functioning and dysfunctioning of autobiographical memory.

Liver-enriched transcription factors uncoupled from expression of hepatic functions in hepatoma cell lines.

PubMed Central

Chaya, D; Fougère-Deschatrette, C; Weiss, M C

1997-01-01

Among the liver-enriched transcription factors identified to date, only expression of hepatocyte nuclear factor 4 (HNF4) and hepatocyte nuclear factor 1 (HNF1) is in strict correlation with hepatic differentiation in cultured rat hepatoma cells. Indeed, differentiated hepatoma cells that stably express an extensive set of adult hepatic functions express liver-enriched transcription factors, while dedifferentiated cells that have lost expression of all these hepatic functions no longer express HNF4 and HNF1. We describe a new heritable phenotype, designated as uncoupled, in which there is a spontaneous dissociation between the expression of these transcription factors and that of the hepatic functions. Cells presenting this phenotype, isolated from differentiated hepatoma cells, cease to accumulate all transcripts coding for hepatic functions but nevertheless maintain expression of HNF4 and HNF1. Transitory transfection experiments indicate that these two factors present in these cells have transcriptional activity similar to that of differentiated hepatoma cells. Characterization of the appropriate intertypic cell hybrids demonstrates that this new phenotype is recessive to the dedifferentiated state and fails to be complemented by differentiated cells. These results indicate the existence of mechanisms that inhibit transcription of genes coding for hepatocyte functions in spite of the presence of functional HNF4 and HNF1. Cells of the uncoupled phenotype present certain properties of oval cells described for pathological states of the liver. PMID:9343392

Liver-enriched transcription factors uncoupled from expression of hepatic functions in hepatoma cell lines.

PubMed

Chaya, D; Fougère-Deschatrette, C; Weiss, M C

1997-11-01

Among the liver-enriched transcription factors identified to date, only expression of hepatocyte nuclear factor 4 (HNF4) and hepatocyte nuclear factor 1 (HNF1) is in strict correlation with hepatic differentiation in cultured rat hepatoma cells. Indeed, differentiated hepatoma cells that stably express an extensive set of adult hepatic functions express liver-enriched transcription factors, while dedifferentiated cells that have lost expression of all these hepatic functions no longer express HNF4 and HNF1. We describe a new heritable phenotype, designated as uncoupled, in which there is a spontaneous dissociation between the expression of these transcription factors and that of the hepatic functions. Cells presenting this phenotype, isolated from differentiated hepatoma cells, cease to accumulate all transcripts coding for hepatic functions but nevertheless maintain expression of HNF4 and HNF1. Transitory transfection experiments indicate that these two factors present in these cells have transcriptional activity similar to that of differentiated hepatoma cells. Characterization of the appropriate intertypic cell hybrids demonstrates that this new phenotype is recessive to the dedifferentiated state and fails to be complemented by differentiated cells. These results indicate the existence of mechanisms that inhibit transcription of genes coding for hepatocyte functions in spite of the presence of functional HNF4 and HNF1. Cells of the uncoupled phenotype present certain properties of oval cells described for pathological states of the liver.

Stimulatory and inhibitory mechanisms of slow muscle-specific myosin heavy chain gene expression in fish: Transient and transgenic analysis of torafugu MYH{sub M86-2} promoter in zebrafish embryos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Asaduzzaman, Md.; Kinoshita, Shigeharu, E-mail: akino@mail.ecc.u-tokyo.ac.jp; Bhuiyan, Sharmin Siddique

The myosin heavy chain gene, MYH{sub M86-2}, exhibited restricted expression in slow muscle fibers of torafugu embryos and larvae, suggesting its functional roles for embryonic and larval muscle development. However, the transcriptional mechanisms involved in its expression are still ambiguous. The present study is the first extensive analysis of slow muscle-specific MYH{sub M86-2} promoter in fish for identifying the cis-elements that are crucial for its expression. Combining both transient transfection and transgenic approaches, we demonstrated that the 2614 bp 5′-flanking sequences of MYH{sub M86-2} contain a sufficient promoter activity to drive gene expression specific to superficial slow muscle fibers. Bymore » cyclopamine treatment, we also demonstrated that the differentiation of such superficial slow muscle fibers depends on hedgehog signaling activity. The deletion analyses defined an upstream fragment necessary for repressing ectopic MYH{sub M86-2} expression in the fast muscle fibers. The transcriptional mechanism that prevents MYH{sub M86-2} expression in the fast muscle fibers is mediated through Sox6 binding elements. We also demonstrated that Sox6 may function as a transcriptional repressor of MYH{sub M86-2} expression. We further discovered that nuclear factor of activated T cells (NFAT) binding elements plays a key role and myocyte enhancer factor-2 (MEF2) binding elements participate in the transcriptional regulation of MYH{sub M86-2} expression. - Highlights: ► MYH{sub M86-2} is highly expressed in slow muscle fibers of torafugu embryos and larvae. ► MYH{sub M86-2} promoter activity depends on the hedgehog signaling. ► Sox6 binding elements inhibits MYH{sub M86-2} expression in fast muscle fibers. ► Sox6 elements function as transcriptional repressor of MYH{sub M86-2} promoter activity. ► NFAT and MEF2 binding elements play a key role for directing MYH{sub M86-2} expression.« less

Modulation of epithelial sodium channel trafficking and function by sodium 4-phenylbutyrate in human nasal epithelial cells.

PubMed

Prulière-Escabasse, Virginie; Planès, Carole; Escudier, Estelle; Fanen, Pascale; Coste, André; Clerici, Christine

2007-11-23

Sodium 4-phenylbutyrate (4-PBA) has been shown to correct the cellular trafficking of several mutant or nonmutant plasma membrane proteins such as cystic fibrosis transmembrane conductance regulator through the expression of 70-kDa heat shock proteins. The objective of the study was to determine whether 4-PBA may influence the functional expression of epithelial sodium channels (ENaC) in human nasal epithelial cells (HNEC). Using primary cultures of HNEC, we demonstrate that 4-PBA (5 mm for 6 h) markedly stimulated amiloride-sensitive sodium channel activity and that this was related to an increased abundance of alpha-, beta-, and gamma-ENaC subunits in the apical membrane. The increase in ENaC cell surface expression (i) was due to insertion of newly ENaC subunits as determined by brefeldin A experiments and (ii) was not associated with cell surface retention of ENaC subunits because endocytosis of ENaC subunits was unchanged. In addition, we find that ENaC co-immunoprecipitated with the heat shock protein constitutively expressed Hsc70, that has been reported to modulate ENaC trafficking, and that 4-PBA decreased Hsc70 protein level. Finally, we report that in cystic fibrosis HNEC obtained from two cystic fibrosis patients, 4-PBA increased functional expression of ENaC as demonstrated by the increase in amiloride-sensitive sodium transport and in alpha-, beta-, and gamma-ENaC subunit expression in the apical membrane. Our results suggest that in HNEC, 4-PBA increases the functional expression of ENaC through the insertion of new alpha-, beta-, and gamma-ENaC subunits into the apical membrane and also suggest that 4-PBA could modify ENaC trafficking by reducing Hsc70 protein expression.

Dimorphous expressions of positive emotion: displays of both care and aggression in response to cute stimuli.

PubMed

Aragón, Oriana R; Clark, Margaret S; Dyer, Rebecca L; Bargh, John A

2015-03-01

Extremely positive experiences, and positive appraisals thereof, produce intense positive emotions that often generate both positive expressions (e.g., smiles) and expressions normatively reserved for negative emotions (e.g., tears). We developed a definition of these dimorphous expressions and tested the proposal that their function is to regulate emotions. We showed that individuals who express emotions in this dimorphous manner do so as a general response across a variety of emotionally provoking situations, which suggests that these expressions are responses to intense positive emotion rather than unique to one particular situation. We used cute stimuli (an elicitor of positive emotion) to demonstrate both the existence of these dimorphous expressions and to provide preliminary evidence of their function as regulators of emotion. © The Author(s) 2015.

Transcriptional and Translational Plasticity in Rodent Urinary Bladder TRP Channels with Urinary Bladder Inflammation, Bladder Dysfunction or Postnatal Maturation

PubMed Central

Merrill, Liana; Girard, Beatrice M.; May, Victor; Vizzard, Margaret A.

2013-01-01

These studies examined transcriptional and translational plasticity of three transient receptor potential (TRP) channels (TRPA1, TRPV1, TRPV4) with established neuronal and non-neuronal expression and functional roles in the lower urinary tract. Mechanosensor and nociceptor roles in either physiological or pathological lower urinary tract states have been suggested for TRPA1, TRPV1 and TRPV4. We have previously demonstrated neurochemical, organizational and functional plasticity in micturition reflex pathways following induction of urinary bladder inflammation using the antineoplastic agent, cyclophosphamide (CYP). More recently, we have characterized similar plasticity in micturition reflex pathways in a transgenic mouse model with chronic urothelial overexpression (OE) of nerve growth factor (NGF) and in a transgenic mouse model with deletion of vasoactive intestinal polypeptide (VIP). In addition, the micturition reflex undergoes postnatal maturation that may also reflect plasticity in urinary bladder TRP channel expression. Thus, we examined plasticity in urinary bladder TRP channel expression in diverse contexts using a combination of quantitative, real-time PCR and western blotting approaches. We demonstrate transcriptional and translational plasticity of urinary bladder TRPA1, TRPV1 and TRVP4 expression. Although the functional significance of urinary bladder TRP channel plasticity awaits further investigation, these studies demonstrate context-(inflammation, postnatal development, NGF-OE, VIP deletion) and tissue-dependent (urothelium + suburothelium, detrusor) plasticity. PMID:22865090

Genome-Level Longitudinal Expression of Signaling Pathways and Gene Networks in Pediatric Septic Shock

PubMed Central

Shanley, Thomas P; Cvijanovich, Natalie; Lin, Richard; Allen, Geoffrey L; Thomas, Neal J; Doctor, Allan; Kalyanaraman, Meena; Tofil, Nancy M; Penfil, Scott; Monaco, Marie; Odoms, Kelli; Barnes, Michael; Sakthivel, Bhuvaneswari; Aronow, Bruce J; Wong, Hector R

2007-01-01

We have conducted longitudinal studies focused on the expression profiles of signaling pathways and gene networks in children with septic shock. Genome-level expression profiles were generated from whole blood-derived RNA of children with septic shock (n = 30) corresponding to day one and day three of septic shock, respectively. Based on sequential statistical and expression filters, day one and day three of septic shock were characterized by differential regulation of 2,142 and 2,504 gene probes, respectively, relative to controls (n = 15). Venn analysis demonstrated 239 unique genes in the day one dataset, 598 unique genes in the day three dataset, and 1,906 genes common to both datasets. Functional analyses demonstrated time-dependent, differential regulation of genes involved in multiple signaling pathways and gene networks primarily related to immunity and inflammation. Notably, multiple and distinct gene networks involving T cell- and MHC antigen-related biology were persistently downregulated on both day one and day three. Further analyses demonstrated large scale, persistent downregulation of genes corresponding to functional annotations related to zinc homeostasis. These data represent the largest reported cohort of patients with septic shock subjected to longitudinal genome-level expression profiling. The data further advance our genome-level understanding of pediatric septic shock and support novel hypotheses. PMID:17932561

Genetic analysis of an overlapping functional requirement for L1- and NCAM-type proteins during sensory axon guidance in Drosophila.

PubMed

Kristiansen, Lars V; Velasquez, Emma; Romani, Susana; Baars, Sigrid; Berezin, Vladimir; Bock, Elisabeth; Hortsch, Michael; Garcia-Alonso, Luis

2005-01-01

L1- and NCAM-type cell adhesion molecules represent distinct protein families that function as specific receptors for different axon guidance cues. However, both L1 and NCAM proteins promote axonal growth by inducing neuronal tyrosine kinase activity and are coexpressed in subsets of axon tracts in arthropods and vertebrates. We have studied the functional requirements for the Drosophila L1- and NCAM-type proteins, Neuroglian (Nrg) and Fasciclin II (FasII), during postembryonic sensory axon guidance. The rescue of the Neuroglian loss-of-function (LOF) phenotype by transgenically expressed L1- and NCAM-type proteins demonstrates a functional interchangeability between these proteins in Drosophila photoreceptor pioneer axons, where both proteins are normally coexpressed. In contrast, the ectopic expression of Fasciclin II in mechanosensory neurons causes a strong enhancement of the axonal misguidance phenotype. Moreover, our findings demonstrate that this functionally redundant specificity to mediate axon guidance has been conserved in their vertebrate homologs, L1-CAM and NCAM.

Functional and molecular evidence for expression of the renin angiotensin system and ADAM17-mediated ACE2 shedding in COS7 cells

PubMed Central

Grobe, Nadja; Di Fulvio, Mauricio; Kashkari, Nada; Chodavarapu, Harshita; Somineni, Hari K.; Singh, Richa

2015-01-01

The renin angiotensin system (RAS) plays a vital role in the regulation of the cardiovascular and renal functions. COS7 is a robust and easily transfectable cell line derived from the kidney of the African green monkey, Cercopithecus aethiops. The aims of this study were to 1) demonstrate the presence of an endogenous and functional RAS in COS7, and 2) investigate the role of a disintegrin and metalloproteinase-17 (ADAM17) in the ectodomain shedding of angiotensin converting enzyme-2 (ACE2). Reverse transcription coupled to gene-specific polymerase chain reaction demonstrated expression of ACE, ACE2, angiotensin II type 1 receptor (AT1R), and renin at the transcript levels in total RNA cell extracts. Western blot and immunohistochemistry identified ACE (60 kDa), ACE2 (75 kDa), AT1R (43 kDa), renin (41 kDa), and ADAM17 (130 kDa) in COS7. At the functional level, a sensitive and selective mass spectrometric approach detected endogenous renin, ACE, and ACE2 activities. ANG-(1–7) formation (m/z 899) from the natural substrate ANG II (m/z 1,046) was detected in lysates and media. COS7 cells stably expressing shRNA constructs directed against endogenous ADAM17 showed reduced ACE2 shedding into the media. This is the first study demonstrating endogenous expression of the RAS and ADAM17 in the widely used COS7 cell line and its utility to study ectodomain shedding of ACE2 mediated by ADAM17 in vitro. The transfectable nature of this cell line makes it an attractive cell model for studying the molecular, functional, and pharmacological properties of the renal RAS. PMID:25740155

Human Eosinophils Express Functional CCR7

PubMed Central

Ueki, Shigeharu; Estanislau, Jessica; Weller, Peter F.

2013-01-01

Human eosinophils display directed chemotactic activity toward an array of soluble chemokines. Eosinophils have been observed to migrate to draining lymph nodes in experimental models of allergic inflammation, yet it is unknown whether eosinophils express CCR7, a key chemokine receptor in coordinating leukocyte trafficking to lymph nodes. The purpose of this study is to demonstrate expression of CCR7 by human eosinophils and functional responses to CCL19 and CCL21, the known ligands of CCR7. Human eosinophils were purified by negative selection from healthy donors. CCR7 expression of freshly purified, unstimulated eosinophils and of IL-5–primed eosinophils was determined by flow cytometry and Western blot. Chemotaxis to CCL19 and CCL21 was measured in transwell assays. Shape changes to CCL19 and CCL21 were analyzed by flow cytometry and microscopy. Calcium fluxes of fluo-4 AM–loaded eosinophils were recorded by flow cytometry after chemokine stimulation. ERK phosphorylation of CCL19- and CCL21-stimulated eosinophils was measured by Western blot and Luminex assay. Human eosinophils expressed CCR7 as demonstrated by flow cytometry and Western blots. Eosinophils exhibited detectable cell surface expression of CCR7. IL-5–primed eosinophils exhibited chemotaxis toward CCL19 and CCL21 in a dose-dependent fashion. Upon stimulation with CCL19 or CCL21, IL-5–primed eosinophils demonstrated dose-dependent shape changes with polarization of F-actin and exhibited calcium influxes. Finally, primed eosinophils stimulated with CCL19 or CCL21 exhibited increased phosphorylation of ERK in response to both CCR7 ligands. We demonstrate that human eosinophils express CCR7 and have multipotent responses to the known ligands of CCR7. PMID:23449735

MEF2‑activated long non‑coding RNA PCGEM1 promotes cell proliferation in hormone‑refractory prostate cancer through downregulation of miR‑148a.

PubMed

Zhang, Shibao; Li, Zongwu; Zhang, Longyang; Xu, Zhonghua

2018-05-04

Prostate cancer gene expression marker 1 (PCGEM1) is a prostate‑specific gene overexpressed in prostate cancer cells that promotes cell proliferation. To study the molecular mechanism of PCGEM1 function in hormone‑refractory prostate cancer, the interaction between myocyte enhancer factor 2 (MEF2) and PCGEM1 was assessed by a luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay. In addition, the underlying mechanism of PCGEM1 regulating expression of microRNA (miR)‑148a in PC3 prostate cancer cells was evaluated. Relative expression levels were measured by reverse transcription‑quantitative polymerase chain reaction, and early apoptosis was measured by flow cytometry. PCGEM1 was demonstrated to be overexpressed in prostate cancer tissues compared with noncancerous tissues. Expression levels of PCGEM1 in PC3 cancer cells were demonstrated to be regulated by MEF2, as PCGME1 mRNA was increased by MEF2 overexpression but decreased by MEF2 silencing. MEF2 was also demonstrated to enhance the activity of PCGEM1 promoter and thus promote PCGEM1 transcription. In addition, downregulation of PCGEM1 expression in PC3 cells increased expression of miR‑148a. By contrast, overexpression of PCGEM1 decreased miR‑148a expression. Finally, PCGME1 silencing by small interfering RNA significantly induced early cell apoptosis but this effect was reduced by a miR‑148a inhibitor. In conclusion, the present study demonstrated a positive regulatory association between MEF2 and PCGEM1, and a reciprocal negative regulatory association between PCGEM1 and miR‑148a that controls cell apoptosis. The present study, therefore, provides new insights into the mechanism of PCGEM1 function in prostate cancer development.

Pathogenic LRRK2 mutations, through increased kinase activity, produce enlarged lysosomes with reduced degradative capacity and increase ATP13A2 expression.

PubMed

Henry, Anastasia G; Aghamohammadzadeh, Soheil; Samaroo, Harry; Chen, Yi; Mou, Kewa; Needle, Elie; Hirst, Warren D

2015-11-01

Lysosomal dysfunction plays a central role in the pathogenesis of several neurodegenerative disorders, including Parkinson's disease (PD). Several genes linked to genetic forms of PD, including leucine-rich repeat kinase 2 (LRRK2), functionally converge on the lysosomal system. While mutations in LRRK2 are commonly associated with autosomal-dominant PD, the physiological and pathological functions of this kinase remain poorly understood. Here, we demonstrate that LRRK2 regulates lysosome size, number and function in astrocytes, which endogenously express high levels of LRRK2. Expression of LRRK2 G2019S, the most common pathological mutation, produces enlarged lysosomes and diminishes the lysosomal capacity of these cells. Enlarged lysosomes appears to be a common phenotype associated with pathogenic LRRK2 mutations, as we also observed this effect in cells expressing other LRRK2 mutations; R1441C or Y1699C. The lysosomal defects associated with these mutations are dependent on both the catalytic activity of the kinase and autophosphorylation of LRRK2 at serine 1292. Further, we demonstrate that blocking LRRK2's kinase activity, with the potent and selective inhibitor PF-06447475, rescues the observed defects in lysosomal morphology and function. The present study also establishes that G2019S mutation leads to a reduction in lysosomal pH and increased expression of the lysosomal ATPase ATP13A2, a gene linked to a parkinsonian syndrome (Kufor-Rakeb syndrome), in brain samples from mouse and human LRRK2 G2019S carriers. Together, these results demonstrate that PD-associated LRRK2 mutations perturb lysosome function in a kinase-dependent manner, highlighting the therapeutic promise of LRRK2 kinase inhibitors in the treatment of PD. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Fab is the most efficient format to express functional antibodies by yeast surface display.

PubMed

Sivelle, Coline; Sierocki, Raphaël; Ferreira-Pinto, Kelly; Simon, Stéphanie; Maillere, Bernard; Nozach, Hervé

2018-04-30

Multiple formats are available for engineering of monoclonal antibodies (mAbs) by yeast surface display, but they do not all lead to efficient expression of functional molecules. We therefore expressed four anti-tumor necrosis factor and two anti-IpaD mAbs as single-chain variable fragment (scFv), antigen-binding fragment (Fab) or single-chain Fabs and compared their expression levels and antigen-binding efficiency. Although the scFv and scFab formats are widely used in the literature, 2 of 6 antibodies were either not or weakly expressed. In contrast, all 6 antibodies expressed as Fab revealed strong binding and high affinity, comparable to that of the soluble form. We also demonstrated that the variations in expression did not affect Fab functionality and were due to variations in light chain display and not to misfolded dimers. Our results suggest that Fab is the most versatile format for the engineering of mAbs.

The antiepileptic medications carbamazepine and phenytoin inhibit native sodium currents in murine osteoblasts.

PubMed

Petty, Sandra J; Milligan, Carol J; Todaro, Marian; Richards, Kay L; Kularathna, Pamuditha K; Pagel, Charles N; French, Chris R; Hill-Yardin, Elisa L; O'Brien, Terence J; Wark, John D; Mackie, Eleanor J; Petrou, Steven

2016-09-01

Fracture risk is a serious comorbidity in epilepsy and may relate to the use of antiepileptic drugs (AEDs). Many AEDs inhibit ion channel function, and the expression of these channels in osteoblasts raises the question of whether altered bone signaling increases bone fragility. We aimed to confirm the expression of voltage-gated sodium (NaV ) channels in mouse osteoblasts, and to investigate the action of carbamazepine and phenytoin on NaV channels. Immunocytochemistry was performed on primary calvarial osteoblasts extracted from neonatal C57BL/6J mice and additional RNA sequencing (RNASeq) was included to confirm expression of NaV . Whole-cell patch-clamp recordings were made to identify the native currents expressed and to assess the actions of carbamazepine (50 μm) or phenytoin (50 μm). NaV expression was demonstrated with immunocytochemistry, RNA sequencing, and functionally, with demonstration of robust tetrodotoxin-sensitive and voltage-activated inward currents. Application of carbamazepine or phenytoin resulted in significant inhibition of current amplitude for carbamazepine (31.6 ± 5.9%, n = 9; p < 0.001), and for phenytoin (35.5 ± 6.9%, n = 7; p < 0.001). Mouse osteoblasts express NaV , and native NaV currents are blocked by carbamazepine and phenytoin, supporting our hypothesis that AEDs can directly influence osteoblast function and potentially affect bone strength. Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.

Local and Systemic CD4+ T Cell Exhaustion Reverses with Clinical Resolution of Pulmonary Sarcoidosis

PubMed Central

Hawkins, Charlene; Shaginurova, Guzel; Shelton, D. Auriel; Herazo-Maya, Jose D.; Oswald-Richter, Kyra A.; Young, Anjuli; Celada, Lindsay J.; Kaminski, Naftali; Sevin, Carla

2017-01-01

Investigation of the Th1 immune response in sarcoidosis CD4+ T cells has revealed reduced proliferative capacity and cytokine expression upon TCR stimulation. In other disease models, such cellular dysfunction has been associated with a step-wise, progressive loss of T cell function that results from chronic antigenic stimulation. T cell exhaustion is defined by decreased cytokine production upon TCR activation, decreased proliferation, increased expression of inhibitory cell surface receptors, and increased susceptibility to apoptosis. We characterized sarcoidosis CD4+ T cell immune function in systemic and local environments among subjects undergoing disease progression compared to those experiencing disease resolution. Spontaneous and TCR-stimulated Th1 cytokine expression and proliferation assays were performed in 53 sarcoidosis subjects and 30 healthy controls. PD-1 expression and apoptosis were assessed by flow cytometry. Compared to healthy controls, sarcoidosis CD4+ T cells demonstrated reductions in Th1 cytokine expression, proliferative capacity (p < 0.05), enhanced apoptosis (p < 0.01), and increased PD-1 expression (p < 0.001). BAL-derived CD4+ T cells also demonstrated multiple facets of T cell exhaustion (p < 0.05). Reversal of CD4+ T cell exhaustion was observed in subjects undergoing spontaneous resolution (p < 0.05). Sarcoidosis CD4+ T cells exhibit loss of cellular function during progressive disease that follows the archetype of T cell exhaustion. PMID:29234685

Identification of Fat Mass and Obesity Associated (FTO) Protein Expression in Cardiomyocytes: Regulation by Leptin and Its Contribution to Leptin-Induced Hypertrophy

PubMed Central

Gan, Xiaohong Tracey; Zhao, Ganjian; Huang, Cathy X.; Rowe, Adrianna C.; Purdham, Daniel M.; Karmazyn, Morris

2013-01-01

The recently-identified fat mass and obesity-associated (FTO) protein is associated with various physiological functions including energy and body weight regulation. Ubiquitously expressed, FTO was identified in heart homogenates although its function is unknown. We studied whether FTO is specifically expressed within the cardiac myocyte and its potential role pertaining to the hypertrophic effect of the adipokine leptin. Most experiments were performed using cultured neonatal rat cardiomyocytes which showed nuclei-specific FTO expression. Leptin significantly increased FTO expression which was associated with myocyte hypertrophy although both events were abrogated by FTO knockdown with siRNA. Administration of a leptin receptor antibody to either normal or obese rats significant reduced myocardial FTO protein expression. Responses in cardiomyocytes were accompanied by JAK2/STAT3 activation whereas JAK2/STAT3 inhibition abolished these effects. Expression of the cut-like homeobox 1(CUX1) transcriptional factor was significantly increased by leptin although this was restricted to the cathepsin L-dependent, proteolytically-derived shorter p110CUX1 isoform whereas the longer p200CUX1 protein was not significantly affected. Cathepsin L expression and activity were both significantly increased by leptin whereas a cathepsin L peptide inhibitor or siRNA specific for CUX1 completely prevented the leptin-induced increase in FTO expression. The cathepsin L peptide inhibitor or siRNA-induced knockdown of either CUX1 or FTO abrogated the hypertrophic response to leptin. Two other pro-hypertrophic factors, endothelin-1 or angiotensin II had no effect on FTO expression and FTO knockdown did not alter the hypertrophic response to either agent. This study demonstrates leptin-induced FTO upregulation in cardiomyocytes via JAK2/STAT3- dependent CUX1 upregulation and suggests an FTO regulatory function of leptin. It also demonstrates for the first time a functional role of FTO in the cardiomyocyte. PMID:24019958

Identification of fat mass and obesity associated (FTO) protein expression in cardiomyocytes: regulation by leptin and its contribution to leptin-induced hypertrophy.

PubMed

Gan, Xiaohong Tracey; Zhao, Ganjian; Huang, Cathy X; Rowe, Adrianna C; Purdham, Daniel M; Karmazyn, Morris

2013-01-01

The recently-identified fat mass and obesity-associated (FTO) protein is associated with various physiological functions including energy and body weight regulation. Ubiquitously expressed, FTO was identified in heart homogenates although its function is unknown. We studied whether FTO is specifically expressed within the cardiac myocyte and its potential role pertaining to the hypertrophic effect of the adipokine leptin. Most experiments were performed using cultured neonatal rat cardiomyocytes which showed nuclei-specific FTO expression. Leptin significantly increased FTO expression which was associated with myocyte hypertrophy although both events were abrogated by FTO knockdown with siRNA. Administration of a leptin receptor antibody to either normal or obese rats significant reduced myocardial FTO protein expression. Responses in cardiomyocytes were accompanied by JAK2/STAT3 activation whereas JAK2/STAT3 inhibition abolished these effects. Expression of the cut-like homeobox 1(CUX1) transcriptional factor was significantly increased by leptin although this was restricted to the cathepsin L-dependent, proteolytically-derived shorter p110CUX1 isoform whereas the longer p200CUX1 protein was not significantly affected. Cathepsin L expression and activity were both significantly increased by leptin whereas a cathepsin L peptide inhibitor or siRNA specific for CUX1 completely prevented the leptin-induced increase in FTO expression. The cathepsin L peptide inhibitor or siRNA-induced knockdown of either CUX1 or FTO abrogated the hypertrophic response to leptin. Two other pro-hypertrophic factors, endothelin-1 or angiotensin II had no effect on FTO expression and FTO knockdown did not alter the hypertrophic response to either agent. This study demonstrates leptin-induced FTO upregulation in cardiomyocytes via JAK2/STAT3- dependent CUX1 upregulation and suggests an FTO regulatory function of leptin. It also demonstrates for the first time a functional role of FTO in the cardiomyocyte.

Massive-scale gene co-expression network construction and robustness testing using random matrix theory.

PubMed

Gibson, Scott M; Ficklin, Stephen P; Isaacson, Sven; Luo, Feng; Feltus, Frank A; Smith, Melissa C

2013-01-01

The study of gene relationships and their effect on biological function and phenotype is a focal point in systems biology. Gene co-expression networks built using microarray expression profiles are one technique for discovering and interpreting gene relationships. A knowledge-independent thresholding technique, such as Random Matrix Theory (RMT), is useful for identifying meaningful relationships. Highly connected genes in the thresholded network are then grouped into modules that provide insight into their collective functionality. While it has been shown that co-expression networks are biologically relevant, it has not been determined to what extent any given network is functionally robust given perturbations in the input sample set. For such a test, hundreds of networks are needed and hence a tool to rapidly construct these networks. To examine functional robustness of networks with varying input, we enhanced an existing RMT implementation for improved scalability and tested functional robustness of human (Homo sapiens), rice (Oryza sativa) and budding yeast (Saccharomyces cerevisiae). We demonstrate dramatic decrease in network construction time and computational requirements and show that despite some variation in global properties between networks, functional similarity remains high. Moreover, the biological function captured by co-expression networks thresholded by RMT is highly robust.

Gene expression links functional networks across cortex and striatum.

PubMed

Anderson, Kevin M; Krienen, Fenna M; Choi, Eun Young; Reinen, Jenna M; Yeo, B T Thomas; Holmes, Avram J

2018-04-12

The human brain is comprised of a complex web of functional networks that link anatomically distinct regions. However, the biological mechanisms supporting network organization remain elusive, particularly across cortical and subcortical territories with vastly divergent cellular and molecular properties. Here, using human and primate brain transcriptional atlases, we demonstrate that spatial patterns of gene expression show strong correspondence with limbic and somato/motor cortico-striatal functional networks. Network-associated expression is consistent across independent human datasets and evolutionarily conserved in non-human primates. Genes preferentially expressed within the limbic network (encompassing nucleus accumbens, orbital/ventromedial prefrontal cortex, and temporal pole) relate to risk for psychiatric illness, chloride channel complexes, and markers of somatostatin neurons. Somato/motor associated genes are enriched for oligodendrocytes and markers of parvalbumin neurons. These analyses indicate that parallel cortico-striatal processing channels possess dissociable genetic signatures that recapitulate distributed functional networks, and nominate molecular mechanisms supporting cortico-striatal circuitry in health and disease.

Sonic hedgehog expression in corticofugal projection neurons directs cortical microcircuit formation.

PubMed

Harwell, Corey C; Parker, Philip R L; Gee, Steven M; Okada, Ami; McConnell, Susan K; Kreitzer, Anatol C; Kriegstein, Arnold R

2012-03-22

The precise connectivity of inputs and outputs is critical for cerebral cortex function; however, the cellular mechanisms that establish these connections are poorly understood. Here, we show that the secreted molecule Sonic Hedgehog (Shh) is involved in synapse formation of a specific cortical circuit. Shh is expressed in layer V corticofugal projection neurons and the Shh receptor, Brother of CDO (Boc), is expressed in local and callosal projection neurons of layer II/III that synapse onto the subcortical projection neurons. Layer V neurons of mice lacking functional Shh exhibit decreased synapses. Conversely, the loss of functional Boc leads to a reduction in the strength of synaptic connections onto layer Vb, but not layer II/III, pyramidal neurons. These results demonstrate that Shh is expressed in postsynaptic target cells while Boc is expressed in a complementary population of presynaptic input neurons, and they function to guide the formation of cortical microcircuitry. Copyright © 2012 Elsevier Inc. All rights reserved.

Obesity-associated variants within FTO form long-range functional connections with IRX3.

PubMed

Smemo, Scott; Tena, Juan J; Kim, Kyoung-Han; Gamazon, Eric R; Sakabe, Noboru J; Gómez-Marín, Carlos; Aneas, Ivy; Credidio, Flavia L; Sobreira, Débora R; Wasserman, Nora F; Lee, Ju Hee; Puviindran, Vijitha; Tam, Davis; Shen, Michael; Son, Joe Eun; Vakili, Niki Alizadeh; Sung, Hoon-Ki; Naranjo, Silvia; Acemel, Rafael D; Manzanares, Miguel; Nagy, Andras; Cox, Nancy J; Hui, Chi-Chung; Gomez-Skarmeta, Jose Luis; Nóbrega, Marcelo A

2014-03-20

Genome-wide association studies (GWAS) have reproducibly associated variants within introns of FTO with increased risk for obesity and type 2 diabetes (T2D). Although the molecular mechanisms linking these noncoding variants with obesity are not immediately obvious, subsequent studies in mice demonstrated that FTO expression levels influence body mass and composition phenotypes. However, no direct connection between the obesity-associated variants and FTO expression or function has been made. Here we show that the obesity-associated noncoding sequences within FTO are functionally connected, at megabase distances, with the homeobox gene IRX3. The obesity-associated FTO region directly interacts with the promoters of IRX3 as well as FTO in the human, mouse and zebrafish genomes. Furthermore, long-range enhancers within this region recapitulate aspects of IRX3 expression, suggesting that the obesity-associated interval belongs to the regulatory landscape of IRX3. Consistent with this, obesity-associated single nucleotide polymorphisms are associated with expression of IRX3, but not FTO, in human brains. A direct link between IRX3 expression and regulation of body mass and composition is demonstrated by a reduction in body weight of 25 to 30% in Irx3-deficient mice, primarily through the loss of fat mass and increase in basal metabolic rate with browning of white adipose tissue. Finally, hypothalamic expression of a dominant-negative form of Irx3 reproduces the metabolic phenotypes of Irx3-deficient mice. Our data suggest that IRX3 is a functional long-range target of obesity-associated variants within FTO and represents a novel determinant of body mass and composition.

Reduced Renal Methylarginine Metabolism Protects against Progressive Kidney Damage

PubMed Central

Caplin, Ben; Boruc, Olga; Bruce-Cobbold, Claire; Cutillas, Pedro; Dormann, Dirk; Faull, Peter; Grossman, Rebecca C.; Khadayate, Sanjay; Mas, Valeria R.; Nitsch, Dorothea D.; Wang, Zhen; Norman, Jill T.; Wilcox, Christopher S.; Wheeler, David C.; Leiper, James

2015-01-01

Nitric oxide (NO) production is diminished in many patients with cardiovascular and renal disease. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthesis, and elevated plasma levels of ADMA are associated with poor outcomes. Dimethylarginine dimethylaminohydrolase-1 (DDAH1) is a methylarginine-metabolizing enzyme that reduces ADMA levels. We reported previously that a DDAH1 gene variant associated with increased renal DDAH1 mRNA transcription and lower plasma ADMA levels, but counterintuitively, a steeper rate of renal function decline. Here, we test the hypothesis that reduced renal-specific ADMA metabolism protects against progressive renal damage. Renal DDAH1 is expressed predominately within the proximal tubule. A novel proximal tubule–specific Ddah1 knockout (Ddah1PT−/−) mouse demonstrated tubular cell accumulation of ADMA and lower NO concentrations, but unaltered plasma ADMA concentrations. Ddah1PT−/− mice were protected from reduced kidney tissue mass, collagen deposition, and profibrotic cytokine expression in two independent renal injury models: folate nephropathy and unilateral ureteric obstruction. Furthermore, a study of two independent kidney transplant cohorts revealed higher levels of human renal allograft methylarginine-metabolizing enzyme gene expression associated with steeper function decline. We also report an association among DDAH1 expression, NO activity, and uromodulin expression supported by data from both animal and human studies, raising the possibility that kidney DDAH1 expression exacerbates renal injury through uromodulin-related mechanisms. Together, these data demonstrate that reduced renal tubular ADMA metabolism protects against progressive kidney function decline. Thus, circulating ADMA may be an imprecise marker of renal methylarginine metabolism, and therapeutic ADMA reduction may even be deleterious to kidney function. PMID:25855779

The essential role of endogenous ghrelin in growth hormone expression during zebrafish adenohypophysis development.

PubMed

Li, Xi; He, Jiangyan; Hu, Wei; Yin, Zhan

2009-06-01

Ghrelin, a multifunctional hormone, including potent GH stimulation activity, has been suggested to be important during embryonic development. Expression of ghrelin has been confirmed in the zebrafish pancreas during embryonic stages. Interfering with ghrelin function using two specific antisense morpholino oligonucleotides causes defects during zebrafish embryonic development. In ghrelin morphants the expression of GH was abolished in zebrafish somatotropes, whereas the expression patterns of the other key molecules involved in hypothalamic-pituitary development and distinct pituitary hormones genes remain largely intact at the appropriate time during zebrafish adenohypophysis development. Effective rescue of the ghrelin morphants with exogenous ghrelin mRNA showed that the correct gene had been targeted. Moreover, by analyzing the efficiencies of the ghrelin morphants rescue experiments with various forms of exogenous mutant ghrelin mRNAs, we also demonstrated the essentiality of the form acyl-ghrelin on GH stimulation during zebrafish adenohypophysis development. Our in vivo experiments, for the first time, also provided evidence of the existence of functional obestatin in the C-terminal part of zebrafish proghrelin peptides. Our research here has demonstrated that zebrafish is a unique model for functional studies of endogenous ghrelin, especially during embryonic development.

Identifying Novel Helix–Loop–Helix Genes in Caenorhabditis elegans through a Classroom Demonstration of Functional Genomics

PubMed Central

Griffin, Vernetta; McMiller, Tracee; Jones, Erika; Johnson, Casonya M.

2003-01-01

A 14-week, undergraduate-level Genetics and Population Biology course at Morgan State University was modified to include a demonstration of functional genomics in the research laboratory. Students performed a rudimentary sequence analysis of the Caenorhabditis elegans genome and further characterized three sequences that were predicted to encode helix–loop–helix proteins. Students then used reverse transcription–polymerase chain reaction to determine which of the three genes is normally expressed in C. elegans. At the end of this laboratory activity, students were 1) to demonstrate a rudimentary knowledge of bioinformatics, including the ability to differentiate between “having” a gene and “expressing” a gene, and 2) to understand basic approaches to functional genomics, including one specific technique for assaying for gene expression. It was also anticipated that students would increase their skills at effectively communicating their research activities through written and/or oral presentation. This article describes the laboratory activity and the assessment of the effectiveness of the activity. PMID:12822036

The role of sialomucin CD164 (MGC-24v or endolyn) in prostate cancer metastasis

PubMed Central

Havens, AM; Jung, Y; Sun, YX; Wang, J; Shah, RB; Bühring, HJ; Pienta, KJ; Taichman, RS

2006-01-01

Background The chemokine stromal derived factor-1 (SDF-1 or CXCL12) and its receptor CXCR4 have been demonstrated to be crucial for the homing of stem cells and prostate cancers to the marrow. While screening prostate cancers for CXCL12-responsive adhesion molecules, we identified CD164 (MGC-24) as a potential regulator of homing. CD164 is known to function as a receptor that regulates stem cell localization to the bone marrow. Results Using prostate cancer cell lines, it was demonstrated that CXCL12 induced both the expression of CD164 mRNA and protein. Functional studies demonstrated that blocking CD164 on prostate cancer cell lines reduced the ability of these cells to adhere to human bone marrow endothelial cells, and invade into extracellular matrices. Human tissue microarrays stained for CD164 demonstrated a positive correlation with prostate-specific antigen levels, while its expression was negatively correlated with the expression of androgen receptor. Conclusion Our findings suggest that CD164 may participate in the localization of prostate cancer cells to the marrow and is further evidence that tumor metastasis and hematopoietic stem cell trafficking may involve similar processes. PMID:16859559

Brown adipose tissue (BAT) specific vaspin expression is increased after obesogenic diets and cold exposure and linked to acute changes in DNA-methylation.

PubMed

Weiner, Juliane; Rohde, Kerstin; Krause, Kerstin; Zieger, Konstanze; Klöting, Nora; Kralisch, Susan; Kovacs, Peter; Stumvoll, Michael; Blüher, Matthias; Böttcher, Yvonne; Heiker, John T

2017-06-01

Several studies have demonstrated anti-diabetic and anti-obesogenic properties of visceral adipose tissue-derived serine protease inhibitor (vaspin) and so evoked its potential use for treatment of obesity-related diseases. The aim of the study was to unravel physiological regulators of vaspin expression and secretion with a particular focus on its role in brown adipose tissue (BAT) biology. We analyzed the effects of obesogenic diets and cold exposure on vaspin expression in liver and white and brown adipose tissue (AT) and plasma levels. Vaspin expression was analyzed in isolated white and brown adipocytes during adipogenesis and in response to adrenergic stimuli. DNA-methylation within the vaspin promoter was analyzed to investigate acute epigenetic changes after cold-exposure in BAT. Our results demonstrate a strong induction of vaspin mRNA and protein expression specifically in BAT of both cold-exposed and high-fat (HF) or high-sugar (HS) fed mice. While obesogenic diets also upregulated hepatic vaspin mRNA levels, cold exposure tended to increase vaspin gene expression of inguinal white adipose tissue (iWAT) depots. Concomitantly, vaspin plasma levels were decreased upon obesogenic or thermogenic triggers. Vaspin expression was increased during adipogenesis but unaffected by sympathetic activation in brown adipocytes. Analysis of vaspin promoter methylation in AT revealed lowest methylation levels in BAT, which were acutely reduced after cold exposure. Our data demonstrate a novel BAT-specific regulation of vaspin gene expression upon physiological stimuli in vivo with acute epigenetic changes that may contribute to cold-induced expression in BAT. We conclude that these findings indicate functional relevance and potentially beneficial effects of vaspin in BAT function.

MicroRNA-542-5p as a Novel Tumor Suppressor in Neuroblastoma

PubMed Central

Bray, Isabella; Tivnan, Amanda; Bryan, Kenneth; Foley, Niamh H; Watters, Karen M; Tracey, Lorraine; Davidoff, Andrew M; Stallings, Raymond L

2011-01-01

Several studies have implicated the dysregulation of microRNAs in neuroblastoma pathogenesis, an often fatal paediatric cancer arising from precursor cells of the sympathetic nervous system. Our group and others have demonstrated that lower expression of miR-542-5p is highly associated with poor patient survival, indicating a potential tumor suppressive function. Here, we demonstrate that ectopic over-expression of this miRNA decreases the invasive potential of neuroblastoma cell lines in vitro, along with primary tumor growth and metastases in an orthotopic mouse xenograft model, providing the first functional evidence for the involvement of miR-542-5p as a tumor suppressor in any type of cancer. PMID:21310526

A P-Norm Robust Feature Extraction Method for Identifying Differentially Expressed Genes

PubMed Central

Liu, Jian; Liu, Jin-Xing; Gao, Ying-Lian; Kong, Xiang-Zhen; Wang, Xue-Song; Wang, Dong

2015-01-01

In current molecular biology, it becomes more and more important to identify differentially expressed genes closely correlated with a key biological process from gene expression data. In this paper, based on the Schatten p-norm and Lp-norm, a novel p-norm robust feature extraction method is proposed to identify the differentially expressed genes. In our method, the Schatten p-norm is used as the regularization function to obtain a low-rank matrix and the Lp-norm is taken as the error function to improve the robustness to outliers in the gene expression data. The results on simulation data show that our method can obtain higher identification accuracies than the competitive methods. Numerous experiments on real gene expression data sets demonstrate that our method can identify more differentially expressed genes than the others. Moreover, we confirmed that the identified genes are closely correlated with the corresponding gene expression data. PMID:26201006

A P-Norm Robust Feature Extraction Method for Identifying Differentially Expressed Genes.

PubMed

Liu, Jian; Liu, Jin-Xing; Gao, Ying-Lian; Kong, Xiang-Zhen; Wang, Xue-Song; Wang, Dong

2015-01-01

In current molecular biology, it becomes more and more important to identify differentially expressed genes closely correlated with a key biological process from gene expression data. In this paper, based on the Schatten p-norm and Lp-norm, a novel p-norm robust feature extraction method is proposed to identify the differentially expressed genes. In our method, the Schatten p-norm is used as the regularization function to obtain a low-rank matrix and the Lp-norm is taken as the error function to improve the robustness to outliers in the gene expression data. The results on simulation data show that our method can obtain higher identification accuracies than the competitive methods. Numerous experiments on real gene expression data sets demonstrate that our method can identify more differentially expressed genes than the others. Moreover, we confirmed that the identified genes are closely correlated with the corresponding gene expression data.

High throughput mutagenesis for identification of residues regulating human prostacyclin (hIP) receptor expression and function.

PubMed

Bill, Anke; Rosethorne, Elizabeth M; Kent, Toby C; Fawcett, Lindsay; Burchell, Lynn; van Diepen, Michiel T; Marelli, Anthony; Batalov, Sergey; Miraglia, Loren; Orth, Anthony P; Renaud, Nicole A; Charlton, Steven J; Gosling, Martin; Gaither, L Alex; Groot-Kormelink, Paul J

2014-01-01

The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structure-function relationship of GPCRs.

High Throughput Mutagenesis for Identification of Residues Regulating Human Prostacyclin (hIP) Receptor Expression and Function

PubMed Central

Kent, Toby C.; Fawcett, Lindsay; Burchell, Lynn; van Diepen, Michiel T.; Marelli, Anthony; Batalov, Sergey; Miraglia, Loren; Orth, Anthony P.; Renaud, Nicole A.; Charlton, Steven J.; Gosling, Martin; Gaither, L. Alex; Groot-Kormelink, Paul J.

2014-01-01

The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structure-function relationship of GPCRs. PMID:24886841

Differential expression of immune-regulatory genes associated with PD-L1 display in melanoma: implications for PD-1 pathway blockade

PubMed Central

Taube, Janis M.; Young, Geoffrey D.; McMiller, Tracee L.; Chen, Shuming; Salas, January T.; Pritchard, Theresa S.; Xu, Haiying; Meeker, Alan K.; Fan, Jinshui; Cheadle, Chris; Berger, Alan E.; Pardoll, Drew M.; Topalian, Suzanne L.

2015-01-01

Purpose Blocking the immunosuppressive PD-1/PD-L1 pathway has anti-tumor activity in multiple cancer types, and PD-L1 expression on tumor cells and infiltrating myeloid cells correlates with the likelihood of response. We previously found that IFNG (interferon-gamma) was over-expressed by TILs in PD-L1+ vs. PD-L1(−) melanomas, creating adaptive immune resistance by promoting PD-L1 display. The current study was undertaken to identify additional factors in the PD-L1+ melanoma microenvironment coordinately contributing to immunosuppression. Experimental design Archived, formalin-fixed paraffin-embedded melanoma specimens were assessed for PD-L1 protein expression at the tumor cell surface with immunohistochemistry (IHC). Whole genome expression analysis, quantitative (q)RT-PCR, immunohistochemistry, and functional in vitro validation studies were employed to assess factors differentially expressed in PD-L1+ versus PD-L1(−) melanomas. Results Functional annotation clustering based on whole genome expression profiling revealed pathways up-regulated in PD-L1+ melanomas, involving immune cell activation, inflammation, and antigen processing and presentation. Analysis by qRT-PCR demonstrated over-expression of functionally related genes in PD-L1+ melanomas, involved in CD8+ T cell activation (CD8A, IFNG, PRF1, CCL5), antigen presentation (CD163, TLR3, CXCL1, LYZ), and immunosuppression [PDCD1 (PD-1), CD274(PD-L1), LAG3, IL10]. Functional studies demonstrated that some factors, including IL-10 and IL-32-gamma, induced PD-L1 expression on monocytes but not tumor cells. Conclusions These studies elucidate the complexity of immune checkpoint regulation in the tumor microenvironment, identifying multiple factors likely contributing to coordinated immunosuppression. These factors may provide tumor escape mechanisms from anti-PD-1/PD-L1 therapy, and should be considered for co-targeting in combinatorial immunomodulation treatment strategies. PMID:25944800

Gene expression profile of the plant pathogen Xylella fastidiosa during biofilm formation in vitro.

PubMed

de Souza, Alessandra A; Takita, Marco A; Coletta-Filho, Helvécio D; Caldana, Camila; Yanai, Giane M; Muto, Nair H; de Oliveira, Regina C; Nunes, Luiz R; Machado, Marcos A

2004-08-15

A biofilm is a community of microorganisms attached to a solid surface. Cells within biofilms differ from planktonic cells, showing higher resistance to biocides, detergent, antibiotic treatments and host defense responses. Even though there are a number of gene expression studies in bacterial biofilm formation, limited information is available concerning plant pathogen. It was previously demonstrated that the plant pathogen Xylella fastidiosa could grow as a biofilm, a possibly important factor for its pathogenicity. In this study we utilized analysis of microarrays to specifically identify genes expressed in X. fastidiosa cells growing in a biofilm, when compared to planktonic cells. About half of the differentially expressed genes encode hypothetical proteins, reflecting the large number of ORFs with unknown functions in bacterial genomes. However, under the biofilm condition we observed an increase in the expression of some housekeeping genes responsible for metabolic functions. We also found a large number of genes from the pXF51 plasmid being differentially expressed. Some of the overexpressed genes in the biofilm condition encode proteins involved in attachment to surfaces. Other genes possibly confer advantages to the bacterium in the environment that it colonizes. This study demonstrates that the gene expression in the biofilm growth condition of the plant pathogen X. fastidiosa is quite similar to other characterized systems.

Intercellular adhesion molecule-1 expression by skeletal muscle cells augments myogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goh, Qingnian; Dearth, Christopher L.; Corbett, Jacob T.

We previously demonstrated that the expression of intercellular adhesion molecule-1 (ICAM-1) by skeletal muscle cells after muscle overload contributes to ensuing regenerative and hypertrophic processes in skeletal muscle. The objective of the present study is to reveal mechanisms through which skeletal muscle cell expression of ICAM-1 augments regenerative and hypertrophic processes of myogenesis. This was accomplished by genetically engineering C2C12 myoblasts to stably express ICAM-1, and by inhibiting the adhesive and signaling functions of ICAM-1 through the use of a neutralizing antibody or cell penetrating peptide, respectively. Expression of ICAM-1 by cultured skeletal muscle cells augmented myoblast–myoblast adhesion, myotube formation,more » myonuclear number, myotube alignment, myotube–myotube fusion, and myotube size without influencing the ability of myoblasts to proliferate or differentiate. ICAM-1 augmented myotube formation, myonuclear accretion, and myotube alignment through a mechanism involving adhesion-induced activation of ICAM-1 signaling, as these dependent measures were reduced via antibody and peptide inhibition of ICAM-1. The adhesive and signaling functions of ICAM-1 also facilitated myotube hypertrophy through a mechanism involving myotube–myotube fusion, protein synthesis, and Akt/p70s6k signaling. Our findings demonstrate that ICAM-1 expression by skeletal muscle cells augments myogenesis, and establish a novel mechanism through which the inflammatory response facilitates growth processes in skeletal muscle. - Highlights: • We examined mechanisms through which skeletal muscle cell expression of ICAM-1 facilitates events of in vitro myogenesis. • Expression of ICAM-1 by cultured myoblasts did not influence their ability to proliferate or differentiate. • Skeletal muscle cell expression of ICAM-1 augmented myoblast fusion, myotube alignment, myotube–myotube fusion, and myotube size. • ICAM-1 augmented myogenic processes through mechanisms involving its adhesive and signaling functions.« less

Biologic consequences of Stat1-independent IFN signaling

PubMed Central

Gil, M. Pilar; Bohn, Erwin; O'Guin, Andrew K.; Ramana, Chilakamarti V.; Levine, Beth; Stark, George R.; Virgin, Herbert W.; Schreiber, Robert D.

2001-01-01

Although Stat1 is required for many IFN-dependent responses, recent work has shown that IFNγ functions independently of Stat1 to affect the growth of tumor cells or immortalized fibroblasts. We now demonstrate that both IFNγ and IFNα/β regulate proliferative responses in cells of the mononuclear phagocyte lineage derived from Stat1-null mice. Using both representational difference analysis and gene arrays, we show that IFNγ exerts its Stat1-independent actions on mononuclear phagocytes by regulating the expression of many genes. This result was confirmed by monitoring changes in expression and function of the corresponding gene products. Regulation of the expression of these genes requires the IFNγ receptor and Jak1. The physiologic relevance of IFN-dependent, Stat1-independent signaling was demonstrated by monitoring antiviral responses in Stat1-null mice. Thus, the IFN receptors engage alternative Stat1-independent signaling pathways that have important physiological consequences. PMID:11390995

Folate receptor targeting silica nanoparticle probe for two-photon fluorescence bioimaging

PubMed Central

Wang, Xuhua; Yao, Sheng; Ahn, Hyo-Yang; Zhang, Yuanwei; Bondar, Mykhailo V.; Torres, Joseph A.; Belfield, Kevin D.

2010-01-01

Narrow dispersity organically modified silica nanoparticles (SiNPs), diameter ~30 nm, entrapping a hydrophobic two-photon absorbing fluorenyl dye, were synthesized by hydrolysis of triethoxyvinylsilane and (3-aminopropyl)triethoxysilane in the nonpolar core of Aerosol-OT micelles. The surface of the SiNPs were functionalized with folic acid, to specifically deliver the probe to folate receptor (FR) over-expressing Hela cells, making these folate two-photon dye-doped SiNPs potential candidates as probes for two-photon fluorescence microscopy (2PFM) bioimaging. In vitro studies using FR over-expressing Hela cells and low FR expressing MG63 cells demonstrated specific cellular uptake of the functionalized nanoparticles. One-photon fluorescence microscopy (1PFM) imaging, 2PFM imaging, and two-photon fluorescence lifetime microscopy (2P-FLIM) imaging of Hela cells incubated with folate-modified two-photon dye-doped SiNPs were demonstrated. PMID:21258480

A differential neural response to threatening and non-threatening negative facial expressions in paranoid and non-paranoid schizophrenics.

PubMed

Phillips, M L; Williams, L; Senior, C; Bullmore, E T; Brammer, M J; Andrew, C; Williams, S C; David, A S

1999-11-08

Several studies have demonstrated impaired facial expression recognition in schizophrenia. Few have examined the neural basis for this; none have compared the neural correlates of facial expression perception in different schizophrenic patient subgroups. We compared neural responses to facial expressions in 10 right-handed schizophrenic patients (five paranoid and five non-paranoid) and five normal volunteers using functional Magnetic Resonance Imaging (fMRI). In three 5-min experiments, subjects viewed alternating 30-s blocks of black-and-white facial expressions of either fear, anger or disgust contrasted with expressions of mild happiness. After scanning, subjects categorised each expression. All patients were less accurate in identifying expressions, and showed less activation to these stimuli than normals. Non-paranoids performed poorly in the identification task and failed to activate neural regions that are normally linked with perception of these stimuli. They categorised disgust as either anger or fear more frequently than paranoids, and demonstrated in response to disgust expressions activation in the amygdala, a region associated with perception of fearful faces. Paranoids were more accurate in recognising expressions, and demonstrated greater activation than non-paranoids to most stimuli. We provide the first evidence for a distinction between two schizophrenic patient subgroups on the basis of recognition of and neural response to different negative facial expressions.

Less Empathic and More Reactive: The Different Impact of Childhood Maltreatment on Facial Mimicry and Vagal Regulation

PubMed Central

Ardizzi, Martina; Umiltà, Maria Alessandra; Evangelista, Valentina; Di Liscia, Alessandra; Ravera, Roberto; Gallese, Vittorio

2016-01-01

Facial mimicry and vagal regulation represent two crucial physiological responses to others’ facial expressions of emotions. Facial mimicry, defined as the automatic, rapid and congruent electromyographic activation to others’ facial expressions, is implicated in empathy, emotional reciprocity and emotions recognition. Vagal regulation, quantified by the computation of Respiratory Sinus Arrhythmia (RSA), exemplifies the autonomic adaptation to contingent social cues. Although it has been demonstrated that childhood maltreatment induces alterations in the processing of the facial expression of emotions, both at an explicit and implicit level, the effects of maltreatment on children’s facial mimicry and vagal regulation in response to facial expressions of emotions remain unknown. The purpose of the present study was to fill this gap, involving 24 street-children (maltreated group) and 20 age-matched controls (control group). We recorded their spontaneous facial electromyographic activations of corrugator and zygomaticus muscles and RSA responses during the visualization of the facial expressions of anger, fear, joy and sadness. Results demonstrated a different impact of childhood maltreatment on facial mimicry and vagal regulation. Maltreated children did not show the typical positive-negative modulation of corrugator mimicry. Furthermore, when only negative facial expressions were considered, maltreated children demonstrated lower corrugator mimicry than controls. With respect to vagal regulation, whereas maltreated children manifested the expected and functional inverse correlation between RSA value at rest and RSA response to angry facial expressions, controls did not. These results describe an early and divergent functional adaptation to hostile environment of the two investigated physiological mechanisms. On the one side, maltreatment leads to the suppression of the spontaneous facial mimicry normally concurring to empathic understanding of others’ emotions. On the other side, maltreatment forces the precocious development of the functional synchronization between vagal regulation and threatening social cues facilitating the recruitment of fight-or-flight defensive behavioral strategies. PMID:27685802

Zebrafish Meis functions to stabilize Pbx proteins and regulate hindbrain patterning.

PubMed

Waskiewicz, A J; Rikhof, H A; Hernandez, R E; Moens, C B

2001-11-01

Homeodomain-containing Hox proteins regulate segmental identity in Drosophila in concert with two partners known as Extradenticle (Exd) and Homothorax (Hth). These partners are themselves DNA-binding, homeodomain proteins, and probably function by revealing the intrinsic specificity of Hox proteins. Vertebrate orthologs of Exd and Hth, known as Pbx and Meis (named for a myeloid ecotropic leukemia virus integration site), respectively, are encoded by multigene families and are present in multimeric complexes together with vertebrate Hox proteins. Previous results have demonstrated that the zygotically encoded Pbx4/Lazarus (Lzr) protein is required for segmentation of the zebrafish hindbrain and proper expression and function of Hox genes. We demonstrate that Meis functions in the same pathway as Pbx in zebrafish hindbrain development, as expression of a dominant-negative mutant Meis results in phenotypes that are remarkably similar to that of lzr mutants. Surprisingly, expression of Meis protein partially rescues the lzr(-) phenotype. Lzr protein levels are increased in embryos overexpressing Meis and are reduced for lzr mutants that cannot bind to Meis. This implies a mechanism whereby Meis rescues lzr mutants by stabilizing maternally encoded Lzr. Our results define two functions of Meis during zebrafish hindbrain segmentation: that of a DNA-binding partner of Pbx proteins, and that of a post-transcriptional regulator of Pbx protein levels.

Biomimetic L-aspartic acid-derived functional poly(ester amide)s for vascular tissue engineering.

PubMed

Knight, Darryl K; Gillies, Elizabeth R; Mequanint, Kibret

2014-08-01

Functionalization of polymeric biomaterials permits the conjugation of cell signaling molecules capable of directing cell function. In this study, l-phenylalanine and l-aspartic acid were used to synthesize poly(ester amide)s (PEAs) with pendant carboxylic acid groups through an interfacial polycondensation approach. Human coronary artery smooth muscle cell (HCASMC) attachment, spreading and proliferation was observed on all PEA films. Vinculin expression at the cell periphery suggested that HCASMCs formed focal adhesions on the functional PEAs, while the absence of smooth muscle α-actin (SMαA) expression implied the cells adopted a proliferative phenotype. The PEAs were also electrospun to yield nanoscale three-dimensional (3-D) scaffolds with average fiber diameters ranging from 130 to 294nm. Immunoblotting studies suggested a potential increase in SMαA and calponin expression from HCASMCs cultured on 3-D fibrous scaffolds when compared to 2-D films. X-ray photoelectron spectroscopy and immunofluorescence demonstrated the conjugation of transforming growth factor-β1 to the surface of the functional PEA through the pendant carboxylic acid groups. Taken together, this study demonstrates that PEAs containing aspartic acid are viable biomaterials for further investigation in vascular tissue engineering. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Tissue-specific expression of transgenic secreted ACE in vasculature can restore normal kidney functions, but not blood pressure, of Ace-/- mice.

PubMed

Chattopadhyay, Saurabh; Kessler, Sean P; Colucci, Juliana Almada; Yamashita, Michifumi; Senanayake, Preenie deS; Sen, Ganes C

2014-01-01

Angiotensin-converting enzyme (ACE) regulates normal blood pressure and fluid homeostasis through its action in the renin-angiotensin-system (RAS). Ace-/- mice are smaller in size, have low blood pressure and defective kidney structure and functions. All of these defects are cured by transgenic expression of somatic ACE (sACE) in vascular endothelial cells of Ace-/- mice. sACE is expressed on the surface of vascular endothelial cells and undergoes a natural cleavage secretion process to generate a soluble form in the body fluids. Both the tissue-bound and the soluble forms of ACE are enzymatically active, and generate the vasoactive octapeptide Angiotensin II (Ang II) with equal efficiency. To assess the relative physiological roles of the secreted and the cell-bound forms of ACE, we expressed, in the vascular endothelial cells of Ace-/- mice, the ectodomain of sACE, which corresponded to only the secreted form of ACE. Our results demonstrated that the secreted form of ACE could normalize kidney functions and RAS integrity, growth and development of Ace-/- mice, but not their blood pressure. This study clearly demonstrates that the secreted form of ACE cannot replace the tissue-bound ACE for maintaining normal blood pressure; a suitable balance between the tissue-bound and the soluble forms of ACE is essential for maintaining all physiological functions of ACE.

Tissue-Specific Expression of Transgenic Secreted ACE in Vasculature Can Restore Normal Kidney Functions, but Not Blood Pressure, of Ace-/- Mice

PubMed Central

Chattopadhyay, Saurabh; Kessler, Sean P.; Colucci, Juliana Almada; Yamashita, Michifumi; Senanayake, Preenie deS; Sen, Ganes C.

2014-01-01

Angiotensin-converting enzyme (ACE) regulates normal blood pressure and fluid homeostasis through its action in the renin-angiotensin-system (RAS). Ace-/- mice are smaller in size, have low blood pressure and defective kidney structure and functions. All of these defects are cured by transgenic expression of somatic ACE (sACE) in vascular endothelial cells of Ace-/- mice. sACE is expressed on the surface of vascular endothelial cells and undergoes a natural cleavage secretion process to generate a soluble form in the body fluids. Both the tissue-bound and the soluble forms of ACE are enzymatically active, and generate the vasoactive octapeptide Angiotensin II (Ang II) with equal efficiency. To assess the relative physiological roles of the secreted and the cell-bound forms of ACE, we expressed, in the vascular endothelial cells of Ace-/- mice, the ectodomain of sACE, which corresponded to only the secreted form of ACE. Our results demonstrated that the secreted form of ACE could normalize kidney functions and RAS integrity, growth and development of Ace-/- mice, but not their blood pressure. This study clearly demonstrates that the secreted form of ACE cannot replace the tissue-bound ACE for maintaining normal blood pressure; a suitable balance between the tissue-bound and the soluble forms of ACE is essential for maintaining all physiological functions of ACE. PMID:24475296

The novel cyst nematode effector protein 30D08 targets host nuclear functions to alter gene expression in feeding sites.

PubMed

Verma, Anju; Lee, Chris; Morriss, Stephanie; Odu, Fiona; Kenning, Charlotte; Rizzo, Nancy; Spollen, William G; Lin, Marriam; McRae, Amanda G; Givan, Scott A; Hewezi, Tarek; Hussey, Richard; Davis, Eric L; Baum, Thomas J; Mitchum, Melissa G

2018-05-04

Cyst nematodes deliver effector proteins into host cells to manipulate cellular processes and establish a metabolically hyperactive feeding site. The novel 30D08 effector protein is produced in the dorsal gland of parasitic juveniles, but its function has remained unknown. We demonstrate that expression of 30D08 contributes to nematode parasitism, the protein is packaged into secretory granules and it is targeted to the plant nucleus where it interacts with SMU2 (homolog of suppressor of mec-8 and unc-52 2), an auxiliary spliceosomal protein. We show that SMU2 is expressed in feeding sites and an smu2 mutant is less susceptible to nematode infection. In Arabidopsis expressing 30D08 under the SMU2 promoter, several genes were found to be alternatively spliced and the most abundant functional classes represented among differentially expressed genes were involved in RNA processing, transcription and binding, as well as in development, and hormone and secondary metabolism, representing key cellular processes known to be important for feeding site formation. In conclusion, we demonstrated that the 30D08 effector is secreted from the nematode and targeted to the plant nucleus where its interaction with a host auxiliary spliceosomal protein may alter the pre-mRNA splicing and expression of a subset of genes important for feeding site formation. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

MicroRNA-372 inhibits endometrial carcinoma development by targeting the expression of the Ras homolog gene family member C (RhoC)

PubMed Central

Liu, Bo-Liang; Sun, Kai-Xuan; Zong, Zhi-Hong; Chen, Shuo; Zhao, Yang

2016-01-01

Here we explore the role of microRNA-372 (miR-372) in tumorigenesis and development of endometrial adenocarcinoma (EC) and analyze the underlying mechanism. We found that miR-372 expression is much lower in EC than normal endometrial specimens. Cell function experiments demonstrated that miR-372 overexpression suppressed cell proliferation, migration, and invasion, and led to a G1 phase arrest and promoted the apoptosis of endometrial carcinoma cells in vitro. The nude mouse xenograft assay demonstrated that miR-372 overexpression suppressed tumor growth. RT-PCR and Western blot assays detected the expression of known targets of miR-372 in other malignant tumors and found Cyclin A1 and Cyclin-dependent Kinase 2 (CDK2) was downregulated by miR-372. Bioinformatic predictions and dual-luciferase reporter assays found that RhoC was a possible target of miR-372. RT-PCR and Western blot assays demonstrated that miR-372 transfection reduced the expression of RhoC, matrix metalloproteinase 2 (MMP2) and MMP9, while it increased the expression of cleaved poly (ADP ribose) polymerase (PARP) and bcl-2-associated X protein (Bax). The cell function experiments that transfected siRNA with RhoC showed the same trend as those which were transfected with miR-372. Taken together, our results demonstrated for the first time that miR-372 suppresses tumorigenesis and the development of EC; RhoC is a new and potentially important therapeutic target. PMID:26673619

The Ras suppressor Rsu-1 binds to the LIM 5 domain of the adaptor protein PINCH1 and participates in adhesion-related functions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dougherty, Gerard W.; Section on Structural Cell Biology, National Institute on Deafness and Communication Disorders; Chopp, Treasa

2005-05-15

Rsu-1 is a highly conserved leucine rich repeat (LRR) protein that is expressed ubiquitously in mammalian cells. Rsu-1 was identified based on its ability to inhibit transformation by Ras, and previous studies demonstrated that ectopic expression of Rsu-1 inhibited anchorage-independent growth of Ras-transformed cells and human tumor cell lines. Using GAL4-based yeast two-hybrid screening, the LIM domain protein, PINCH1, was identified as the binding partner of Rsu-1. PINCH1 is an adaptor protein that localizes to focal adhesions and it has been implicated in the regulation of adhesion functions. Subdomain mapping in yeast revealed that Rsu-1 binds to the LIM 5more » domain of PINCH1, a region not previously identified as a specific binding domain for any other protein. Additional testing demonstrated that PINCH2, which is highly homologous to PINCH1, except in the LIM 5 domain, does not interact with Rsu-1. Glutathione transferase fusion protein binding studies determined that the LRR region of Rsu-1 interacts with PINCH1. Transient expression studies using epitope-tagged Rsu-1 and PINCH1 revealed that Rsu-1 co-immunoprecipitated with PINCH1 and colocalized with vinculin at sites of focal adhesions in mammalian cells. In addition, endogenous P33 Rsu-1 from 293T cells co-immunoprecipitated with transiently expressed myc-tagged PINCH1. Furthermore, RNAi-induced reduction in Rsu-1 RNA and protein inhibited cell attachment, and while previous studies demonstrated that ectopic expression of Rsu-1 inhibited Jun kinase activation, the depletion of Rsu-1 resulted in activation of Jun and p38 stress kinases. These studies demonstrate that Rsu-1 interacts with PINCH1 in mammalian cells and functions, in part, by altering cell adhesion.« less

Parcellation of left parietal tool representations by functional connectivity

PubMed Central

Garcea, Frank E.; Z. Mahon, Bradford

2014-01-01

Manipulating a tool according to its function requires the integration of visual, conceptual, and motor information, a process subserved in part by left parietal cortex. How these different types of information are integrated and how their integration is reflected in neural responses in the parietal lobule remains an open question. Here, participants viewed images of tools and animals during functional magnetic resonance imaging (fMRI). K-means clustering over time series data was used to parcellate left parietal cortex into subregions based on functional connectivity to a whole brain network of regions involved in tool processing. One cluster, in the inferior parietal cortex, expressed privileged functional connectivity to the left ventral premotor cortex. A second cluster, in the vicinity of the anterior intraparietal sulcus, expressed privileged functional connectivity with the left medial fusiform gyrus. A third cluster in the superior parietal lobe expressed privileged functional connectivity with dorsal occipital cortex. Control analyses using Monte Carlo style permutation tests demonstrated that the clustering solutions were outside the range of what would be observed based on chance ‘lumpiness’ in random data, or mere anatomical proximity. Finally, hierarchical clustering analyses were used to formally relate the resulting parcellation scheme of left parietal tool representations to previous work that has parcellated the left parietal lobule on purely anatomical grounds. These findings demonstrate significant heterogeneity in the functional organization of manipulable object representations in left parietal cortex, and outline a framework that generates novel predictions about the causes of some forms of upper limb apraxia. PMID:24892224

Impaired osteoblast differentiation in Annexin A2- and -A5-deficient cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Genetos, Damian C.; Wong, Alice; Weber, Thomas J.

Annexins are a class of calcium-binding proteins with diverse functions in the regulation of lipid rafts inflammation,fibrinolysis, transcriptional programming and ion transport. Within bone, they are well-characterized as components of mineralizing matrix vesicles, although little else is known as to their function during osteogenesis. We generated annexin A2 (AnxA2)- or annexin A5 (AnxA5)-knockdown pre-osteoblasts, and asked whether proliferation or osteogenic differentiation was altered in knockdown cells, compared to vector controls. We report that DNA content, a marker of proliferation, was significantly reduced in both AnxA2 and AnxA5 knockdown cells. Alkaline phosphatase expression and staining activity were also suppressed in AnxA2-more » or AnxA5-knockdown after 14 days of culture. The pattern of osteogenic gene expression was altered in knockdown cells, with Col1a1 expressed more rapidly in knock-down cells, compared to controls. In contrast, Runx2, Ibsp, and Bglap all revealed decreased expression after 14 days of culture. Using a murine fracture model, we demonstrate that AnxA2 and AnxA5 are rapidly expressed within the fracture callus. These data demonstrate that AnxA2 and AnxA5 can influence bone formation via regulation of osteoprogenitor proliferation and differentiation in addition to their well-studied function in matrix vesicles.« less

Autocrine stimulation of VEGFR-2 activates human leukemic cell growth and migration

PubMed Central

Dias, Sergio; Hattori, Koichi; Zhu, Zhenping; Heissig, Beate; Choy, Margaret; Lane, William; Wu, Yan; Chadburn, Amy; Hyjek, Elizabeth; Gill, Muhammad; Hicklin, Daniel J.; Witte, Larry; Moore, M.A.S.; Rafii, Shahin

2000-01-01

Emerging data suggest that VEGF receptors are expressed by endothelial cells as well as hematopoietic stem cells. Therefore, we hypothesized that functional VEGF receptors may also be expressed in malignant counterparts of hematopoietic stem cells such as leukemias. We demonstrate that certain leukemias not only produce VEGF but also express functional VEGFR-2 in vivo and in vitro, resulting in the generation of an autocrine loop that may support leukemic cell survival and proliferation. Approximately 50% of freshly isolated leukemias expressed mRNA and protein for VEGFR-2. VEGF165 induced phosphorylation of VEGFR-2 and increased proliferation of leukemic cells, demonstrating these receptors were functional. VEGF165 also induced the expression of MMP-9 by leukemic cells and promoted their migration through reconstituted basement membrane. The neutralizing mAb IMC-1C11, specific to human VEGFR-2, inhibited leukemic cell survival in vitro and blocked VEGF165-mediated proliferation of leukemic cells and VEGF-induced leukemic cell migration. Xenotransplantation of primary leukemias and leukemic cell lines into immunocompromised nonobese diabetic mice resulted in significant elevation of human, but not murine, VEGF in plasma and death of inoculated mice within 3 weeks. Injection of IMC-1C11 inhibited proliferation of xenotransplanted human leukemias and significantly increased the survival of inoculated mice. Interruption of signaling by VEGFRs, particularly VEGFR-2, may provide a novel strategy for inhibiting leukemic cell proliferation. PMID:10953026

Ryk receptor regulates hematopoietic stem and progenitor sensitivity to myelosuppressive injury in mice

PubMed Central

Povinelli, Benjamin J.; Srivastava, Pragya; Nemeth, Michael J.

2017-01-01

Maintaining a careful balance between quiescence and proliferation of hematopoietic stem and progenitor cells (HSPCs) is necessary for lifelong blood formation. Previously, we demonstrated that the Wnt5a ligand inhibits HSPC proliferation through a functional interaction with a non-canonical Wnt ligand receptor termed Ryk. Expression of Ryk on HSPCs in vivo is associated with a lower rate of proliferation and following treatment with fluorouracil (5-FU), the percentage of Ryk+/high HSPCs increased while the percent of Ryk−/low HSPCs decreased. Based on these data, we hypothesized that one function of the Ryk receptor is to protect HSPCs from the effects of myeloablative agents. We found that Ryk expression on HSPCs is associated with lower rates of apoptosis following 5-FU and radiation. Transient inhibition of Ryk signaling in vivo resulted in increased HSC proliferation and decreased HSC function in bone marrow transplant assays. Furthermore, inhibition of Ryk signaling sensitized HSPCs to 5-FU treatment in association with increased levels of reactive oxygen species. Together, these results demonstrated an association between Ryk expression and survival of HSPCs following suppressive injury. PMID:25461251

Distinct protein domains and expression patterns confer divergent axon guidance functions for Drosophila Robo receptors.

PubMed

Spitzweck, Bettina; Brankatschk, Marko; Dickson, Barry J

2010-02-05

The orthogonal array of axon pathways in the Drosophila CNS is constructed in part under the control of three Robo family axon guidance receptors: Robo1, Robo2 and Robo3. Each of these receptors is responsible for a distinct set of guidance decisions. To determine the molecular basis for these functional specializations, we used homologous recombination to create a series of 9 "robo swap" alleles: expressing each of the three Robo receptors from each of the three robo loci. We demonstrate that the lateral positioning of longitudinal axon pathways relies primarily on differences in gene regulation, not distinct combinations of Robo proteins as previously thought. In contrast, specific features of the Robo1 and Robo2 proteins contribute to their distinct functions in commissure formation. These specializations allow Robo1 to prevent crossing and Robo2 to promote crossing. These data demonstrate how diversification of expression and structure within a single family of guidance receptors can shape complex patterns of neuronal wiring. 2010 Elsevier Inc. All rights reserved.

Expression and function of CD8 alpha/beta chains on rat and human mast cells.

PubMed

Kim, Mi-Sun; Kim, Sung-Hoon; Lee, Hye-Jung; Kim, Hyung-Min

2004-03-01

The expression and functional role of CD8 glycoprotein, a marker of cytotoxic/suppressor T lymphocytes and NK cells, were not studied on freshly isolated connective tissue type rat peritoneal mast cells, a rat mucosal type mast cell line (RBL 2H3), or human mast cell line (HMC-1). We used the reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, immunohistochemistry and enzyme-linked immunosorbent assay. RT-PCR and Western blot analysis identified the presence of CD8 alpha/beta chains on the mast cells, and immunohistochemistry confirmed CD8alpha expression on rat or human mast cells. Functional studies demonstrated that stimulation of CD8 alpha/beta chains on rat mast cells induced the secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), which are regarded as important mediators during infection. However, co-stimulation with stem cell factor had no effect on CD8-induced mediator secretion. Our findings demonstrate novel biological roles of CD8 molecules in mast cells.

Feasibility of an expressive-disclosure group intervention for post-treatment colorectal cancer patients: results of the Healthy Expressions study.

PubMed

Carmack, Cindy L; Basen-Engquist, Karen; Yuan, Ying; Greisinger, Anthony; Rodriguez-Bigas, Miguel; Wolff, Robert A; Barker, Trina; Baum, George; Pennebaker, James W

2011-11-01

Adjusting to cancer requires effective cognitive and emotional processing. Written and verbal disclosure facilitate processing and have been studied independently in cancer survivors. Combined written and verbal expression may be more effective than either alone, particularly for patients with difficult to discuss or embarrassing side effects. Thus, the authors developed and tested the efficacy of a 12-session combined written and verbal expression group program for psychologically distressed colorectal cancer (CRC) patients. Forty post-treatment patients with CRC (stages I-III) identified as psychologically distressed using the Brief Symptom Inventory (BSI) were randomized to an intervention group (Healthy Expressions; n = 25) or standard care (control group; n = 15). Assessments were completed at baseline, Month 2, and Month 4 (postintervention). Primary outcomes were psychological functioning and quality of life (QOL). Most participants were women (63%), white (63%), and non-Hispanic (75%). The Healthy Expressions group demonstrated significantly greater changes in distress compared with the control group at Month 2 on the BSI Global Severity Index (GSI) and the Centers for Epidemiologic Studies Depression scale (CES-D) scores (P < .05 for each); differences in the European Organization for Research and Treatment of Cancer (EORTC) global QOL scores approached significance (P = .063). The BSI GSI and Positive Symptom Total, CES-D, and EORTC emotional functioning subscale scores were all significant at Month 4 (P < .05 for each). The Healthy Expressions program improved psychological functioning in CRC patients who reported experiencing distress. Findings demonstrate the program's feasibility and provide strong support for conducting a larger randomized trial. Copyright © 2011 American Cancer Society.

Identification of the long non‑coding RNA LET as a novel tumor suppressor in gastric cancer.

PubMed

Tian, Jingjing; Hu, Xibao; Gao, Wei; Zhang, Jie; Chen, Ming; Zhang, Xinrong; Ma, Junhong; Yuan, Hongxia

2017-04-01

Long non-coding RNAs (lncRNAs) have emerged recently as important factors in regulating fundamental biological processes. Alterations in the expression and function of lncRNAs have been observed to promote tumor formation, progression and metastasis. Although downregulation of the expression levels of LET lncRNA in several tumors has been reported, its role in gastric cancer remains unknown. The aim of the present study was to investigate the expression and function of LET in gastric cancer development. The expression levels of LET in 37 pairs of gastric cancer and adjacent non‑tumor tissues were detected by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). In addition, LET expression in gastric cancer cell lines was analyzed by RT‑qPCR assay analysis. Furthermore, the impact of LET on cell proliferation, migration and apoptosis were detected using the cell counting kit‑8, wound scratch and ELISA assays, respectively. The results demonstrated that the expression level of LET was downregulated in gastric cancer tissues and cell lines (SGC‑7901 and MGC‑803) compared with normal tissues and a normal human gastric epithelial cell line (GES‑1). Restoration of LET expression using a synthesized recombinant overexpression vector transfected into SGC‑7901 and MGC‑803 cells, significantly inhibited cell proliferation and migration, and promoted cell apoptosis in vitro. The present study is the first to demonstrate that LET may function as a tumor suppressor in gastric cancer. The results indicate that LET may be a promising biomarker and/or a therapeutic target for gastric cancer.

Altering sensorimotor feedback disrupts visual discrimination of facial expressions.

PubMed

Wood, Adrienne; Lupyan, Gary; Sherrin, Steven; Niedenthal, Paula

2016-08-01

Looking at another person's facial expression of emotion can trigger the same neural processes involved in producing the expression, and such responses play a functional role in emotion recognition. Disrupting individuals' facial action, for example, interferes with verbal emotion recognition tasks. We tested the hypothesis that facial responses also play a functional role in the perceptual processing of emotional expressions. We altered the facial action of participants with a gel facemask while they performed a task that involved distinguishing target expressions from highly similar distractors. Relative to control participants, participants in the facemask condition demonstrated inferior perceptual discrimination of facial expressions, but not of nonface stimuli. The findings suggest that somatosensory/motor processes involving the face contribute to the visual perceptual-and not just conceptual-processing of facial expressions. More broadly, our study contributes to growing evidence for the fundamentally interactive nature of the perceptual inputs from different sensory modalities.

Expression of the myeloid-associated marker CD33 is not an exclusive factor for leukemic plasmacytoid dendritic cells.

PubMed

Garnache-Ottou, Francine; Chaperot, Laurence; Biichle, Sabeha; Ferrand, Christophe; Remy-Martin, Jean-Paul; Deconinck, Eric; de Tailly, Patrick Darodes; Bulabois, Bénédicte; Poulet, Jacqueline; Kuhlein, Emilienne; Jacob, Marie-Christine; Salaun, Véronique; Arock, Michel; Drenou, Bernard; Schillinger, Françoise; Seilles, Estelle; Tiberghien, Pierre; Bensa, Jean-Claude; Plumas, Joel; Saas, Philippe

2005-02-01

A new entity of acute leukemia coexpressing CD4(+)CD56(+) markers without any other lineage-specific markers has been identified recently as arising from lymphoid-related plasmacytoid dendritic cells (pDCs). In our laboratory, cells from a patient with such CD4(+)CD56(+) lineage-negative leukemia were unexpectedly found to also express the myeloid marker CD33. To confirm the diagnosis of pDC leukemia despite the CD33 expression, we demonstrated that the leukemic cells indeed exhibited pDC phenotypic and functional properties. In 7 of 8 other patients with CD4(+)CD56(+) pDC malignancies, we were able to confirm that the tumor cells expressed CD33 although with variable expression levels. CD33 expression was shown by flow cytometry, reverse transcriptase-polymerase chain reaction, and immunoblot analysis. Furthermore, CD33 monoclonal antibody stimulation of purified CD4(+)CD56(+) leukemic cells led to cytokine secretion, thus confirming the presence of a functional CD33 on these leukemic cells. Moreover, we found that circulating pDCs in healthy individuals also weakly express CD33. Overall, our results demonstrate that the expression of CD33 on CD4(+)CD56(+) lineage-negative cells should not exclude the diagnosis of pDC leukemia and underline that pDC-specific markers should be used at diagnosis for CD4(+)CD56(+) malignancies.

An essential role for IL-2 receptor in regulatory T cell function

PubMed Central

Levine, Andrew G; Fan, Xiying; Klein, Ulf; Zheng, Ye; Gasteiger, Georg; Feng, Yongqiang; Fontenot, Jason D.; Rudensky, Alexander Y.

2016-01-01

Regulatory T (Treg) cells, expressing abundant amounts of the IL-2 receptor (IL-2R), are reliant on IL-2 produced by activated T cells. This feature implied a key role for a simple network based on IL-2 consumption by Treg cells in their suppressor function. However, congenital deficiency in IL-2R results in reduced expression of the Treg cell lineage specification factor Foxp3, confounding experimental efforts to understand the role of IL-2R expression and signaling in Treg suppressor function. Using genetic gain and loss of function approaches, we demonstrate that IL-2 capture is dispensable for control of CD4+ T cells, but is important for limiting CD8+ T cell activation, and that IL-2R dependent STAT5 transcription factor activation plays an essential role in Treg cell suppressor function separable from T cell receptor signaling. PMID:27595233

Selecting antagonistic antibodies that control differentiation through inducible expression in embryonic stem cells

PubMed Central

Melidoni, Anna N.; Dyson, Michael R.; Wormald, Sam; McCafferty, John

2013-01-01

Antibodies that modulate receptor function have great untapped potential in the control of stem cell differentiation. In contrast to many natural ligands, antibodies are stable, exquisitely specific, and are unaffected by the regulatory mechanisms that act on natural ligands. Here we describe an innovative system for identifying such antibodies by introducing and expressing antibody gene populations in ES cells. Following induced antibody expression and secretion, changes in differentiation outcomes of individual antibody-expressing ES clones are monitored using lineage-specific gene expression to identify clones that encode and express signal-modifying antibodies. This in-cell expression and reporting system was exemplified by generating blocking antibodies to FGF4 and its receptor FGFR1β, identified through delayed onset of ES cell differentiation. Functionality of the selected antibodies was confirmed by addition of exogenous antibodies to three different ES reporter cell lines, where retained expression of pluripotency markers Oct4, Nanog, and Rex1 was observed. This work demonstrates the potential for discovery and utility of functional antibodies in stem cell differentiation. This work is also unique in constituting an example of ES cells carrying an inducible antibody that causes a functional protein “knock-down” and allows temporal control of stable signaling components at the protein level. PMID:24082130

Massive-Scale Gene Co-Expression Network Construction and Robustness Testing Using Random Matrix Theory

PubMed Central

Isaacson, Sven; Luo, Feng; Feltus, Frank A.; Smith, Melissa C.

2013-01-01

The study of gene relationships and their effect on biological function and phenotype is a focal point in systems biology. Gene co-expression networks built using microarray expression profiles are one technique for discovering and interpreting gene relationships. A knowledge-independent thresholding technique, such as Random Matrix Theory (RMT), is useful for identifying meaningful relationships. Highly connected genes in the thresholded network are then grouped into modules that provide insight into their collective functionality. While it has been shown that co-expression networks are biologically relevant, it has not been determined to what extent any given network is functionally robust given perturbations in the input sample set. For such a test, hundreds of networks are needed and hence a tool to rapidly construct these networks. To examine functional robustness of networks with varying input, we enhanced an existing RMT implementation for improved scalability and tested functional robustness of human (Homo sapiens), rice (Oryza sativa) and budding yeast (Saccharomyces cerevisiae). We demonstrate dramatic decrease in network construction time and computational requirements and show that despite some variation in global properties between networks, functional similarity remains high. Moreover, the biological function captured by co-expression networks thresholded by RMT is highly robust. PMID:23409071

Smad4 suppresses the tumorigenesis and aggressiveness of neuroblastoma through repressing the expression of heparanase.

PubMed

Qu, Hongxia; Zheng, Liduan; Jiao, Wanju; Mei, Hong; Li, Dan; Song, Huajie; Fang, Erhu; Wang, Xiaojing; Li, Shiwang; Huang, Kai; Tong, Qiangsong

2016-09-06

Heparanase (HPSE) is the only endo-β-D-glucuronidase that is correlated with the progression of neuroblastoma (NB), the most common extracranial malignancy in childhood. However, the mechanisms underlying HPSE expression in NB still remain largely unknown. Herein, through analyzing cis-regulatory elements and mining public microarray datasets, we identified SMAD family member 4 (Smad4) as a crucial transcription regulator of HPSE in NB. We demonstrated that Smad4 repressed the HPSE expression at the transcriptional levels in NB cells. Mechanistically, Smad4 suppressed the HPSE expression through directly binding to its promoter and repressing the lymphoid enhancer binding factor 1 (LEF1)-facilitated transcription of HPSE via physical interaction. Gain- and loss-of-function studies demonstrated that Smad4 inhibited the growth, invasion, metastasis, and angiogenesis of NB cells in vitro and in vivo. Restoration of HPSE expression prevented the NB cells from changes in these biological features induced by Smad4. In clinical NB specimens, Smad4 was under-expressed and inversely correlated with HPSE levels, while LEF1 was highly expressed and positively correlated with HPSE expression. Patients with high Smad4 expression, low LEF1 or HPSE levels had greater survival probability. These results demonstrate that Smad4 suppresses the tumorigenesis and aggressiveness of NB through repressing the HPSE expression.

Characterisation of human tubular cell monolayers as a model of proximal tubular xenobiotic handling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, Colin D.A.; Sayer, Rachel; Windass, Amy S.

2008-12-15

The aim of this study was to determine whether primary human tubular cell monolayers could provide a powerful tool with which to investigate the renal proximal tubular handling of xenobiotics. Human proximal and distal tubule/collecting duct cells were grown as monolayers on permeable filter supports. After 10 days in culture, proximal tubule cells remained differentiated and expressed a wide palette of transporters at the mRNA level including NaPi-IIa, SGLT1, SGLT2, OCT2, OCTN2, OAT1, OAT3, OAT4, MDR1, MRP2 and BCRP. At the protein level, the expression of a subset of transporters including NaPi-IIa, OAT1 and OAT3 was demonstrated using immunohistochemistry. Analysismore » of the expression of the ATP binding cassette efflux pumps MDR1, MRP2 and BCRP confirmed their apical membrane localisation. At the functional level, tubule cell monolayers retain the necessary machinery to mediate the net secretion of the prototypic substrates; PAH and creatinine. PAH secretion across the monolayer consisted of the uptake of PAH across the basolateral membrane by OAT1 and OAT3 and the apical exit of PAH by a probenecid and MK571-sensitive route consistent with actions of MRP2 or MRP4. Creatinine secretion was by OCT2-mediated uptake at the basolateral membrane and via MDR1 at the apical membrane. Functional expression of MDR1 and BCRP at the apical membrane was also demonstrated using a Hoechst 33342 dye. Similarly, measurement of calcein efflux demonstrated the functional expression of MRP2 at the apical membrane of cell monolayers. In conclusion, human tubular cell monolayers provide a powerful tool to investigate renal xenobiotic handling.« less

Differential Effect of Active Smoking on Gene Expression in Male and Female Smokers

PubMed Central

Paul, Sunirmal; Amundson, Sally A

2015-01-01

Smoking is the second leading cause of preventable death in the United States. Cohort epidemiological studies have demonstrated that women are more vulnerable to cigarette-smoking induced diseases than their male counterparts, however, the molecular basis of these differences has remained unknown. In this study, we explored if there were differences in the gene expression patterns between male and female smokers, and how these patterns might reflect different sex-specific responses to the stress of smoking. Using whole genome microarray gene expression profiling, we found that a substantial number of oxidant related genes were expressed in both male and female smokers, however, smoking-responsive genes did indeed differ greatly between male and female smokers. Gene set enrichment analysis (GSEA) against reference oncogenic signature gene sets identified a large number of oncogenic pathway gene-sets that were significantly altered in female smokers compared to male smokers. In addition, functional annotation with Ingenuity Pathway Analysis (IPA) identified smoking-correlated genes associated with biological functions in male and female smokers that are directly relevant to well-known smoking related pathologies. However, these relevant biological functions were strikingly overrepresented in female smokers compared to male smokers. IPA network analysis with the functional categories of immune and inflammatory response gene products suggested potential interactions between smoking response and female hormones. Our results demonstrate a striking dichotomy between male and female gene expression responses to smoking. This is the first genome-wide expression study to compare the sex-specific impacts of smoking at a molecular level and suggests a novel potential connection between sex hormone signaling and smoking-induced diseases in female smokers. PMID:25621181

Cell-free Co-expression of Functional Membrane Proteins and Apolipoprotein, Forming Soluble Nanolipoprotein Particles*S⃞

PubMed Central

Cappuccio, Jenny A.; Blanchette, Craig D.; Sulchek, Todd A.; Arroyo, Erin S.; Kralj, Joel M.; Hinz, Angela K.; Kuhn, Edward A.; Chromy, Brett A.; Segelke, Brent W.; Rothschild, Kenneth J.; Fletcher, Julia E.; Katzen, Federico; Peterson, Todd C.; Kudlicki, Wieslaw A.; Bench, Graham; Hoeprich, Paul D.; Coleman, Matthew A.

2008-01-01

Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cell-free extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein Δ1–49 apolipoprotein A-I fragment (Δ49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR → M transition. Importantly the functional bR was solubilized in discoidal bR·NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with Δ49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies. PMID:18603642

Reduced Renal Methylarginine Metabolism Protects against Progressive Kidney Damage.

PubMed

Tomlinson, James A P; Caplin, Ben; Boruc, Olga; Bruce-Cobbold, Claire; Cutillas, Pedro; Dormann, Dirk; Faull, Peter; Grossman, Rebecca C; Khadayate, Sanjay; Mas, Valeria R; Nitsch, Dorothea D; Wang, Zhen; Norman, Jill T; Wilcox, Christopher S; Wheeler, David C; Leiper, James

2015-12-01

Nitric oxide (NO) production is diminished in many patients with cardiovascular and renal disease. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthesis, and elevated plasma levels of ADMA are associated with poor outcomes. Dimethylarginine dimethylaminohydrolase-1 (DDAH1) is a methylarginine-metabolizing enzyme that reduces ADMA levels. We reported previously that a DDAH1 gene variant associated with increased renal DDAH1 mRNA transcription and lower plasma ADMA levels, but counterintuitively, a steeper rate of renal function decline. Here, we test the hypothesis that reduced renal-specific ADMA metabolism protects against progressive renal damage. Renal DDAH1 is expressed predominately within the proximal tubule. A novel proximal tubule-specific Ddah1 knockout (Ddah1(PT-/-)) mouse demonstrated tubular cell accumulation of ADMA and lower NO concentrations, but unaltered plasma ADMA concentrations. Ddah1(PT-/-) mice were protected from reduced kidney tissue mass, collagen deposition, and profibrotic cytokine expression in two independent renal injury models: folate nephropathy and unilateral ureteric obstruction. Furthermore, a study of two independent kidney transplant cohorts revealed higher levels of human renal allograft methylarginine-metabolizing enzyme gene expression associated with steeper function decline. We also report an association among DDAH1 expression, NO activity, and uromodulin expression supported by data from both animal and human studies, raising the possibility that kidney DDAH1 expression exacerbates renal injury through uromodulin-related mechanisms. Together, these data demonstrate that reduced renal tubular ADMA metabolism protects against progressive kidney function decline. Thus, circulating ADMA may be an imprecise marker of renal methylarginine metabolism, and therapeutic ADMA reduction may even be deleterious to kidney function. Copyright © 2015 by the American Society of Nephrology.

Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs

PubMed Central

Yin, Linlin; Maddison, Lisette A.; Li, Mingyu; Kara, Nergis; LaFave, Matthew C.; Varshney, Gaurav K.; Burgess, Shawn M.; Patton, James G.; Chen, Wenbiao

2015-01-01

Determining the mechanism of gene function is greatly enhanced using conditional mutagenesis. However, generating engineered conditional alleles is inefficient and has only been widely used in mice. Importantly, multiplex conditional mutagenesis requires extensive breeding. Here we demonstrate a system for one-generation multiplex conditional mutagenesis in zebrafish (Danio rerio) using transgenic expression of both cas9 and multiple single guide RNAs (sgRNAs). We describe five distinct zebrafish U6 promoters for sgRNA expression and demonstrate efficient multiplex biallelic inactivation of tyrosinase and insulin receptor a and b, resulting in defects in pigmentation and glucose homeostasis. Furthermore, we demonstrate temporal and tissue-specific mutagenesis using transgenic expression of Cas9. Heat-shock-inducible expression of cas9 allows temporal control of tyr mutagenesis. Liver-specific expression of cas9 disrupts insulin receptor a and b, causing fasting hypoglycemia and postprandial hyperglycemia. We also show that delivery of sgRNAs targeting ascl1a into the eye leads to impaired damage-induced photoreceptor regeneration. Our findings suggest that CRISPR/Cas9-based conditional mutagenesis in zebrafish is not only feasible but rapid and straightforward. PMID:25855067

Genome-wide methylation and gene expression changes in newborn rats following maternal protein restriction and reversal by folic acid.

PubMed

Altobelli, Gioia; Bogdarina, Irina G; Stupka, Elia; Clark, Adrian J L; Langley-Evans, Simon

2013-01-01

A large body of evidence from human and animal studies demonstrates that the maternal diet during pregnancy can programme physiological and metabolic functions in the developing fetus, effectively determining susceptibility to later disease. The mechanistic basis of such programming is unclear but may involve resetting of epigenetic marks and fetal gene expression. The aim of this study was to evaluate genome-wide DNA methylation and gene expression in the livers of newborn rats exposed to maternal protein restriction. On day one postnatally, there were 618 differentially expressed genes and 1183 differentially methylated regions (FDR 5%). The functional analysis of differentially expressed genes indicated a significant effect on DNA repair/cycle/maintenance functions and of lipid, amino acid metabolism and circadian functions. Enrichment for known biological functions was found to be associated with differentially methylated regions. Moreover, these epigenetically altered regions overlapped genetic loci associated with metabolic and cardiovascular diseases. Both expression changes and DNA methylation changes were largely reversed by supplementing the protein restricted diet with folic acid. Although the epigenetic and gene expression signatures appeared to underpin largely different biological processes, the gene expression profile of DNA methyl transferases was altered, providing a potential link between the two molecular signatures. The data showed that maternal protein restriction is associated with widespread differential gene expression and DNA methylation across the genome, and that folic acid is able to reset both molecular signatures.

Dermal Stem Cells Can Differentiate Down an Endothelial Lineage

PubMed Central

Bell, Emma; Richardson, Gavin D.; Jahoda, Colin A.; Gledhill, Karl; Phillips, Helen M.; Henderson, Deborah; Owens, W. Andrew

2012-01-01

In this study, we have demonstrated that cells of neural crest origin located in the dermal papilla (DP) exhibit endothelial marker expression and a functional activity. When grown in endothelial growth media, DP primary cultures upregulate expression of vascular endothelial growth factor receptor 1 (FLT1) mRNA and downregulate expression of the dermal stem cell marker α-smooth muscle actin. DP cells have demonstrated functional characteristics of endothelial cells, including the ability to form capillary-like structures on Matrigel, increase uptake of low-density lipoprotein and upregulate ICAM1 (CD54) in response to tumour necrosis factor alpha (TNF-α) stimulation. We confirmed that these observations were not due to contaminating endothelial cells, by using DP clones. We have also used the WNT1cre/ROSA26R and WNT1cre/YFP lineage-tracing mouse models to identify a population of neural crest-derived cells in DP cultures that express the endothelial marker PECAM (CD31); these cells also form capillary-like structures on Matrigel. Importantly, cells of neural crest origin that express markers of endothelial and mesenchymal lineages exist within the dermal sheath of the vibrissae follicle. PMID:22571645

Functional expression of the TMEM16 family of calcium-activated chloride channels in airway smooth muscle

PubMed Central

Remy, Kenneth E.; Danielsson, Jennifer; Funayama, Hiromi; Fu, Xiao Wen; Chang, Herng-Yu Sucie; Yim, Peter; Xu, Dingbang; Emala, Charles W.

2013-01-01

Airway smooth muscle hyperresponsiveness is a key component in the pathophysiology of asthma. Although calcium-activated chloride channel (CaCC) flux has been described in many cell types, including human airway smooth muscle (HASM), the true molecular identity of the channels responsible for this chloride conductance remains controversial. Recently, a new family of proteins thought to represent the true CaCCs was identified as the TMEM16 family. This led us to question whether members of this family are functionally expressed in native and cultured HASM. We further questioned whether expression of these channels contributes to the contractile function of HASM. We identified the mRNA expression of eight members of the TMEM16 family in HASM cells and show immunohistochemical evidence of TMEM16A in both cultured and native HASM. Functionally, we demonstrate that the classic chloride channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), inhibited halide flux in cultured HASM cells. Moreover, HASM cells displayed classical electrophysiological properties of CaCCs during whole cell electrophysiological recordings, which were blocked by using an antibody selective for TMEM16A. Furthermore, two distinct TMEM16A antagonists (tannic acid and benzbromarone) impaired a substance P-induced contraction in isolated guinea pig tracheal rings. These findings demonstrate that multiple members of this recently described family of CaCCs are expressed in HASM cells, they display classic electrophysiological properties of CaCCs, and they modulate contractile tone in airway smooth muscle. The TMEM16 family may provide a novel therapeutic target for limiting airway constriction in asthma. PMID:23997176

Functional expression of the TMEM16 family of calcium-activated chloride channels in airway smooth muscle.

PubMed

Gallos, George; Remy, Kenneth E; Danielsson, Jennifer; Funayama, Hiromi; Fu, Xiao Wen; Chang, Herng-Yu Sucie; Yim, Peter; Xu, Dingbang; Emala, Charles W

2013-11-01

Airway smooth muscle hyperresponsiveness is a key component in the pathophysiology of asthma. Although calcium-activated chloride channel (CaCC) flux has been described in many cell types, including human airway smooth muscle (HASM), the true molecular identity of the channels responsible for this chloride conductance remains controversial. Recently, a new family of proteins thought to represent the true CaCCs was identified as the TMEM16 family. This led us to question whether members of this family are functionally expressed in native and cultured HASM. We further questioned whether expression of these channels contributes to the contractile function of HASM. We identified the mRNA expression of eight members of the TMEM16 family in HASM cells and show immunohistochemical evidence of TMEM16A in both cultured and native HASM. Functionally, we demonstrate that the classic chloride channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), inhibited halide flux in cultured HASM cells. Moreover, HASM cells displayed classical electrophysiological properties of CaCCs during whole cell electrophysiological recordings, which were blocked by using an antibody selective for TMEM16A. Furthermore, two distinct TMEM16A antagonists (tannic acid and benzbromarone) impaired a substance P-induced contraction in isolated guinea pig tracheal rings. These findings demonstrate that multiple members of this recently described family of CaCCs are expressed in HASM cells, they display classic electrophysiological properties of CaCCs, and they modulate contractile tone in airway smooth muscle. The TMEM16 family may provide a novel therapeutic target for limiting airway constriction in asthma.

A chronological expression profile of gene activity during embryonic mouse brain development.

PubMed

Goggolidou, P; Soneji, S; Powles-Glover, N; Williams, D; Sethi, S; Baban, D; Simon, M M; Ragoussis, I; Norris, D P

2013-12-01

The brain is a functionally complex organ, the patterning and development of which are key to adult health. To help elucidate the genetic networks underlying mammalian brain patterning, we conducted detailed transcriptional profiling during embryonic development of the mouse brain. A total of 2,400 genes were identified as showing differential expression between three developmental stages. Analysis of the data identified nine gene clusters to demonstrate analogous expression profiles. A significant group of novel genes of as yet undiscovered biological function were detected as being potentially relevant to brain development and function, in addition to genes that have previously identified roles in the brain. Furthermore, analysis for genes that display asymmetric expression between the left and right brain hemispheres during development revealed 35 genes as putatively asymmetric from a combined data set. Our data constitute a valuable new resource for neuroscience and neurodevelopment, exposing possible functional associations between genes, including novel loci, and encouraging their further investigation in human neurological and behavioural disorders.

Use of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) to Monitor Compound Effects on Cardiac Myocyte Signaling Pathways.

PubMed

Guo, Liang; Eldridge, Sandy; Furniss, Mike; Mussio, Jodie; Davis, Myrtle

2015-09-01

There is a need to develop mechanism-based assays to better inform risk of cardiotoxicity. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are rapidly gaining acceptance as a biologically relevant in vitro model for use in drug discovery and cardiotoxicity screens. Utilization of hiPSC-CMs for mechanistic investigations would benefit from confirmation of the expression and activity of cellular pathways that are known to regulate cardiac myocyte viability and function. This unit describes an approach to demonstrate the presence and function of signaling pathways in hiPSC-CMs and the effects of treatments on these pathways. We present a workflow that employs protocols to demonstrate protein expression and functional integrity of signaling pathway(s) of interest and to characterize biological consequences of signaling modulation. These protocols utilize a unique combination of structural, functional, and biochemical endpoints to interrogate compound effects on cardiomyocytes. Copyright © 2015 John Wiley & Sons, Inc.

Feline immunodeficiency virus OrfA alters gene expression of splicing factors and proteasome-ubiquitination proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sundstrom, Magnus; Chatterji, Udayan; Schaffer, Lana

2008-02-20

Expression of the feline immunodeficiency virus (FIV) accessory protein OrfA (or Orf2) is critical for efficient viral replication in lymphocytes, both in vitro and in vivo. OrfA has been reported to exhibit functions in common with the human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) accessory proteins Vpr and Tat, although the function of OrfA has not been fully explained. Here, we use microarray analysis to characterize how OrfA modulates the gene expression profile of T-lymphocytes. The primary IL-2-dependent T-cell line 104-C1 was transduced to express OrfA. Functional expression of OrfA was demonstrated by trans complementation of the OrfA-defectivemore » clone, FIV-34TF10. OrfA-expressing cells had a slightly reduced cell proliferation rate but did not exhibit any significant alteration in cell cycle distribution. Reverse-transcribed RNA from cells expressing green fluorescent protein (GFP) or GFP + OrfA were hybridized to Affymetrix HU133 Plus 2.0 microarray chips representing more than 47,000 genome-wide transcripts. By using two statistical approaches, 461 (Rank Products) and 277 (ANOVA) genes were identified as modulated by OrfA expression. The functional relevance of the differentially expressed genes was explored by Ingenuity Pathway Analysis. The analyses revealed alterations in genes critical for RNA post-transcriptional modifications and protein ubiquitination as the two most significant functional outcomes of OrfA expression. In these two groups, several subunits of the spliceosome, cellular splicing factors and family members of the proteasome-ubiquitination system were identified. These findings provide novel information on the versatile function of OrfA during FIV infection and indicate a fine-tuning mechanism of the cellular environment by OrfA to facilitate efficient FIV replication.« less

Functional expression of Ca²⁺ dependent mammalian transmembrane gap junction protein Cx43 in slime mold Dictyostelium discoideum.

PubMed

Kaufmann, Stefan; Weiss, Ingrid M; Eckstein, Volker; Tanaka, Motomu

2012-03-09

In this paper, we expressed murine gap junction protein Cx43 in Dictyostelium discoideum by introducing the specific vector pDXA. In the first step, the successful expression of Cx43 and Cx43-eGFP was verified by (a) Western blot (anti-Cx43, anti-GFP), (b) fluorescence microscopy (eGFP-Cx43 co-expression, Cx43 immunostaining), and (c) flow cytometry analysis (eGFP-Cx43 co-expression). Although the fluorescence signals from cells expressing Cx43-eGFP detected by fluorescence microscopy seem relatively low, analysis by flow cytometry demonstrated that more than 60% of cells expressed Cx43-eGFP. In order to evaluate the function of expressed Cx43 in D. discoideum, we examined the hemi-channel function of Cx43. In this series of experiments, the passive uptake of carboxyfluorescein was monitored using flow cytometric analysis. A significant number of the transfected cells showed a prominent dye uptake in the absence of Ca(2+). The dye uptake by transfected cells in the presence of Ca(2+) was even lower than the non-specific dye uptake by non-transformed Ax3 orf+ cells, confirming that Cx43 expressed in D. discoideum retains its Ca(2+)-dependent, specific gating function. The expression of gap junction proteins expressed in slime molds opens a possibility to the biological significance of intercellular communications in development and maintenance of multicellular organisms. Copyright © 2012 Elsevier Inc. All rights reserved.

REEPs Are Membrane Shaping Adapter Proteins That Modulate Specific G Protein-Coupled Receptor Trafficking by Affecting ER Cargo Capacity

PubMed Central

Ho, Vincent K.; Angelotti, Timothy

2013-01-01

Receptor expression enhancing proteins (REEPs) were identified by their ability to enhance cell surface expression of a subset of G protein-coupled receptors (GPCRs), specifically GPCRs that have proven difficult to express in heterologous cell systems. Further analysis revealed that they belong to the Yip (Ypt-interacting protein) family and that some REEP subtypes affect ER structure. Yip family comparisons have established other potential roles for REEPs, including regulation of ER-Golgi transport and processing/neuronal localization of cargo proteins. However, these other potential REEP functions and the mechanism by which they selectively enhance GPCR cell surface expression have not been clarified. By utilizing several REEP family members (REEP1, REEP2, and REEP6) and model GPCRs (α2A and α2C adrenergic receptors), we examined REEP regulation of GPCR plasma membrane expression, intracellular processing, and trafficking. Using a combination of immunolocalization and biochemical methods, we demonstrated that this REEP subset is localized primarily to ER, but not plasma membranes. Single cell analysis demonstrated that these REEPs do not specifically enhance surface expression of all GPCRs, but affect ER cargo capacity of specific GPCRs and thus their surface expression. REEP co-expression with α2 adrenergic receptors (ARs) revealed that this REEP subset interacts with and alter glycosidic processing of α2C, but not α2A ARs, demonstrating selective interaction with cargo proteins. Specifically, these REEPs enhanced expression of and interacted with minimally/non-glycosylated forms of α2C ARs. Most importantly, expression of a mutant REEP1 allele (hereditary spastic paraplegia SPG31) lacking the carboxyl terminus led to loss of this interaction. Thus specific REEP isoforms have additional intracellular functions besides altering ER structure, such as enhancing ER cargo capacity, regulating ER-Golgi processing, and interacting with select cargo proteins. Therefore, some REEPs can be further described as ER membrane shaping adapter proteins. PMID:24098485

Conservation of transcription factor binding events predicts gene expression across species

PubMed Central

Hemberg, Martin; Kreiman, Gabriel

2011-01-01

Recent technological advances have made it possible to determine the genome-wide binding sites of transcription factors (TFs). Comparisons across species have suggested a relatively low degree of evolutionary conservation of experimentally defined TF binding events (TFBEs). Using binding data for six different TFs in hepatocytes and embryonic stem cells from human and mouse, we demonstrate that evolutionary conservation of TFBEs within orthologous proximal promoters is closely linked to function, defined as expression of the target genes. We show that (i) there is a significantly higher degree of conservation of TFBEs when the target gene is expressed in both species; (ii) there is increased conservation of binding events for groups of TFs compared to individual TFs; and (iii) conserved TFBEs have a greater impact on the expression of their target genes than non-conserved ones. These results link conservation of structural elements (TFBEs) to conservation of function (gene expression) and suggest a higher degree of functional conservation than implied by previous studies. PMID:21622661

CD161 Defines a Functionally Distinct Subset of Pro-Inflammatory Natural Killer Cells

PubMed Central

Kurioka, Ayako; Cosgrove, Cormac; Simoni, Yannick; van Wilgenburg, Bonnie; Geremia, Alessandra; Björkander, Sophia; Sverremark-Ekström, Eva; Thurnheer, Christine; Günthard, Huldrych F.; Khanna, Nina; Aubert, V; Arancibia-Cárcamo, CV; Walker, Lucy Jane; Arancibia-Cárcamo, Carolina V.; Newell, Evan W.; Willberg, Christian B.; Klenerman, Paul

2018-01-01

CD161 is a C-type lectin-like receptor expressed on the majority of natural killer (NK) cells; however, the significance of CD161 expression on NK cells has not been comprehensively investigated. Recently, we found that CD161 expression identifies a transcriptional and innate functional phenotype that is shared across various T cell populations. Using mass cytometry and microarray experiments, we demonstrate that this functional phenotype extends to NK cells. CD161 marks NK cells that have retained the ability to respond to innate cytokines during their differentiation, and is lost upon cytomegalovirus-induced maturation in both healthy and human immunodeficiency virus (HIV)-infected patients. These pro-inflammatory NK cells are present in the inflamed lamina propria where they are enriched for integrin CD103 expression. Thus, CD161 expression identifies NK cells that may contribute to inflammatory disease pathogenesis and correlates with an innate responsiveness to cytokines in both T and NK cells. PMID:29686665

Module discovery by exhaustive search for densely connected, co-expressed regions in biomolecular interaction networks.

PubMed

Colak, Recep; Moser, Flavia; Chu, Jeffrey Shih-Chieh; Schönhuth, Alexander; Chen, Nansheng; Ester, Martin

2010-10-25

Computational prediction of functionally related groups of genes (functional modules) from large-scale data is an important issue in computational biology. Gene expression experiments and interaction networks are well studied large-scale data sources, available for many not yet exhaustively annotated organisms. It has been well established, when analyzing these two data sources jointly, modules are often reflected by highly interconnected (dense) regions in the interaction networks whose participating genes are co-expressed. However, the tractability of the problem had remained unclear and methods by which to exhaustively search for such constellations had not been presented. We provide an algorithmic framework, referred to as Densely Connected Biclustering (DECOB), by which the aforementioned search problem becomes tractable. To benchmark the predictive power inherent to the approach, we computed all co-expressed, dense regions in physical protein and genetic interaction networks from human and yeast. An automatized filtering procedure reduces our output which results in smaller collections of modules, comparable to state-of-the-art approaches. Our results performed favorably in a fair benchmarking competition which adheres to standard criteria. We demonstrate the usefulness of an exhaustive module search, by using the unreduced output to more quickly perform GO term related function prediction tasks. We point out the advantages of our exhaustive output by predicting functional relationships using two examples. We demonstrate that the computation of all densely connected and co-expressed regions in interaction networks is an approach to module discovery of considerable value. Beyond confirming the well settled hypothesis that such co-expressed, densely connected interaction network regions reflect functional modules, we open up novel computational ways to comprehensively analyze the modular organization of an organism based on prevalent and largely available large-scale datasets. Software and data sets are available at http://www.sfu.ca/~ester/software/DECOB.zip.

Biological Function of Ribosomal Protein L10 on Cell Behavior in Human Epithelial Ovarian Cancer

PubMed Central

Shi, Jimin; Zhang, Lingyun; Zhou, Daibing; Zhang, Jinguo; Lin, Qunbo; Guan, Wencai; Zhang, Jihong; Ren, Weimin; Xu, Guoxiong

2018-01-01

Ribosomal protein L10 (RPL10) is one of large ribosomal proteins and plays a role in Wilms' tumor and premature ovarian failure. However, the function of RPL10 in human epithelial ovarian cancer (EOC) remains unknown. The purpose of this study was to examine the expression level and function of RPL10 in EOC. RPL10 protein expression was detected by immunohistochemistry and Western blot. The association RPL10 expression with clinical features was analyzed. Loss-of-function and gain-of-function approaches were applied in cellular assays, including cell viability, migration, invasion, and apoptosis. Our study demonstrated for the first time that RPL10 was upregulated in human EOC compared with normal ovarian tissues. Knockdown of RPL10 inhibited cell viability, migration, and invasion, and increased cell apoptosis. On the contrary, upregulation of RPL10 increased cell viability, migration, invasion, and decreased cell apoptosis. Furthermore, miR-143-3p regulated RPL10 expression. Our data indicate that RPL10 is a potential tissue biomarker of patients with EOC and may be a therapeutic target of ovarian cancer. PMID:29556332

Characterization of a Distinct Population of Circulating Human Non-Adherent Endothelial Forming Cells and Their Recruitment via Intercellular Adhesion Molecule-3

PubMed Central

Thompson, Emma J.; Barrett, Jeffrey M.; Tooley, Katie; Sen, Shaundeep; Sun, Wai Yan; Grose, Randall; Nicholson, Ian; Levina, Vitalina; Cooke, Ira; Talbo, Gert; Lopez, Angel F.; Bonder, Claudine S.

2012-01-01

Circulating vascular progenitor cells contribute to the pathological vasculogenesis of cancer whilst on the other hand offer much promise in therapeutic revascularization in post-occlusion intervention in cardiovascular disease. However, their characterization has been hampered by the many variables to produce them as well as their described phenotypic and functional heterogeneity. Herein we have isolated, enriched for and then characterized a human umbilical cord blood derived CD133+ population of non-adherent endothelial forming cells (naEFCs) which expressed the hematopoietic progenitor cell markers (CD133, CD34, CD117, CD90 and CD38) together with mature endothelial cell markers (VEGFR2, CD144 and CD31). These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8) or myeloid markers (CD11b and CD14) which distinguishes them from ‘early’ endothelial progenitor cells (EPCs). Functional studies demonstrated that these naEFCs (i) bound Ulex europaeus lectin, (ii) demonstrated acetylated-low density lipoprotein uptake, (iii) increased vascular cell adhesion molecule (VCAM-1) surface expression in response to tumor necrosis factor and (iv) in co-culture with mature endothelial cells increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human endothelial cells (ECs). Extensive genomic and proteomic analyses of the naEFCs showed that intercellular adhesion molecule (ICAM)-3 is expressed on their cell surface but not on mature endothelial cells. Furthermore, functional analysis demonstrated that ICAM-3 mediated the rolling and adhesive events of the naEFCs under shear stress. We suggest that the distinct population of naEFCs identified and characterized here represents a new valuable therapeutic target to control aberrant vasculogenesis. PMID:23144795

Versatile control of Plasmodium falciparum gene expression with an inducible protein-RNA interaction

PubMed Central

Goldfless, Stephen J.; Wagner, Jeffrey C.; Niles, Jacquin C.

2014-01-01

The available tools for conditional gene expression in Plasmodium falciparum are limited. Here, to enable reliable control of target gene expression, we build a system to efficiently modulate translation. We overcame several problems associated with other approaches for regulating gene expression in P. falciparum. Specifically, our system functions predictably across several native and engineered promoter contexts, and affords control over reporter and native parasite proteins irrespective of their subcellular compartmentalization. Induction and repression of gene expression are rapid, homogeneous, and stable over prolonged periods. To demonstrate practical application of our system, we used it to reveal direct links between antimalarial drugs and their native parasite molecular target. This is an important out come given the rapid spread of resistance, and intensified efforts to efficiently discover and optimize new antimalarial drugs. Overall, the studies presented highlight the utility of our system for broadly controlling gene expression and performing functional genetics in P. falciparum. PMID:25370483

Improving membrane protein expression and function using genomic edits

DOE PAGES

Jensen, Heather M.; Eng, Thomas; Chubukov, Victor; ...

2017-10-12

Expression of membrane proteins often leads to growth inhibition and perturbs central metabolism and this burden varies with the protein being overexpressed. There are also known strain backgrounds that allow greater expression of membrane proteins but that differ in efficacy across proteins. Here, we hypothesized that for any membrane protein, it may be possible to identify a modified strain background where its expression can be accommodated with less burden. To directly test this hypothesis, we used a bar-coded transposon insertion library in tandem with cell sorting to assess genome-wide impact of gene deletions on membrane protein expression. The expression ofmore » five membrane proteins (CyoB, CydB, MdlB, YidC, and LepI) and one soluble protein (GST), each fused to GFP, was examined. We identified Escherichia coli mutants that demonstrated increased membrane protein expression relative to that in wild type. For two of the proteins (CyoB and CydB), we conducted functional assays to confirm that the increase in protein expression also led to phenotypic improvement in function. This study represents a systematic approach to broadly identify genetic loci that can be used to improve membrane protein expression, and our method can be used to improve expression of any protein that poses a cellular burden.« less

Improving membrane protein expression and function using genomic edits

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jensen, Heather M.; Eng, Thomas; Chubukov, Victor

Expression of membrane proteins often leads to growth inhibition and perturbs central metabolism and this burden varies with the protein being overexpressed. There are also known strain backgrounds that allow greater expression of membrane proteins but that differ in efficacy across proteins. Here, we hypothesized that for any membrane protein, it may be possible to identify a modified strain background where its expression can be accommodated with less burden. To directly test this hypothesis, we used a bar-coded transposon insertion library in tandem with cell sorting to assess genome-wide impact of gene deletions on membrane protein expression. The expression ofmore » five membrane proteins (CyoB, CydB, MdlB, YidC, and LepI) and one soluble protein (GST), each fused to GFP, was examined. We identified Escherichia coli mutants that demonstrated increased membrane protein expression relative to that in wild type. For two of the proteins (CyoB and CydB), we conducted functional assays to confirm that the increase in protein expression also led to phenotypic improvement in function. This study represents a systematic approach to broadly identify genetic loci that can be used to improve membrane protein expression, and our method can be used to improve expression of any protein that poses a cellular burden.« less

Evidence-Based Annotation of Gene Function in Shewanella oneidensis MR-1 Using Genome-Wide Fitness Profiling across 121 Conditions

PubMed Central

Deutschbauer, Adam; Price, Morgan N.; Wetmore, Kelly M.; Shao, Wenjun; Baumohl, Jason K.; Xu, Zhuchen; Nguyen, Michelle; Tamse, Raquel; Davis, Ronald W.; Arkin, Adam P.

2011-01-01

Most genes in bacteria are experimentally uncharacterized and cannot be annotated with a specific function. Given the great diversity of bacteria and the ease of genome sequencing, high-throughput approaches to identify gene function experimentally are needed. Here, we use pools of tagged transposon mutants in the metal-reducing bacterium Shewanella oneidensis MR-1 to probe the mutant fitness of 3,355 genes in 121 diverse conditions including different growth substrates, alternative electron acceptors, stresses, and motility. We find that 2,350 genes have a pattern of fitness that is significantly different from random and 1,230 of these genes (37% of our total assayed genes) have enough signal to show strong biological correlations. We find that genes in all functional categories have phenotypes, including hundreds of hypotheticals, and that potentially redundant genes (over 50% amino acid identity to another gene in the genome) are also likely to have distinct phenotypes. Using fitness patterns, we were able to propose specific molecular functions for 40 genes or operons that lacked specific annotations or had incomplete annotations. In one example, we demonstrate that the previously hypothetical gene SO_3749 encodes a functional acetylornithine deacetylase, thus filling a missing step in S. oneidensis metabolism. Additionally, we demonstrate that the orphan histidine kinase SO_2742 and orphan response regulator SO_2648 form a signal transduction pathway that activates expression of acetyl-CoA synthase and is required for S. oneidensis to grow on acetate as a carbon source. Lastly, we demonstrate that gene expression and mutant fitness are poorly correlated and that mutant fitness generates more confident predictions of gene function than does gene expression. The approach described here can be applied generally to create large-scale gene-phenotype maps for evidence-based annotation of gene function in prokaryotes. PMID:22125499

Turning gene function ON and OFF using sense and antisense photo-morpholinos in zebrafish

PubMed Central

Tallafuss, Alexandra; Gibson, Dan; Morcos, Paul; Li, Yongfu; Seredick, Steve; Eisen, Judith; Washbourne, Philip

2012-01-01

To understand the molecular mechanisms of development it is essential to be able to turn genes on and off at will and in a spatially restricted fashion. Morpholino oligonucleotides (MOs) are very common tools used in several model organisms with which it is possible to block gene expression. Recently developed photo-activated MOs allow control over the onset of MO activity. However, deactivation of photo-cleavable MO activity has remained elusive. Here, we describe photo-cleavable MOs with which it is possible to activate or de-activate MO function by UV exposure in a temporal and spatial manner. We show, using several different genes as examples, that it is possible to turn gene expression on or off both in the entire zebrafish embryo and in single cells. We use these tools to demonstrate the sufficiency of no tail expression as late as tailbud stage to drive medial precursor cells towards the notochord cell fate. As a broader approach for the use of photo-cleavable MOs, we show temporal control over gal4 function, which has many potential applications in multiple transgenic lines. We demonstrate temporal manipulation of Gal4 transgene expression in only primary motoneurons and not secondary motoneurons, heretofore impossible with conventional transgenic approaches. In another example, we follow and analyze neural crest cells that regained sox10 function after deactivation of a photo-cleavable sox10-MO at different time points. Our results suggest that sox10 function might not be critical during neural crest formation. PMID:22492359

Distinct Functions of CCAAT Enhancer-binding Protein Isoforms in the Regulation of Manganese Superoxide Dismutase during Interleukin-1β Stimulation*

PubMed Central

Qiu, Xiaolei; Aiken, Kimberly J.; Chokas, Ann L.; Beachy, Dawn E.; Nick, Harry S.

2008-01-01

The mitochondrial antioxidant enzyme manganese superoxide dismutase (Mn-SOD) is crucial in maintaining cellular and organismal homeostasis. Mn-SOD expression is tightly regulated in a manner that synchronizes its cytoprotective functions during inflammatory challenges. Induction of Mn-SOD gene expression by the proinflammatory cytokine IL-1β is mediated through a complex intronic enhancer element. To identify and characterize the transcription factors required for Mn-SOD enhancer function, a yeast one-hybrid assay was utilized, and two CCAAT enhancer-binding protein (C/EBP) members, C/EBP β and C/EBP δ, were identified. These two transcription factors responded to IL-1β treatment with distinct expression profiles, different temporal yet inducible interactions with the endogenous Mn-SOD enhancer, and also opposite effects on Mn-SOD transcription. C/EBP β is expressed as three isoforms, LAP* (liver-activating protein), LAP, and LIP (liver-inhibitory protein). Our functional analysis demonstrated that only the full-length C/EBP β/LAP* served as a true activator for Mn-SOD, whereas LAP, LIP, and C/EBP δ functioned as potential repressors. Finally, our systematic mutagenesis of the unique N-terminal 21 amino acids further solidified the importance of LAP* in the induction of Mn-SOD and emphasized the crucial role of this isoform. Our data demonstrating the physiological relevance of the N-terminal peptide also provide a rationale for revisiting the role of LAP* in the regulation of other genes and in pathways such as lipogenesis and development. PMID:18559338

Dusp5 negatively regulates IL-33-mediated eosinophil survival and function

PubMed Central

Holmes, Derek A; Yeh, Jung-Hua; Yan, Donghong; Xu, Min; Chan, Andrew C

2015-01-01

Mitogen-activated protein kinase (MAPK) activation controls diverse cellular functions including cellular survival, proliferation, and apoptosis. Tuning of MAPK activation is counter-regulated by a family of dual-specificity phosphatases (DUSPs). IL-33 is a recently described cytokine that initiates Th2 immune responses through binding to a heterodimeric IL-33Rα (ST2L)/IL-1α accessory protein (IL-1RAcP) receptor that coordinates activation of ERK and NF-κB pathways. We demonstrate here that DUSP5 is expressed in eosinophils, is upregulated following IL-33 stimulation and regulates IL-33 signaling. Dusp5−/− mice have prolonged eosinophil survival and enhanced eosinophil effector functions following infection with the helminth Nippostrongylus brasiliensis. IL-33-activated Dusp5−/− eosinophils exhibit increased cellular ERK1/2 activation and BCL-XL expression that results in enhanced eosinophil survival. In addition, Dusp5−/− eosinophils demonstrate enhanced IL-33-mediated activation and effector functions. Together, these data support a role for DUSP5 as a novel negative regulator of IL-33-dependent eosinophil function and survival. PMID:25398911

Novel functions for glycosyltransferases Jhp0562 and GalT in Lewis antigen synthesis and variation in Helicobacter pylori.

PubMed

Pohl, Mary Ann; Kienesberger, Sabine; Blaser, Martin J

2012-04-01

Lewis (Le) antigens are fucosylated oligosaccharides present in the Helicobacter pylori lipopolysaccharide. Expression of these antigens is believed to be important for H. pylori colonization, since Le antigens also are expressed on the gastric epithelia in humans. A galactosyltransferase encoded by β-(1,3)galT is essential for production of type 1 (Le(a) and Le(b)) antigens. The upstream gene jhp0562, which is present in many but not all H. pylori strains, is homologous to β-(1,3)galT but is of unknown function. Because H. pylori demonstrates extensive intragenomic recombination, we hypothesized that these two genes could undergo DNA rearrangement. A PCR screen and subsequent sequence analyses revealed that the two genes can recombine at both the 5' and 3' ends. Chimeric β-(1,3)galT-like alleles can restore function in a β-(1,3)galT null mutant, but neither native nor recombinant jhp0562 can. Mutagenesis of jhp0562 revealed that it is essential for synthesis of both type 1 and type 2 Le antigens. Transcriptional analyses of both loci showed β-(1,3)galT expression in all wild-type (WT) and mutant strains tested, whereas jhp0562 was not expressed in jhp0562 null mutants, as expected. Since jhp0562 unexpectedly displayed functions in both type 1 and type 2 Le synthesis, we asked whether galT, part of the type 2 synthesis pathway, had analogous functions in type 1 synthesis. Mutagenesis and complementation analysis confirmed that galT is essential for Le(b) production. In total, these results demonstrate that galT and jhp0562 have functions that cross the expected Le synthesis pathways and that jhp0562 provides a substrate for intragenomic recombination to generate diverse Le synthesis enzymes.

Novel Functions for Glycosyltransferases Jhp0562 and GalT in Lewis Antigen Synthesis and Variation in Helicobacter pylori

PubMed Central

Kienesberger, Sabine; Blaser, Martin J.

2012-01-01

Lewis (Le) antigens are fucosylated oligosaccharides present in the Helicobacter pylori lipopolysaccharide. Expression of these antigens is believed to be important for H. pylori colonization, since Le antigens also are expressed on the gastric epithelia in humans. A galactosyltransferase encoded by β-(1,3)galT is essential for production of type 1 (Lea and Leb) antigens. The upstream gene jhp0562, which is present in many but not all H. pylori strains, is homologous to β-(1,3)galT but is of unknown function. Because H. pylori demonstrates extensive intragenomic recombination, we hypothesized that these two genes could undergo DNA rearrangement. A PCR screen and subsequent sequence analyses revealed that the two genes can recombine at both the 5′ and 3′ ends. Chimeric β-(1,3)galT-like alleles can restore function in a β-(1,3)galT null mutant, but neither native nor recombinant jhp0562 can. Mutagenesis of jhp0562 revealed that it is essential for synthesis of both type 1 and type 2 Le antigens. Transcriptional analyses of both loci showed β-(1,3)galT expression in all wild-type (WT) and mutant strains tested, whereas jhp0562 was not expressed in jhp0562 null mutants, as expected. Since jhp0562 unexpectedly displayed functions in both type 1 and type 2 Le synthesis, we asked whether galT, part of the type 2 synthesis pathway, had analogous functions in type 1 synthesis. Mutagenesis and complementation analysis confirmed that galT is essential for Leb production. In total, these results demonstrate that galT and jhp0562 have functions that cross the expected Le synthesis pathways and that jhp0562 provides a substrate for intragenomic recombination to generate diverse Le synthesis enzymes. PMID:22290141

Knockdown of long non-coding RNA HOTAIR increases miR-454-3p by targeting Stat3 and Atg12 to inhibit chondrosarcoma growth.

PubMed

Bao, Xing; Ren, Tingting; Huang, Yi; Sun, Kunkun; Wang, Shidong; Liu, Kuisheng; Zheng, Bingxin; Guo, Wei

2017-02-09

Current practices for the therapy of chondrosarcoma, including wide-margin surgical resection and chemotherapy, are less than satisfactory. Recently, emerging evidence has demonstrated that long non-coding RNAs (lncRNAs) have an essential role in the initiation and progression of tumors. As a typical lncRNA, HOTAIR is significantly overexpressed in various tumors. However, the function and potential biological mechanisms of HOTAIR in human chondrosarcoma remain unknown. Quantitative RT-PCR demonstrated that HOTAIR expression was upregulated in chondrosarcoma tissues and cell lines. High HOTAIR expression is correlated with tumor stage and poor prognosis. Functional experiments reveal that HOTAIR knockdown leads to growth inhibition of human chondrosarcoma cells in vitro and in vivo. In addition to cycle arrest and apoptosis, knockdown of HOTAIR inhibits autophagy, which favors cell death. Mechanistically, we demonstrated that HOTAIR induced DNA methylation of miR-454-3p by recruiting EZH2 and DNMT1 to the miR-454-3p promoter regions, which markedly silences miR-454-3p expression. Further analysis revealed that STAT3 and ATG12 are targets of miR-454-3p, initiate HOTAIR deficiency-induced apoptosis and reduce autophagy. Collectively, our data reveal the roles and functional mechanisms of HOTAIR in human chondrosarcoma and suggest that HOTAIR may act as a prognostic biomarker and potential therapeutic target for chondrosarcoma.

Health-related quality of life for patients with rheumatoid arthritis foot involvement.

PubMed

Wickman, Amy M; Pinzur, Michael S; Kadanoff, Ruth; Juknelis, Dainius

2004-01-01

Rheumatoid arthritis is a common disabling form of arthritis that frequently affects the hands and feet. With time, the majority of affected individuals will become disabled. Sixty-nine consecutively selected mild to moderately affected individuals with rheumatoid arthritis provided demographic data and agreed to complete the Short Musculoskeletal Function Assessment (SFMA) instrument. Focus group subjects selectively used minimally adaptive nonprescription footwear. Control subjects had similar disease expression, but did not alter their choice of footwear due to their disease. Adult patients with rheumatoid arthritis demonstrate a significant negative impact on their quality of life with mild or moderate disease expression, as evidenced by poor scores in all six domains of the SFMA. Subjects who used even mildly adaptive nonprescription footwear demonstrated a statistically significant negative impact in mobility (p < .044) and functional index (p < .052) domains as compared with the control population having similar overall disease expression. Focus subjects also demonstrated a trend to less favorable scores in the arm and hand domain. Mean scores of the daily activity, emotional status, and bother index domains fared worse than population norms, but there was no statistical difference between subjects using, or not using, adaptive footwear. Individuals affected with mild to moderate rheumatoid arthritis are disabled as compared with the general population. There is a severe negative impact on mobility and functional capacity when the disease process begins to affect their feet.

Genome-wide characterization of mammalian promoters with distal enhancer functions.

PubMed

Dao, Lan T M; Galindo-Albarrán, Ariel O; Castro-Mondragon, Jaime A; Andrieu-Soler, Charlotte; Medina-Rivera, Alejandra; Souaid, Charbel; Charbonnier, Guillaume; Griffon, Aurélien; Vanhille, Laurent; Stephen, Tharshana; Alomairi, Jaafar; Martin, David; Torres, Magali; Fernandez, Nicolas; Soler, Eric; van Helden, Jacques; Puthier, Denis; Spicuglia, Salvatore

2017-07-01

Gene expression in mammals is precisely regulated by the combination of promoters and gene-distal regulatory regions, known as enhancers. Several studies have suggested that some promoters might have enhancer functions. However, the extent of this type of promoters and whether they actually function to regulate the expression of distal genes have remained elusive. Here, by exploiting a high-throughput enhancer reporter assay, we unravel a set of mammalian promoters displaying enhancer activity. These promoters have distinct genomic and epigenomic features and frequently interact with other gene promoters. Extensive CRISPR-Cas9 genomic manipulation demonstrated the involvement of these promoters in the cis regulation of expression of distal genes in their natural loci. Our results have important implications for the understanding of complex gene regulation in normal development and disease.

Phenotypic Checkpoints Regulate Neuronal Development

PubMed Central

Ben-Ari, Yehezkel; Spitzer, Nicholas C.

2010-01-01

Nervous system development proceeds by sequential gene expression mediated by cascades of transcription factors in parallel with sequences of patterned network activity driven by receptors and ion channels. These sequences are cell type- and developmental stage-dependent and modulated by paracrine actions of substances released by neurons and glia. How and to what extent these sequences interact to enable neuronal network development is not understood. Recent evidence demonstrates that CNS development requires intermediate stages of differentiation providing functional feedback that influences gene expression. We suggest that embryonic neuronal functions constitute a series of phenotypic checkpoint signatures; neurons failing to express these functions are delayed or developmentally arrested. Such checkpoints are likely to be a general feature of neuronal development and may constitute presymptomatic signatures of neurological disorders when they go awry. PMID:20864191

Functional Heterologous Protein Expression by Genetically Engineered Probiotic Yeast Saccharomyces boulardii

PubMed Central

Hudson, Lauren E.; Fasken, Milo B.; McDermott, Courtney D.; McBride, Shonna M.; Kuiper, Emily G.; Guiliano, David B.; Corbett, Anita H.; Lamb, Tracey J.

2014-01-01

Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT) S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders. PMID:25391025

Functional heterologous protein expression by genetically engineered probiotic yeast Saccharomyces boulardii.

PubMed

Hudson, Lauren E; Fasken, Milo B; McDermott, Courtney D; McBride, Shonna M; Kuiper, Emily G; Guiliano, David B; Corbett, Anita H; Lamb, Tracey J

2014-01-01

Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT) S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders.

Dopamine D2-like receptors (DRD2 and DRD4) in chickens: Tissue distribution, functional analysis, and their involvement in dopamine inhibition of pituitary prolactin expression.

PubMed

Lv, Can; Mo, Chunheng; Liu, Haikun; Wu, Chao; Li, Zhengyang; Li, Juan; Wang, Yajun

2018-04-20

Dopamine (DA) D2-like (and D1-like) receptors are suggested to mediate the dopamine actions in the anterior pituitary and/or CNS of birds. However, the information regarding the structure, functionality, and expression of avian D2-like receptors have not been fully characterized. In this study, we cloned two D2-like receptors (cDRD2, cDRD4) from chicken brain using RACE PCR. The cloned cDRD4 is a 378-amino acid receptor, which shows 57% amino acid (a.a.) identity with mouse DRD4. As in mammals, two cDRD2 isoforms, cDRD2L (long isoform, 437 a.a.) and cDRD2S (short isoform, 408 a.a.), which differ in their third intracellular loop, were identified in chickens. Using cell-based luciferase reporter assays or Western blot, we demonstrated that cDRD4, cDRD2L and cDRD2S could be activated by dopamine and quinpirole (a D2-like receptor agonist) dose-dependently, and their activation inhibits cAMP signaling pathway and stimulates MAPK/ERK signaling cascade, indicating that they are functional receptors capable of mediating dopamine actions. Quantitative real-time PCR revealed that cDRD2 and cDRD4 are widely expressed in chicken tissues with abundant expression noted in anterior pituitary, and their expressions are likely controlled by their promoters near exon 1, as demonstrated by dual-luciferase reporter assays in DF-1 cells. In accordance with cDRD2/cDRD4 expression in the pituitary, DA or quinpirole could partially inhibit vasoactive intestinal peptide-induced prolactin expression in cultured chick pituitary cells. Together, our data proves the functionality of DRD2 and DRD4 in birds and aids to uncover the conserved roles of DA/D2-like receptor system in vertebrates, such as its action on the pituitary. Copyright © 2018. Published by Elsevier B.V.

Promoting Expressive Language in Young Children with or At-Risk for Autism Spectrum Disorder in a Preschool Classroom.

PubMed

Lane, Justin D; Shepley, Collin; Lieberman-Betz, Rebecca

2016-10-01

Young children with autism spectrum disorder (ASD) often demonstrate delays in expressive communication, impacting their ability to independently function in typical environments. Individuals with ASD who develop expressive language during early childhood experience better outcomes later in life; therefore, examination of naturalistic language interventions (NLIs) remain an important area of investigation. The current study used a multiple probe design across participants to examine the effects of a classroom-based NLI on various expressive language targets in three preschool-aged children demonstrating characteristics of ASD. Findings suggest the intervention had positive and maintained effects on trial-based use of language targets, as well as concomitant changes in commenting, requesting, and phrase complexity. Implications regarding implementation of NLIs within typical classroom play activities are discussed.

The activity of spontaneous action potentials in developing hair cells is regulated by Ca(2+)-dependence of a transient K+ current.

PubMed

Levic, Snezana; Lv, Ping; Yamoah, Ebenezer N

2011-01-01

Spontaneous action potentials have been described in developing sensory systems. These rhythmic activities may have instructional roles for the functional development of synaptic connections. The importance of spontaneous action potentials in the developing auditory system is underpinned by the stark correlation between the time of auditory system functional maturity, and the cessation of spontaneous action potentials. A prominent K(+) current that regulates patterning of action potentials is I(A). This current undergoes marked changes in expression during chicken hair cell development. Although the properties of I(A) are not normally classified as Ca(2+)-dependent, we demonstrate that throughout the development of chicken hair cells, I(A) is greatly reduced by acute alterations of intracellular Ca(2+). As determinants of spike timing and firing frequency, intracellular Ca(2+) buffers shift the activation and inactivation properties of the current to more positive potentials. Our findings provide evidence to demonstrate that the kinetics and functional expression of I(A) are tightly regulated by intracellular Ca(2+). Such feedback mechanism between the functional expression of I(A) and intracellular Ca(2+) may shape the activity of spontaneous action potentials, thus potentially sculpting synaptic connections in an activity-dependent manner in the developing cochlea. © 2011 Levic et al.

Functional genomics analysis of low concentration of ethanol in human hepatocellular carcinoma (HepG2) cells. Role of genes involved in transcriptional and translational processes.

PubMed

Castaneda, Francisco; Rosin-Steiner, Sigrid; Jung, Klaus

2006-12-21

We previously found that ethanol at millimolar level (1 mM) activates the expression of transcription factors with subsequent regulation of apoptotic genes in human hepatocellular carcinoma (HCC) HepG2 cells. However, the role of ethanol on the expression of genes implicated in transcriptional and translational processes remains unknown. Therefore, the aim of this study was to characterize the effect of low concentration of ethanol on gene expression profiling in HepG2 cells using cDNA microarrays with especial interest in genes with transcriptional and translational function. The gene expression pattern observed in the ethanol-treated HepG2 cells revealed a relatively similar pattern to that found in the untreated control cells. The pairwise comparison analysis demonstrated four significantly up-regulated (COBRA1, ITGB4, STAU2, and HMGN3) genes and one down-regulated (ANK3) gene. All these genes exert their function on transcriptional and translational processes and until now none of these genes have been associated with ethanol. This functional genomic analysis demonstrates the reported interaction between ethanol and ethanol-regulated genes. Moreover, it confirms the relationship between ethanol-regulated genes and various signaling pathways associated with ethanol-induced apoptosis. The data presented in this study represents an important contribution toward the understanding of the molecular mechanisms of ethanol at low concentration in HepG2 cells, a HCC-derived cell line.

Functional genomics analysis of low concentration of ethanol in human hepatocellular carcinoma (HepG2) cells. Role of genes involved in transcriptional and translational processes

PubMed Central

Castaneda, Francisco; Rosin-Steiner, Sigrid; Jung, Klaus

2007-01-01

We previously found that ethanol at millimolar level (1 mM) activates the expression of transcription factors with subsequent regulation of apoptotic genes in human hepatocellular carcinoma (HCC) HepG2 cells. However, the role of ethanol on the expression of genes implicated in transcriptional and translational processes remains unknown. Therefore, the aim of this study was to characterize the effect of low concentration of ethanol on gene expression profiling in HepG2 cells using cDNA microarrays with especial interest in genes with transcriptional and translational function. The gene expression pattern observed in the ethanol-treated HepG2 cells revealed a relatively similar pattern to that found in the untreated control cells. The pairwise comparison analysis demonstrated four significantly up-regulated (COBRA1, ITGB4, STAU2, and HMGN3) genes and one down-regulated (ANK3) gene. All these genes exert their function on transcriptional and translational processes and until now none of these genes have been associated with ethanol. This functional genomic analysis demonstrates the reported interaction between ethanol and ethanol-regulated genes. Moreover, it confirms the relationship between ethanol-regulated genes and various signaling pathways associated with ethanol-induced apoptosis. The data presented in this study represents an important contribution toward the understanding of the molecular mechanisms of ethanol at low concentration in HepG2 cells, a HCC-derived cell line. PMID:17211498

Role of Wnt Signaling in the Control of Adult Hippocampal Functioning in Health and Disease: Therapeutic Implications

PubMed Central

Ortiz-Matamoros, Abril; Salcedo-Tello, Pamela; Avila-Muñoz, Evangelina; Zepeda, Angélica; Arias, Clorinda

2013-01-01

It is well recognized the role of the Wnt pathway in many developmental processes such as neuronal maturation, migration, neuronal connectivity and synaptic formation. Growing evidence is also demonstrating its function in the mature brain where is associated with modulation of axonal remodeling, dendrite outgrowth, synaptic activity, neurogenesis and behavioral plasticity. Proteins involved in Wnt signaling have been found expressed in the adult hippocampus suggesting that Wnt pathway plays a role in the hippocampal function through life. Indeed, Wnt ligands act locally to regulate neurogenesis, neuronal cell shape and pre- and postsynaptic assembly, events that are thought to underlie changes in synaptic function associated with long-term potentiation and with cognitive tasks such as learning and memory. Recent data have demonstrated the increased expression of the Wnt antagonist Dickkopf-1 (DKK1) in brains of Alzheimer´s disease (AD) patients suggesting that dysfunction of Wnt signaling could also contribute to AD pathology. We review here evidence of Wnt-associated molecules expression linked to physiological and pathological hippocampal functioning in the adult brain. The basic aspects of Wnt related mechanisms underlying hippocampal plasticity as well as evidence of how hippocampal dysfunction may rely on Wnt dysregulation is analyzed. This information would provide some clues about the possible therapeutic targets for developing treatments for neurodegenerative diseases associated with aberrant brain plasticity. PMID:24403870

Role of wnt signaling in the control of adult hippocampal functioning in health and disease: therapeutic implications.

PubMed

Ortiz-Matamoros, Abril; Salcedo-Tello, Pamela; Avila-Muñoz, Evangelina; Zepeda, Angélica; Arias, Clorinda

2013-09-01

It is well recognized the role of the Wnt pathway in many developmental processes such as neuronal maturation, migration, neuronal connectivity and synaptic formation. Growing evidence is also demonstrating its function in the mature brain where is associated with modulation of axonal remodeling, dendrite outgrowth, synaptic activity, neurogenesis and behavioral plasticity. Proteins involved in Wnt signaling have been found expressed in the adult hippocampus suggesting that Wnt pathway plays a role in the hippocampal function through life. Indeed, Wnt ligands act locally to regulate neurogenesis, neuronal cell shape and pre- and postsynaptic assembly, events that are thought to underlie changes in synaptic function associated with long-term potentiation and with cognitive tasks such as learning and memory. Recent data have demonstrated the increased expression of the Wnt antagonist Dickkopf-1 (DKK1) in brains of Alzheimer´s disease (AD) patients suggesting that dysfunction of Wnt signaling could also contribute to AD pathology. We review here evidence of Wnt-associated molecules expression linked to physiological and pathological hippocampal functioning in the adult brain. The basic aspects of Wnt related mechanisms underlying hippocampal plasticity as well as evidence of how hippocampal dysfunction may rely on Wnt dysregulation is analyzed. This information would provide some clues about the possible therapeutic targets for developing treatments for neurodegenerative diseases associated with aberrant brain plasticity.

HIERARCHICAL ORGANIZATION OF OSTEOBLASTS REVEALS THE SIGNIFICANT ROLE OF CD166 IN HEMATOPOIETIC STEM CELL MAINTANANCE AND FUNCTION

PubMed Central

Chitteti, Brahmananda R.; Cheng, Ying-Hua; Kacena, Melissa A.; Srour, Edward F.

2013-01-01

The role of osteoblasts (OB) in maintaining hematopoietic stem cells (HSC) in their niche is well elucidated, but the exact definition, both phenotypically and hierarchically of OB responsible for these functions is not clearly known. We previously demonstrated that OB maturational status influences HSC function whereby immature OB with high Runx2 expression promote hematopoietic expansion. Here, we show that Activated Leukocyte Cell Adhesion Molecule (ALCAM) or CD166 expression on OB is directly correlated with Runx2 expression and high hematopoiesis enhancing activity (HEA). Fractionation of OB with lineage markers: Sca1, osteopontin (OPN), CD166, CD44, and CD90 revealed that Lin-Sca1-OPN+CD166+ cells (CD166+) and their subpopulations fractionated with CD44 and CD90 expressed high levels of Runx2 and low levels of osteocalcin (OC) demonstrating the relatively immature status of these cells. Conversely, the majority of the Lin-Sca1-OPN+CD166− cells (CD166−) expressed high OC levels suggesting that CD166− OB are more mature. In vitro hematopoietic potential of LSK cells co-cultured for 7 days with fresh OB or OB pre-cultured for 1, 2, or 3 weeks declined precipitously with increasing culture duration concomitant with loss of CD166 expression. Importantly, LSK cells co-cultured with CD166+CD44+CD90+ OB maintained their in vivo repopulating potential through primary and secondary transplantation, suggesting that robust HEA activity is best mediated by immature CD166+ OB with high Runx2 and low OC expression. These studies begin to define the hierarchical organization of osteoblastic cells and provide a more refined definition of OB that can mediate HEA. PMID:23369988

Functional and prognostic significance of long non-coding RNA MALAT1 as a metastasis driver in ER negative lymph node negative breast cancer

PubMed Central

Jadaliha, Mahdieh; Zong, Xinying; Malakar, Pushkar; Ray, Tania; Singh, Deepak K.; Freier, Susan M.; Jensen, Tor; Prasanth, Supriya G.; Karni, Rotem; Ray, Partha S.; Prasanth, Kannanganattu V.

2016-01-01

MALAT1 (metastasis associated lung adenocarcinoma transcript1) is a conserved long non-coding RNA, known to regulate gene expression by modulating transcription and post-transcriptional pre-mRNA processing of a large number of genes. MALAT1 expression is deregulated in various tumors, including breast cancer. However, the significance of such abnormal expression is yet to be fully understood. In this study, we demonstrate that regulation of aggressive breast cancer cell traits by MALAT1 is not predicted solely based on an elevated expression level but is context specific. By performing loss- and gain-of-function studies, both under in vitro and in vivo conditions, we demonstrate that MALAT1 facilitates cell proliferation, tumor progression and metastasis of triple-negative breast cancer (TNBC) cells despite having a comparatively lower expression level than ER or HER2-positive breast cancer cells. Furthermore, MALAT1 regulates the expression of several cancer metastasis-related genes, but displays molecular subtype specific correlations with such genes. Assessment of the prognostic significance of MALAT1 in human breast cancer (n=1992) revealed elevated MALAT1 expression was associated with decreased disease-specific survival in ER negative, lymph node negative patients of the HER2 and TNBC molecular subtypes. Multivariable analysis confirmed MALAT1 to have independent prognostic significance in the TNBC lymph node negative patient subset (HR=2.64, 95%CI 1.35 − 5.16, p=0.005). We propose that the functional significance of MALAT1 as a metastasis driver and its potential use as a prognostic marker is most promising for those patients diagnosed with ER negative, lymph node negative breast cancer who might otherwise mistakenly be stratified to have low recurrence risk. PMID:27250026

Functional and prognostic significance of long non-coding RNA MALAT1 as a metastasis driver in ER negative lymph node negative breast cancer.

PubMed

Jadaliha, Mahdieh; Zong, Xinying; Malakar, Pushkar; Ray, Tania; Singh, Deepak K; Freier, Susan M; Jensen, Tor; Prasanth, Supriya G; Karni, Rotem; Ray, Partha S; Prasanth, Kannanganattu V

2016-06-28

MALAT1 (metastasis associated lung adenocarcinoma transcript1) is a conserved long non-coding RNA, known to regulate gene expression by modulating transcription and post-transcriptional pre-mRNA processing of a large number of genes. MALAT1 expression is deregulated in various tumors, including breast cancer. However, the significance of such abnormal expression is yet to be fully understood. In this study, we demonstrate that regulation of aggressive breast cancer cell traits by MALAT1 is not predicted solely based on an elevated expression level but is context specific. By performing loss- and gain-of-function studies, both under in vitro and in vivo conditions, we demonstrate that MALAT1 facilitates cell proliferation, tumor progression and metastasis of triple-negative breast cancer (TNBC) cells despite having a comparatively lower expression level than ER or HER2-positive breast cancer cells. Furthermore, MALAT1 regulates the expression of several cancer metastasis-related genes, but displays molecular subtype specific correlations with such genes. Assessment of the prognostic significance of MALAT1 in human breast cancer (n=1992) revealed elevated MALAT1 expression was associated with decreased disease-specific survival in ER negative, lymph node negative patients of the HER2 and TNBC molecular subtypes. Multivariable analysis confirmed MALAT1 to have independent prognostic significance in the TNBC lymph node negative patient subset (HR=2.64, 95%CI 1.35- 5.16, p=0.005). We propose that the functional significance of MALAT1 as a metastasis driver and its potential use as a prognostic marker is most promising for those patients diagnosed with ER negative, lymph node negative breast cancer who might otherwise mistakenly be stratified to have low recurrence risk.

The norepinephrine transporter and its regulation.

PubMed

Mandela, Prashant; Ordway, Gregory A

2006-04-01

For many years, the norepinephrine transporter (NET) was considered a 'static' protein that contributed to the termination of the action of norepinephrine in the synapse of noradrenergic neurons. The concept that the NET is dynamically regulated, adjusting noradrenergic transmission by changing its function and/or expression, was considered initially in the mid 1980s. Since that time, a plethora of studies demonstrate that the NET is regulated by several intracellular and extracellular signaling molecules, and that phosphorylation of the NET is a major pathway regulating its cell surface expression and thereby its function. The NET is a target of action of a number of drugs that are used long-term therapeutically or abused chronically. This has driven numerous investigations of how the NET and its function are regulated by long-term exposure to drugs. While repeated exposure to many drugs has been shown to affect NET function and expression, the intracellular mechanisms for these effects remains elusive.

Drosophila bunched integrates opposing DPP and EGF signals to set the operculum boundary.

PubMed

Dobens, L L; Peterson, J S; Treisman, J; Raftery, L A

2000-02-01

The Drosophila BMP homolog DPP can function as a morphogen, inducing multiple cell fates across a developmental field. However, it is unknown how graded levels of extracellular DPP are interpreted to organize a sharp boundary between different fates. Here we show that opposing DPP and EGF signals set the boundary for an ovarian follicle cell fate. First, DPP regulates gene expression in the follicle cells that will create the operculum of the eggshell. DPP induces expression of the enhancer trap reporter A359 and represses expression of bunched, which encodes a protein similar to the mammalian transcription factor TSC-22. Second, DPP signaling indirectly regulates A359 expression in these cells by downregulating expression of bunched. Reduced bunched function restores A359 expression in cells that lack the Smad protein MAD; ectopic expression of BUNCHED suppresses A359 expression in this region. Importantly, reduction of bunched function leads to an expansion of the operculum and loss of the collar at its boundary. Third, EGF signaling upregulates expression of bunched. We previously demonstrated that the bunched expression pattern requires the EGF receptor ligand GURKEN. Here we show that activated EGF receptor is sufficient to induce ectopic bunched expression. Thus, the balance of DPP and EGF signals sets the boundary of bunched expression. We propose that the juxtaposition of cells with high and low BUNCHED activity organizes a sharp boundary for the operculum fate.

Proof of Concept Study to Assess Fetal Gene Expression in Amniotic Fluid by NanoArray PCR

PubMed Central

Massingham, Lauren J.; Johnson, Kirby L.; Bianchi, Diana W.; Pei, Shermin; Peter, Inga; Cowan, Janet M.; Tantravahi, Umadevi; Morrison, Tom B.

2011-01-01

Microarray analysis of cell-free RNA in amniotic fluid (AF) supernatant has revealed differential fetal gene expression as a function of gestational age and karyotype. Once informative genes are identified, research moves to a more focused platform such as quantitative reverse transcriptase-PCR. Standardized NanoArray PCR (SNAP) is a recently developed gene profiling technology that enables the measurement of transcripts from samples containing reduced quantities or degraded nucleic acids. We used a previously developed SNAP gene panel as proof of concept to determine whether fetal functional gene expression could be ascertained from AF supernatant. RNA was extracted and converted to cDNA from 19 AF supernatant samples of euploid fetuses between 15 to 20 weeks of gestation, and transcript abundance of 21 genes was measured. Statistically significant differences in expression, as a function of advancing gestational age, were observed for 5 of 21 genes. ANXA5, GUSB, and PPIA showed decreasing gene expression over time, whereas CASC3 and ZNF264 showed increasing gene expression over time. Statistically significantly increased expression of MTOR and STAT2 was seen in female compared with male fetuses. This study demonstrates the feasibility of focused fetal gene expression analysis using SNAP technology. In the future, this technique could be optimized to examine specific genes instrumental in fetal organ system function, which could be a useful addition to prenatal care. PMID:21827969

Expression of BMP-2 in Vascular Endothelial Cells of Recipient May Predict Delayed Graft Function After Renal Transplantation.

PubMed

Basic-Jukic, Nikolina; Gulin, Marijana; Hudolin, Tvrtko; Kastelan, Zeljko; Katalinic, Lea; Coric, Marijana; Veda, Marija Varnai; Ivkovic, Vanja; Kes, Petar; Jelakovic, Bojan

2016-01-01

Delayed graft function (DGF) is associated with adverse outcomes after renal transplantation. Bone morphogenetic protein-2 (BMP-2) is involved in both endothelial function and immunological events. We compared expression of BMP-2 in epigastric artery of renal transplant recipients with immediate graft function (IGF) and DGF. 79 patients were included in this prospective study. Patients were divided in IGF group (64 patients) and DGF group (15 patients). BMP-2 expression in intima media (BMP2m) and endothelium (BMP2e) of epigastric artery was assessed by immunohistochemistry. Lower intensity of BMP2e staining was recorded in DGF compared to IGF. In DGF patients, 93% had no expression of BMP2e and 7% had 1st grade expression, compared to 45% and 41% in IGF group, respectively (P=0.001) (P<0.001 for no expression and P = 0.015 for 1st grade expression). Patients who had BMP2e staining positive had lower odds for DGF (OR 0.059 [0.007, 0.477]) and this remained significant even after adjustment for donor and recipient variables, cold ischemia time, and immunological matching (OR 0.038 [0.003, 0.492]). Our results demonstrate that BMP-2 expression in endothelial cells of epigastric arteries may predict development of DGF. © 2016 The Author(s) Published by S. Karger AG, Basel.

Smad4 suppresses the tumorigenesis and aggressiveness of neuroblastoma through repressing the expression of heparanase

PubMed Central

Qu, Hongxia; Zheng, Liduan; Jiao, Wanju; Mei, Hong; Li, Dan; Song, Huajie; Fang, Erhu; Wang, Xiaojing; Li, Shiwang; Huang, Kai; Tong, Qiangsong

2016-01-01

Heparanase (HPSE) is the only endo-β-D-glucuronidase that is correlated with the progression of neuroblastoma (NB), the most common extracranial malignancy in childhood. However, the mechanisms underlying HPSE expression in NB still remain largely unknown. Herein, through analyzing cis-regulatory elements and mining public microarray datasets, we identified SMAD family member 4 (Smad4) as a crucial transcription regulator of HPSE in NB. We demonstrated that Smad4 repressed the HPSE expression at the transcriptional levels in NB cells. Mechanistically, Smad4 suppressed the HPSE expression through directly binding to its promoter and repressing the lymphoid enhancer binding factor 1 (LEF1)-facilitated transcription of HPSE via physical interaction. Gain- and loss-of-function studies demonstrated that Smad4 inhibited the growth, invasion, metastasis, and angiogenesis of NB cells in vitro and in vivo. Restoration of HPSE expression prevented the NB cells from changes in these biological features induced by Smad4. In clinical NB specimens, Smad4 was under-expressed and inversely correlated with HPSE levels, while LEF1 was highly expressed and positively correlated with HPSE expression. Patients with high Smad4 expression, low LEF1 or HPSE levels had greater survival probability. These results demonstrate that Smad4 suppresses the tumorigenesis and aggressiveness of NB through repressing the HPSE expression. PMID:27595937

Expression profiles of urbilaterian genes uniquely shared between honey bee and vertebrates

PubMed Central

Matsui, Toshiaki; Yamamoto, Toshiyuki; Wyder, Stefan; Zdobnov, Evgeny M; Kadowaki, Tatsuhiko

2009-01-01

Background Large-scale comparison of metazoan genomes has revealed that a significant fraction of genes of the last common ancestor of Bilateria (Urbilateria) is lost in each animal lineage. This event could be one of the underlying mechanisms involved in generating metazoan diversity. However, the present functions of these ancient genes have not been addressed extensively. To understand the functions and evolutionary mechanisms of such ancient Urbilaterian genes, we carried out comprehensive expression profile analysis of genes shared between vertebrates and honey bees but not with the other sequenced ecdysozoan genomes (honey bee-vertebrate specific, HVS genes) as a model. Results We identified 30 honey bee and 55 mouse HVS genes. Many HVS genes exhibited tissue-selective expression patterns; intriguingly, the expression of 60% of honey bee HVS genes was found to be brain enriched, and 24% of mouse HVS genes were highly expressed in either or both the brain and testis. Moreover, a minimum of 38% of mouse HVS genes demonstrated neuron-enriched expression patterns, and 62% of them exhibited expression in selective brain areas, particularly the forebrain and cerebellum. Furthermore, gene ontology (GO) analysis of HVS genes predicted that 35% of genes are associated with DNA transcription and RNA processing. Conclusion These results suggest that HVS genes include genes that are biased towards expression in the brain and gonads. They also demonstrate that at least some of Urbilaterian genes retained in the specific animal lineage may be selectively maintained to support the species-specific phenotypes. PMID:19138430

Expression profiles of urbilaterian genes uniquely shared between honey bee and vertebrates.

PubMed

Matsui, Toshiaki; Yamamoto, Toshiyuki; Wyder, Stefan; Zdobnov, Evgeny M; Kadowaki, Tatsuhiko

2009-01-12

Large-scale comparison of metazoan genomes has revealed that a significant fraction of genes of the last common ancestor of Bilateria (Urbilateria) is lost in each animal lineage. This event could be one of the underlying mechanisms involved in generating metazoan diversity. However, the present functions of these ancient genes have not been addressed extensively. To understand the functions and evolutionary mechanisms of such ancient Urbilaterian genes, we carried out comprehensive expression profile analysis of genes shared between vertebrates and honey bees but not with the other sequenced ecdysozoan genomes (honey bee-vertebrate specific, HVS genes) as a model. We identified 30 honey bee and 55 mouse HVS genes. Many HVS genes exhibited tissue-selective expression patterns; intriguingly, the expression of 60% of honey bee HVS genes was found to be brain enriched, and 24% of mouse HVS genes were highly expressed in either or both the brain and testis. Moreover, a minimum of 38% of mouse HVS genes demonstrated neuron-enriched expression patterns, and 62% of them exhibited expression in selective brain areas, particularly the forebrain and cerebellum. Furthermore, gene ontology (GO) analysis of HVS genes predicted that 35% of genes are associated with DNA transcription and RNA processing. These results suggest that HVS genes include genes that are biased towards expression in the brain and gonads. They also demonstrate that at least some of Urbilaterian genes retained in the specific animal lineage may be selectively maintained to support the species-specific phenotypes.

A functional role for CD28 costimulation in tumor recognition by single-chain receptor-modified T cells.

PubMed

Moeller, Maria; Haynes, Nicole M; Trapani, Joseph A; Teng, Michele W L; Jackson, Jacob T; Tanner, Jane E; Cerutti, Loretta; Jane, Stephen M; Kershaw, Michael H; Smyth, Mark J; Darcy, Phillip K

2004-05-01

T cells engineered to express single-chain antibody receptors that incorporate TCR-zeta and cluster designation (CD)28 signaling domains (scFv-alpha-erbB2-CD28-zeta) can be redirected in vivo to cancer cells that lack triggering costimulatory molecules. To assess the contribution of CD28 signaling to the function of the scFv-CD28-zeta receptor, we expressed a series of mutated scFv-CD28-zeta receptors directed against erbB2. Residues known to be critical for CD28 signaling were mutated from tyrosine to phenylalanine at position 170 or proline to alanine at positions 187 and 190. Primary mouse T cells expressing either of the mutant receptors demonstrated impaired cytokine (IFN-gamma and GM-CSF) production and decreased proliferation after antigen ligation in vitro and decreased antitumor efficacy in vivo compared with T cells expressing the wild-type scFv-CD28-zeta receptor, suggesting a key signaling role for the CD28 component of the scFv-CD28-zeta receptor. Importantly, cell surface expression, binding capacity and cytolytic activity mediated by the scFv-CD28-zeta receptor were not diminished by either mutation. Overall, this study has definitively demonstrated a functional role for the CD28 component of the scFv-CD28-zeta receptor and has shown that incorporation of costimulatory activity in chimeric scFv receptors is a powerful approach for improving adoptive cancer immunotherapy.

A Viral Receptor Complementation Strategy to Overcome CAV-2 Tropism for Efficient Retrograde Targeting of Neurons.

PubMed

Li, Shu-Jing; Vaughan, Alexander; Sturgill, James Fitzhugh; Kepecs, Adam

2018-06-06

Retrogradely transported neurotropic viruses enable genetic access to neurons based on their long-range projections and have become indispensable tools for linking neural connectivity with function. A major limitation of viral techniques is that they rely on cell-type-specific molecules for uptake and transport. Consequently, viruses fail to infect variable subsets of neurons depending on the complement of surface receptors expressed (viral tropism). We report a receptor complementation strategy to overcome this by potentiating neurons for the infection of the virus of interest-in this case, canine adenovirus type-2 (CAV-2). We designed AAV vectors for expressing the coxsackievirus and adenovirus receptor (CAR) throughout candidate projection neurons. CAR expression greatly increased retrograde-labeling rates, which we demonstrate for several long-range projections, including some resistant to other retrograde-labeling techniques. Our results demonstrate a receptor complementation strategy to abrogate endogenous viral tropism and thereby facilitate efficient retrograde targeting for functional analysis of neural circuits. Copyright © 2018 Elsevier Inc. All rights reserved.

Improving Written Expression of Seventh Grade Mildly Intellectually Disabled Students Utilizing a Basal Reading Program, Journal Writing and Computer Applications.

ERIC Educational Resources Information Center

Wimberly, Sabrenai R.

A practicum was designed to increase mildly intellectually disabled students' written communication skills by demonstrating functional written expression skills in daily assignments and in social communication. A sequenced reading and language program with the integration of journal writing and computer applications was utilized. Seventh- and…

Functional expression of cysteinyl leukotriene receptors on human platelets.

PubMed

Hasegawa, Shunji; Ichiyama, Takashi; Hashimoto, Kunio; Suzuki, Yasuo; Hirano, Reiji; Fukano, Reiji; Furukawa, Susumu

2010-01-01

Normal peripheral blood leukocytes, such as basophils, eosinophils, B lymphocytes and monocytes/macrophages, have a cysteinyl leukotriene 1 (CysLT1) receptor, while the cysteinyl leukotriene 2 (CysLT2) receptor is expressed in cardiac Purkinje cells, endothelium, brain and leukocytes. However, it is unknown whether or not platelets express the CysLT1 or CysLT2 receptor. In this study we identify and characterize the biological function of the CysLT receptor of human platelets. We determined the CysLT1 or CysLT2 receptor mRNA expression in normal human platelets by RT-PCR and determined protein expression by Western blotting and flow cytometry. Moreover, we examined the effect of cysteinyl leukotrienes (CysLTs) in platelets on the induction of RANTES (Regulated on Activation, Normal T Expressed, and presumably Secreted). We also investigated whether the CysLT1 receptor antagonist pranlukast inhibits CysLT-induced RANTES release. In conclusion, we showed the functional expression of CysLT receptors on human platelets and demonstrated that CysLTs induced the release of significant amounts of RANTES, which suggests a novel role for human platelets in CysLT-mediated allergic inflammation.

Human T lymphocytes express N-methyl-D-aspartate receptors functionally active in controlling T cell activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miglio, Gianluca; Varsaldi, Federica; Lombardi, Grazia

2005-12-30

The aim of this study was to investigate the expression and the functional role of N-methyl-D-aspartate (NMDA) receptors in human T cells. RT-PCR analysis showed that human resting peripheral blood lymphocytes (PBL) and Jurkat T cells express genes encoding for both NR1 and NR2B subunits: phytohemagglutinin (PHA)-activated PBL also expresses both these genes and the NR2A and NR2D genes. Cytofluorimetric analysis showed that NR1 expression increases as a consequence of PHA (10 {mu}g/ml) treatment. D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5), and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine [(+)-MK 801], competitive and non-competitive NMDA receptor antagonists, respectively, inhibited PHA-induced T cell proliferation, whereas they did not affect IL-2 (10more » U/ml)-induced proliferation of PHA blasts. These effects were due to the prevention of T cell activation (inhibition of cell aggregate formation and CD25 expression), but not to cell cycle arrest or death. These results demonstrate that human T lymphocytes express NMDA receptors, which are functionally active in controlling cell activation.« less

Hyperglycemia and Diabetes Downregulate the Functional Expression of TRPV4 Channels in Retinal Microvascular Endothelium

PubMed Central

Monaghan, Kevin; McNaughten, Jennifer; McGahon, Mary K.; Kelly, Catriona; Kyle, Daniel; Yong, Phaik Har

2015-01-01

Retinal endothelial cell dysfunction is believed to play a key role in the etiology and pathogenesis of diabetic retinopathy. Numerous studies have shown that TRPV4 channels are critically involved in maintaining normal endothelial cell function. In the current paper, we demonstrate that TRPV4 is functionally expressed in the endothelium of the retinal microcirculation and that both channel expression and activity is downregulated by hyperglycaemia. Quantitative PCR and immunostaining demonstrated molecular expression of TRPV4 in cultured bovine retinal microvascular endothelial cells (RMECs). Functional TRPV4 activity was assessed in cultured RMECs from endothelial Ca2+-responses recorded using fura-2 microfluorimetry and electrophysiological recordings of membrane currents. The TRPV4 agonist 4α-phorbol 12,13-didecanoate (4-αPDD) increased [Ca2+]i in RMECs and this response was largely abolished using siRNA targeted against TRPV4. These Ca2+-signals were completely inhibited by removal of extracellular Ca2+, confirming their dependence on influx of extracellular Ca2+. The 4-αPDD Ca2+-response recorded in the presence of cyclopiazonic acid (CPA), which depletes the intracellular stores preventing any signal amplification through store release, was used as a measure of Ca2+-influx across the cell membrane. This response was blocked by HC067047, a TRPV4 antagonist. Under voltage clamp conditions, the TRPV4 agonist GSK1016790A stimulated a membrane current, which was again inhibited by HC067047. Following incubation with 25mM D-glucose TRPV4 expression was reduced in comparison with RMECs cultured under control conditions, as were 4αPDD-induced Ca2+-responses in the presence of CPA and ion currents evoked by GSK1016790A. Molecular expression of TRPV4 in the retinal vascular endothelium of 3 months’ streptozotocin-induced diabetic rats was also reduced in comparison with that in age-matched controls. We conclude that hyperglycaemia and diabetes reduce the molecular and functional expression of TRPV4 channels in retinal microvascular endothelial cells. These changes may contribute to diabetes induced endothelial dysfunction and retinopathy. PMID:26047504

Downregulation of miR‑135a predicts poor prognosis in acute myeloid leukemia and regulates leukemia progression via modulating HOXA10 expression.

PubMed

Xu, Hongwei; Wen, Quan

2018-05-23

MicroRNA‑135a (miR‑135a) has been shown to exert important roles in various human cancer types, such as glioblastoma, thyroid carcinoma and renal carcinoma. However, the function of miR‑135a in acute myeloid leukemia (AML) remains largely unknown. In the present study, it was demonstrated that miR‑135a expression was significantly downregulated in AML cells compared with normal control cells. Furthermore, the downregulation of miR‑135a in patients with AML predicted poor prognosis. Through functional experiments, overexpression of miR‑135a was demonstrated to significantly inhibit the proliferation and cell cycle of AML cells, while it promoted cellular apoptosis. miR‑135a directly targeted HOXA10 in AML cells. miR‑135a overexpression significantly suppressed the mRNA and protein levels of HOXA10 in AML cells. Moreover, there was an inverse association between miR‑135a expression and HOXA10 level in AML samples. Additionally, by ectopic expression of HOXA10, restoration of HOXA10 significantly abolished the effects of miR‑135a overexpression on AML cell proliferation, cell cycle and apoptosis. In conclusion, the present study demonstrated that miR‑135a serves as a tumor suppressor in AML by targeting HOXA10, and miR‑135a may be a promising prognostic biomarker for AML patients.

Cobalt Chloride Upregulates Impaired HIF-1α Expression to Restore Sevoflurane Post-conditioning-Dependent Myocardial Protection in Diabetic Rats.

PubMed

Wu, Jianjiang; Yang, Long; Xie, Peng; Yu, Jin; Yu, Tian; Wang, Haiying; Maimaitili, Yiliyaer; Wang, Jiang; Ma, Haiping; Yang, Yining; Zheng, Hong

2017-01-01

Previous studies from our group have demonstrated that sevoflurane post-conditioning (SPC) protects against myocardial ischemia reperfusion injury via elevating the intranuclear expression of hypoxia inducible factor-1 alpha (HIF-1α). However, diabetic SPC is associated with decreased myocardial protection and disruption of the HIF-1 signaling pathway. Previous studies have demonstrated that cobalt chloride (CoCl 2 ) can upregulate HIF-1α expression under diabetic conditions, but whether myocardial protection by SPC can be restored afterward remains unclear. We established a rat model of type 2 diabetes and a Langendorff isolated heart model of ischemia-reperfusion injury. Prior to reperfusion, 2.4% sevoflurane was used as a post-conditioning treatment. The diabetic rats were treated with CoCl 2 24 h before the experiment. At the end of reperfusion, tests were performed to assess myocardial function, infarct size, mitochondrial morphology, nitric oxide (NO), Mitochondrial reactive oxygen species (ROS), mitochondrial respiratory function and enzyme activity, HIF-1α, vascular endothelial growth factor (VEGF) and endothelial NO synthase (eNOS) protein levels. In addition, myocardial protection by SPC was monitored after the blood glucose levels were lowered by insulin. The diabetic state was associated with deficient SPC protection and decreased HIF-1α expression. After treating the diabetic rats with CoCl 2 , SPC significantly upregulated the expression of HIF-1α, VEGF and eNOS, which markedly improved cardiac function, NO, mitochondrial respiratory function, and enzyme activity and decreased the infarction areas and ROS. In addition, these effects were not influenced by blood glucose levels. This study proved that CoCl 2 activates the HIF-1α signaling pathway, which restores SPC-dependent myocardial protection under diabetic conditions, and the protective effects of SPC were independent of blood glucose levels.

Laminin γ3 plays an important role in retinal lamination, photoreceptor organisation and ganglion cell differentiation.

PubMed

Dorgau, Birthe; Felemban, Majed; Sharpe, Alexander; Bauer, Roman; Hallam, Dean; Steel, David H; Lindsay, Susan; Mellough, Carla; Lako, Majlinda

2018-05-23

Laminins are heterotrimeric glycoproteins of the extracellular matrix. Eleven different laminin chains have been identified in vertebrates. They are ubiquitously expressed in the human body, with a distinct tissue distribution. Laminin expression in neural retina and their functional role during human retinogenesis is still unknown. This study investigated the laminin expression in human developing and adult retina, showing laminin α1, α5, β1, β2 and γ1 to be predominantly expressed in Bruch's membrane and the inner limiting membrane. Laminin-332 and laminin γ3 expression were mainly observed in the neural retina during retinal histogenesis. These expression patterns were largely conserved in pluripotent stem cell-derived retinal organoids. Blocking of laminin γ3 function in retinal organoids resulted in the disruption of laminar organisation and synapse formation, the loss of photoreceptor organisation and retinal ganglion cells. Our data demonstrate a unique temporal and spatial expression for laminins and reveal a novel role for laminin γ3 during human retinogenesis.

Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs.

PubMed

Yin, Linlin; Maddison, Lisette A; Li, Mingyu; Kara, Nergis; LaFave, Matthew C; Varshney, Gaurav K; Burgess, Shawn M; Patton, James G; Chen, Wenbiao

2015-06-01

Determining the mechanism of gene function is greatly enhanced using conditional mutagenesis. However, generating engineered conditional alleles is inefficient and has only been widely used in mice. Importantly, multiplex conditional mutagenesis requires extensive breeding. Here we demonstrate a system for one-generation multiplex conditional mutagenesis in zebrafish (Danio rerio) using transgenic expression of both cas9 and multiple single guide RNAs (sgRNAs). We describe five distinct zebrafish U6 promoters for sgRNA expression and demonstrate efficient multiplex biallelic inactivation of tyrosinase and insulin receptor a and b, resulting in defects in pigmentation and glucose homeostasis. Furthermore, we demonstrate temporal and tissue-specific mutagenesis using transgenic expression of Cas9. Heat-shock-inducible expression of cas9 allows temporal control of tyr mutagenesis. Liver-specific expression of cas9 disrupts insulin receptor a and b, causing fasting hypoglycemia and postprandial hyperglycemia. We also show that delivery of sgRNAs targeting ascl1a into the eye leads to impaired damage-induced photoreceptor regeneration. Our findings suggest that CRISPR/Cas9-based conditional mutagenesis in zebrafish is not only feasible but rapid and straightforward. Copyright © 2015 by the Genetics Society of America.

Increased TRPV4 expression in urinary bladder and lumbosacral dorsal root ganglia in mice with chronic overexpression of NGF in urothelium.

PubMed

Girard, Beatrice M; Merrill, Liana; Malley, Susan; Vizzard, Margaret A

2013-10-01

Transient receptor potential vanilloid (TRPV) family member 4 (TRPV4) expression has been demonstrated in urothelial cells and dorsal root ganglion (DRG) neurons, and roles in normal micturition reflexes as well as micturition dysfunction have been suggested. TRP channel expression and function is dependent upon target tissue expression of growth factors. These studies expand upon the target tissue dependence of TRPV4 expression in the urinary bladder and lumbosacral DRG using a recently characterized transgenic mouse model with chronic overexpression of nerve growth factor (NGF-OE) in the urothelium. Immunohistochemistry with image analyses, real-time quantitative polymerase chain reaction, and Western blotting were used to determine TRPV4 protein and transcript expression in the urinary bladder (urothelium + suburothelium, detrusor) and lumbosacral DRG from littermate wild-type (WT) and NGF-OE mice. Antibody specificity controls were performed in TRPV4(-/-) mice. TRPV4 transcript and protein expression was significantly (p ≤ 0.001) increased in the urothelium + suburothelium and suburothelial nerve plexus of the urinary bladder and in small- and medium-sized lumbosacral (L1, L2, L6-S1) DRG cells from NGF-OE mice compared to littermate WT mice. NGF-OE mice exhibit significant (p ≤ 0.001) increases in NGF transcript and protein in the urothelium + suburothelium and lumbosacral DRG. These studies demonstrate regulation of TRPV4 expression by NGF in lower urinary tract tissues. Ongoing studies are characterizing the functional roles of TRPV4 expression in the sensory limb (DRG, urothelium) of the micturition reflex.

Transplant of insulin-like growth factor-1 expressing bone marrow stem cells improves functional regeneration of injured rat uterus by NF-κB pathway.

PubMed

Wang, Lei; Yang, Mengnan; Jin, Minfei; Wu, Yuelin; Zheng, Tao; Gu, Shengyi; Hua, Xiaolin

2018-05-01

To investigate the potential beneficial effect of insulin-like growth factor-1 (IGF-1) in BMSC transplantation therapy of uterus injury and the underlying molecular mechanisms, rat BMSCs were isolated and cultured. The relative expressions of IGF-1 and IL-10 were determined by RT-PCR and immunoblotting. The secretory IL-10 and released E2 were measured using ELISA kits. The relative vWF and α-SMA expressions were determined by immunohistochemistry. The direct binding of NF-κB subunit p50 with IL-10 promoter was analysed by chromatin immunoprecipitation assay. The regulation of IL-10 expression by p50 was interrogated by luciferase reporter assay. Our data demonstrated that IGF-1 expression in BMSCs induced IL-10 expression and secretion, which was further enhanced by E2-PLGA. IGF-1 overexpression improved BMSCs transplantation therapy in rat uterus injury. We further demonstrated that both inhibition and knockdown of p50 abolished IGF-1-induced expression and secretion of IL-10 in BMSCs, which consequently compromised the IGF-1 conferred therapeutic benefits against uterus injury. Furthermore, we elucidated that p50 regulated IL-10 expression via direct association with its promoter. Our data suggested that transplantation of IGF-1 overexpressing BMSCs improved functional regeneration of injured uterus by inducing IL-10 expression and secretion via activation of NF-κB signalling. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Ectopic expression of a novel CD22 splice-variant regulates survival and proliferation in malignant T cells from cutaneous T cell lymphoma (CTCL) patients

PubMed Central

Bagdonaite, Ieva; Wandall, Hans H.; Litvinov, Ivan V.; Nastasi, Claudia; Becker, Jürgen C.; Dabelsteen, Sally; Geisler, Carsten; Bonefeld, Charlotte M.; Zhang, Qian; Wasik, Mariusz A.; Zhou, Youwen; Sasseville, Denis; Ødum, Niels; Woetmann, Anders

2015-01-01

CD22 is a member of the Sialic acid-binding Ig-like lectin (Siglec) family of lectins described to be exclusively present in B lymphocytes and B cell-derived neoplasms. Here, we describe a novel splice form of CD22 (designated CD22ΔN), which lacks the N-terminal domain as demonstrated by exon-specific RT-PCR and differential recognition by anti-CD22 antibodies. Importantly, CD22ΔN mRNA is expressed in skin lesions from 39 out of 60 patients with cutaneous T cell lymphoma (CTCL), whereas few patients (6 out of 60) expresses full-length, wild type CD22 (CD22wt). In addition, IHC staining of tumor biopsies confirmed the expression of CD22 in CD4+ T cells. Moreover, four out of four malignant T cell lines express CD22: Two cell lines express CD22ΔN (MyLa2059 and PB2B) and two express CD22wt (MAC-1 and MAC-2A). siRNA-mediated silencing of CD22 impairs proliferation and survival of malignant T cells, demonstrating a functional role for both CD22ΔN and CD22wt in these cells. In conclusion, we provide the first evidence for an ectopic expression of CD22 and a novel splice variant regulating malignant proliferation and survival in CTCL. Analysis of expression and function of CD22 in cutaneous lymphomas may form the basis for development of novel targeted therapies for our patients. PMID:25957418

Ectopic expression of a novel CD22 splice-variant regulates survival and proliferation in malignant T cells from cutaneous T cell lymphoma (CTCL) patients.

PubMed

Bagdonaite, Ieva; Wandall, Hans H; Litvinov, Ivan V; Nastasi, Claudia; Becker, Jürgen C; Dabelsteen, Sally; Geisler, Carsten; Bonefeld, Charlotte M; Zhang, Qian; Wasik, Mariusz A; Zhou, Youwen; Sasseville, Denis; Ødum, Niels; Woetmann, Anders

2015-06-10

CD22 is a member of the Sialic acid-binding Ig-like lectin (Siglec) family of lectins described to be exclusively present in B lymphocytes and B cell-derived neoplasms. Here, we describe a novel splice form of CD22 (designated CD22∆N), which lacks the N-terminal domain as demonstrated by exon-specific RT-PCR and differential recognition by anti-CD22 antibodies. Importantly, CD22∆N mRNA is expressed in skin lesions from 39 out of 60 patients with cutaneous T cell lymphoma (CTCL), whereas few patients (6 out of 60) expresses full-length, wild type CD22 (CD22wt). In addition, IHC staining of tumor biopsies confirmed the expression of CD22 in CD4+ T cells. Moreover, four out of four malignant T cell lines express CD22: Two cell lines express CD22∆N (MyLa2059 and PB2B) and two express CD22wt (MAC-1 and MAC-2A). siRNA-mediated silencing of CD22 impairs proliferation and survival of malignant T cells, demonstrating a functional role for both CD22∆N and CD22wt in these cells.In conclusion, we provide the first evidence for an ectopic expression of CD22 and a novel splice variant regulating malignant proliferation and survival in CTCL. Analysis of expression and function of CD22 in cutaneous lymphomas may form the basis for development of novel targeted therapies for our patients.

INCREASED TRPV4 EXPRESSION IN URINARY BLADDER AND LUMBOSACRAL DORSAL ROOT GANGLIA IN MICE WITH CHRONIC OVEREXPRESSION OF NGF IN UROTHELIUM

PubMed Central

Girard, Beatrice M.; Merrill, Liana; Malley, Susan; Vizzard, Margaret A.

2013-01-01

Transient receptor potential vanilloid (TRPV) family member 4 (TRPV4) expression has been demonstrated in urothelial cells and dorsal root ganglion (DRG) neurons and roles in normal micturition reflexes as well as micturition dysfunction have been suggested. TRP channel expression and function is dependent upon target tissue expression of growth factors. These studies expand upon the target tissue dependence of TRPV4 expression in the urinary bladder and lumbosacral DRG using a recently characterized transgenic mouse model with chronic overexpression of nerve growth factor (NGF-OE) in the urothelium. Immunohistochemistry with image analyses, real-time quantitative polymerase chain reaction (Q-PCR) and western blotting were used to determine TRPV4 protein and transcript expression in the urinary bladder (urothelium + suburothelium, detrusor) and lumbosacral DRG from littermate wildtype (WT) and NGF-OE mice. Antibody specificity controls were performed in TRPV4-/- mice. TRPV4 transcript and protein expression was significantly (p ≤ 0.001) increased in the urothelium + suburothelium and suburothelial nerve plexus of the urinary bladder and in small- and medium-sized lumbosacral (L1, L2, L6-S1) DRG cells from NGF-OE mice compared to littermate WT mice. NGF-OE mice exhibit significant (p ≤ 0.001) increases in NGF transcript and protein in the urothelium + suburothelium and lumbosacral DRG. These studies demonstrate regulation of TRPV4 expression by NGF in lower urinary tract tissues. Ongoing studies are characterizing the functional roles of TRPV4 expression in the sensory limb (DRG, urothelium) of the micturition reflex. PMID:23690258

An essential role for LPA signalling in telencephalon development.

PubMed

Geach, Timothy J; Faas, Laura; Devader, Christelle; Gonzalez-Cordero, Anai; Tabler, Jacqueline M; Brunsdon, Hannah; Isaacs, Harry V; Dale, Leslie

2014-02-01

Lysophosphatidic acid (LPA) has wide-ranging effects on many different cell types, acting through G-protein-coupled receptors such as LPAR6. We show that Xenopus lpar6 is expressed from late blastulae and is enriched in the mesoderm and dorsal ectoderm of early gastrulae. Expression in gastrulae is an early response to FGF signalling. Transcripts for lpar6 are enriched in the neural plate of Xenopus neurulae and loss of function caused forebrain defects, with reduced expression of telencephalic markers (foxg1, emx1 and nkx2-1). Midbrain (en2) and hindbrain (egr2) markers were unaffected. Foxg1 expression requires LPAR6 within ectoderm and not mesoderm. Head defects caused by LPAR6 loss of function were enhanced by co-inhibiting FGF signalling, with defects extending into the hindbrain (en2 and egr2 expression reduced). This is more severe than expected from simple summation of individual defects, suggesting that LPAR6 and FGF have overlapping or partially redundant functions in the anterior neural plate. We observed similar defects in forebrain development in loss-of-function experiments for ENPP2, an enzyme involved in the synthesis of extracellular LPA. Our study demonstrates a role for LPA in early forebrain development.

Functional Expression of Two Neuronal Nicotinic Acetylcholine Receptors from cDNA Clones Identifies a Gene Family

NASA Astrophysics Data System (ADS)

Boulter, Jim; Connolly, John; Deneris, Evan; Goldman, Dan; Heinemann, Steven; Patrick, Jim

1987-11-01

A family of genes coding for proteins homologous to the α subunit of the muscle nicotinic acetylcholine receptor has been identified in the rat genome. These genes are transcribed in the central and peripheral nervous systems in areas known to contain functional nicotinic receptors. In this paper, we demonstrate that three of these genes, which we call alpha3, alpha4, and beta2, encode proteins that form functional nicotinic acetylcholine receptors when expressed in Xenopus oocytes. Oocytes expressing either alpha3 or alpha4 protein in combination with the beta2 protein produced a strong response to acetylcholine. Oocytes expressing only the alpha4 protein gave a weak response to acetylcholine. These receptors are activated by acetylcholine and nicotine and are blocked by Bungarus toxin 3.1. They are not blocked by α -bungarotoxin, which blocks the muscle nicotinic acetylcholine receptor. Thus, the receptors formed by the alpha3, alpha4, and beta2 subunits are pharmacologically similar to the ganglionic-type neuronal nicotinic acetylcholine receptor. These results indicate that the alpha3, alpha4, and beta2 genes encode functional nicotinic acetylcholine receptor subunits that are expressed in the brain and peripheral nervous system.

Facial emotion recognition, socio-occupational functioning and expressed emotions in schizophrenia versus bipolar disorder.

PubMed

Thonse, Umesh; Behere, Rishikesh V; Praharaj, Samir Kumar; Sharma, Podila Sathya Venkata Narasimha

2018-06-01

Facial emotion recognition deficits have been consistently demonstrated in patients with severe mental disorders. Expressed emotion is found to be an important predictor of relapse. However, the relationship between facial emotion recognition abilities and expressed emotions and its influence on socio-occupational functioning in schizophrenia versus bipolar disorder has not been studied. In this study we examined 91 patients with schizophrenia and 71 with bipolar disorder for psychopathology, socio occupational functioning and emotion recognition abilities. Primary caregivers of 62 patients with schizophrenia and 49 with bipolar disorder were assessed on Family Attitude Questionnaire to assess their expressed emotions. Patients of schizophrenia and bipolar disorder performed similarly on the emotion recognition task. Patients with schizophrenia group experienced higher critical comments and had a poorer socio-occupational functioning as compared to patients with bipolar disorder. Poorer socio-occupational functioning in patients with schizophrenia was significantly associated with greater dissatisfaction in their caregivers. In patients with bipolar disorder, poorer emotion recognition scores significantly correlated with poorer adaptive living skills and greater hostility and dissatisfaction in their caregivers. The findings of our study suggest that emotion recognition abilities in patients with bipolar disorder are associated with negative expressed emotions leading to problems in adaptive living skills. Copyright © 2018 Elsevier B.V. All rights reserved.

Functional Erythropoietin Autocrine Loop in Melanoma

PubMed Central

Kumar, Suresh M.; Acs, Geza; Fang, Dong; Herlyn, Meenhard; Elder, David E.; Xu, Xiaowei

2005-01-01

Although erythropoietin (Epo) is a known stimulator of erythropoiesis, recent evidence suggests that its biological functions are not confined to hematopoietic cells. To elucidate the role of Epo and erythropoietin receptor (EpoR) in melanoma, we examined the expression and function of these proteins in melanocytes and melanoma cells. We found increased expression of Epo in melanoma cells compared to melanocyte in vitro. EpoR was also strongly expressed in all of the melanoma cell lines and two of the three melanocyte cell lines examined. Epo expression was significantly higher in melanoma than in benign nevi as determined by immunohistochemistry. Although melanoma cells secreted Epo in normoxic condition in vitro, hypoxia and CoCl2 treatment increased Epo secretion. EpoR in melanoma cells was functional, because exogenous Epo increased melanoma resistance to hypoxic stress, pretreatment of melanoma cells with Epo significantly increased resistance to dacarbazine treatment, and Epo increased the phosphorylation of EpoR, RAF, and MEK. In conclusion, we demonstrated constitutive expression of Epo and EpoR as well as autonomous secretion of Epo by melanoma cells, indicating a novel autocrine loop of Epo in melanoma. The results suggest that the autocrine and paracrine functions of Epo might play a role in malignant transformation of melanocytes and in the survival of melanoma cells in hypoxia and other adverse conditions. PMID:15743794

Neutralization of Bacterial YoeBSpn Toxicity and Enhanced Plant Growth in Arabidopsis thaliana via Co-Expression of the Toxin-Antitoxin Genes

PubMed Central

Abu Bakar, Fauziah; Yeo, Chew Chieng; Harikrishna, Jennifer Ann

2016-01-01

Bacterial toxin-antitoxin (TA) systems have various cellular functions, including as part of the general stress response. The genome of the Gram-positive human pathogen Streptococcus pneumoniae harbors several putative TA systems, including yefM-yoeBSpn, which is one of four systems that had been demonstrated to be biologically functional. Overexpression of the yoeBSpn toxin gene resulted in cell stasis and eventually cell death in its native host, as well as in Escherichia coli. Our previous work showed that induced expression of a yoeBSpn toxin-Green Fluorescent Protein (GFP) fusion gene apparently triggered apoptosis and was lethal in the model plant, Arabidopsis thaliana. In this study, we investigated the effects of co-expression of the yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic A. thaliana. When co-expressed in Arabidopsis, the YefMSpn antitoxin was found to neutralize the toxicity of YoeBSpn-GFP. Interestingly, the inducible expression of both yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic hybrid Arabidopsis resulted in larger rosette leaves and taller plants with a higher number of inflorescence stems and increased silique production. To our knowledge, this is the first demonstration of a prokaryotic antitoxin neutralizing its cognate toxin in plant cells. PMID:27104531

Spatial and temporal expression of the Grainyhead-like transcription factor family during murine development.

PubMed

Auden, Alana; Caddy, Jacinta; Wilanowski, Tomasz; Ting, Stephen B; Cunningham, John M; Jane, Stephen M

2006-10-01

The Drosophila transcription factor Grainyhead (grh) is expressed in ectoderm-derived tissues where it regulates several key developmental events including cuticle formation, tracheal elongation and dorsal closure. Our laboratory has recently identified three novel mammalian homologues of the grh gene, Grainyhead-like 1, -2 and -3 (Grhl1-3) that rewrite the phylogeny of this family. Using gene targeting in mice, we have shown that Grhl3 is essential for neural tube closure, skin barrier formation and wound healing. Despite their extensive sequence homology, Grhl1 and Grhl2 are unable to compensate for loss of Grhl3 in these developmental processes. To explore this lack of redundancy, and to gain further insights into the functions of this gene family in mammalian development we have performed an extensive in situ hybridisation analysis. We demonstrate that, although all three Grhl genes are highly expressed in the developing epidermis, they display subtle differences in the timing and level of expression. Surprisingly, we also demonstrate differential expression patterns in non-ectoderm-derived tissues, including the heart, the lung, and the metanephric kidney. These findings expand our understanding of the unique role of Grhl3 in neurulation and epidermal morphogenesis, and provide a focus for further functional analysis of the Grhl genes during mouse embryogenesis.

Murine cell glycolipids customization by modular expression of glycosyltransferases.

PubMed

Cid, Emili; Yamamoto, Miyako; Buschbeck, Marcus; Yamamoto, Fumiichiro

2013-01-01

Functional analysis of glycolipids has been hampered by their complex nature and combinatorial expression in cells and tissues. We report an efficient and easy method to generate cells with specific glycolipids. In our proof of principle experiments we have demonstrated the customized expression of two relevant glycosphingolipids on murine fibroblasts, stage-specific embryonic antigen 3 (SSEA-3), a marker for stem cells, and Forssman glycolipid, a xenoantigen. Sets of genes encoding glycosyltansferases were transduced by viral infection followed by multi-color cell sorting based on coupled expression of fluorescent proteins.

Cloning and expression of prion protein encoding gene of flounder ( Paralichthys olivaceus)

NASA Astrophysics Data System (ADS)

Zhang, Zhiwen; Sun, Xiuqin; Zhang, Jinxing; Zan, Jindong

2008-02-01

The prion protein (PrP) encoding gene of flounder ( Paralichthys olivaceus) was cloned. It was not interrupted by an intron. This gene has two promoters in its 5' upstream, indicating that its transcription may be intensive, and should have an important function. It was expressed in all 14 tissues tested, demonstrating that it is a house-keeping gene. Its expression in digestion and reproduction systems implies that the possible prions of fish may transfer horizontally.

Intercellular adhesion molecule-1 expression by skeletal muscle cells augments myogenesis.

PubMed

Goh, Qingnian; Dearth, Christopher L; Corbett, Jacob T; Pierre, Philippe; Chadee, Deborah N; Pizza, Francis X

2015-02-15

We previously demonstrated that the expression of intercellular adhesion molecule-1 (ICAM-1) by skeletal muscle cells after muscle overload contributes to ensuing regenerative and hypertrophic processes in skeletal muscle. The objective of the present study is to reveal mechanisms through which skeletal muscle cell expression of ICAM-1 augments regenerative and hypertrophic processes of myogenesis. This was accomplished by genetically engineering C2C12 myoblasts to stably express ICAM-1, and by inhibiting the adhesive and signaling functions of ICAM-1 through the use of a neutralizing antibody or cell penetrating peptide, respectively. Expression of ICAM-1 by cultured skeletal muscle cells augmented myoblast-myoblast adhesion, myotube formation, myonuclear number, myotube alignment, myotube-myotube fusion, and myotube size without influencing the ability of myoblasts to proliferate or differentiate. ICAM-1 augmented myotube formation, myonuclear accretion, and myotube alignment through a mechanism involving adhesion-induced activation of ICAM-1 signaling, as these dependent measures were reduced via antibody and peptide inhibition of ICAM-1. The adhesive and signaling functions of ICAM-1 also facilitated myotube hypertrophy through a mechanism involving myotube-myotube fusion, protein synthesis, and Akt/p70s6k signaling. Our findings demonstrate that ICAM-1 expression by skeletal muscle cells augments myogenesis, and establish a novel mechanism through which the inflammatory response facilitates growth processes in skeletal muscle. Copyright © 2014 Elsevier Inc. All rights reserved.

Intercellular Adhesion Molecule-1 Expression by Skeletal Muscle Cells Augments Myogenesis

PubMed Central

Goh, Qingnian; Dearth, Christopher L.; Corbett, Jacob T.; Pierre, Philippe; Chadee, Deborah N.; Pizza, Francis X.

2014-01-01

We previously demonstrated that the expression of intercellular adhesion molecule-1 (ICAM-1) by skeletal muscle cells after muscle overload contributes to ensuing regenerative and hypertrophic processes in skeletal muscle. The objective of the present study is to reveal mechanisms through which skeletal muscle cell expression of ICAM-1 augments regenerative and hypertrophic processes of myogenesis. This was accomplished by genetically engineering C2C12 myoblasts to stably express ICAM-1, and by inhibiting the adhesive and signaling functions of ICAM-1 through the use of a neutralizing antibody or cell penetrating peptide, respectively. Expression of ICAM-1 by cultured skeletal muscle cells augmented myoblast-myoblast adhesion, myotube formation, myonuclear number, myotube alignment, myotube-myotube fusion, and myotube size without influencing the ability of myoblasts to proliferate or differentiate. ICAM-1 augmented myotube formation, myonuclear accretion, and myotube alignment through a mechanism involving adhesion-induced activation of ICAM-1 signaling, as these dependent measures were reduced via antibody and peptide inhibition of ICAM-1. The adhesive and signaling functions of ICAM-1 also facilitated myotube hypertrophy through a mechanism involving myotube-myotube fusion, protein synthesis, and Akt/p70s6k signaling. Our findings demonstrate that ICAM-1 expression by skeletal muscle cells augments myogenesis, and establish a novel mechanism through which the inflammatory response facilitates growth processes in skeletal muscle. PMID:25281303

Genetic Ablation of CD68 Results in Mice with Increased Bone and Dysfunctional Osteoclasts

PubMed Central

Ashley, Jason W.; Shi, Zhenqi; Zhao, Haibo; Li, Xingsheng; Kesterson, Robert A.; Feng, Xu

2011-01-01

CD68 is a member of the lysosome associated membrane protein (LAMP) family that is restricted in its expression to cells of the monocyte/macrophage lineage. This lineage restriction includes osteoclasts, and, while previous studies of CD68 in macrophages and dendritic cells have proposed roles in lipid metabolism, phagocytosis, and antigen presentation, the expression and function of CD68 in osteoclasts have not been explored. In this study, we investigated the expression and localization of CD68 in macrophages and osteoclasts in response to the monocyte/macrophage-colony stimulating factor (M-CSF) and the receptor activator of NF-κB ligand (RANKL). We found that M-CSF stimulates CD68 expression and RANKL alters the apparent molecular weight of CD68 as measured by Western immunoblotting. In addition, we explored the significance of CD68 expression in osteoclasts by generating mice that lack expression of CD68. These mice have increased trabecular bone, and in vitro assessment of CD68−/− osteoclasts revealed that, in the absence of CD68, osteoclasts demonstrate an accumulation of intracellular vesicle-like structures, and do not efficiently resorb bone. These findings demonstrate a role for CD68 in the function of osteoclasts, and future studies will determine the mechanistic nature of the defects seen in CD68−/− osteoclasts. PMID:21991369

Human Antiviral Protein IFIX Suppresses Viral Gene Expression during Herpes Simplex Virus 1 (HSV-1) Infection and Is Counteracted by Virus-induced Proteasomal Degradation.

PubMed

Crow, Marni S; Cristea, Ileana M

2017-04-01

The interferon-inducible protein X (IFIX), a member of the PYHIN family, was recently recognized as an antiviral factor against infection with herpes simplex virus 1 (HSV-1). IFIX binds viral DNA upon infection and promotes expression of antiviral cytokines. How IFIX exerts its host defense functions and whether it is inhibited by the virus remain unknown. Here, we integrated live cell microscopy, proteomics, IFIX domain characterization, and molecular virology to investigate IFIX regulation and antiviral functions during HSV-1 infection. We find that IFIX has a dynamic localization during infection that changes from diffuse nuclear and nucleoli distribution in uninfected cells to discrete nuclear puncta early in infection. This is rapidly followed by a reduction in IFIX protein levels. Indeed, using immunoaffinity purification and mass spectrometry, we define IFIX interactions during HSV-1 infection, finding an association with a proteasome subunit and proteins involved in ubiquitin-proteasome processes. Using synchronized HSV-1 infection, microscopy, and proteasome-inhibition experiments, we demonstrate that IFIX co-localizes with nuclear proteasome puncta shortly after 3 h of infection and that its pyrin domain is rapidly degraded in a proteasome-dependent manner. We further demonstrate that, in contrast to several other host defense factors, IFIX degradation is not dependent on the E3 ubiquitin ligase activity of the viral protein ICP0. However, we show IFIX degradation requires immediate-early viral gene expression, suggesting a viral host suppression mechanism. The IFIX interactome also demonstrated its association with transcriptional regulatory proteins, including the 5FMC complex. We validate this interaction using microscopy and reciprocal isolations and determine it is mediated by the IFIX HIN domain. Finally, we show IFIX suppresses immediate-early and early viral gene expression during infection. Altogether, our study demonstrates that IFIX antiviral functions work in part via viral transcriptional suppression and that HSV-1 has acquired mechanisms to block its functions via proteasome-dependent degradation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Mitochondrial and Chloroplast Stress Responses Are Modulated in Distinct Touch and Chemical Inhibition Phases1[OPEN

PubMed Central

Ivanova, Aneta; Millar, A. Harvey; Whelan, James

2016-01-01

Previous studies have identified a range of transcription factors that modulate retrograde regulation of mitochondrial and chloroplast functions in Arabidopsis (Arabidopsis thaliana). However, the relative importance of these regulators and whether they act downstream of separate or overlapping signaling cascades is still unclear. Here, we demonstrate that multiple stress-related signaling pathways, with distinct kinetic signatures, converge on overlapping gene sets involved in energy organelle function. The transcription factor ANAC017 is almost solely responsible for transcript induction of marker genes around 3 to 6 h after chemical inhibition of organelle function and is a key regulator of mitochondrial and specific types of chloroplast retrograde signaling. However, an independent and highly transient gene expression phase, initiated within 10 to 30 min after treatment, also targets energy organelle functions, and is related to touch and wounding responses. Metabolite analysis demonstrates that this early response is concurrent with rapid changes in tricarboxylic acid cycle intermediates and large changes in transcript abundance of genes encoding mitochondrial dicarboxylate carrier proteins. It was further demonstrated that transcription factors AtWRKY15 and AtWRKY40 have repressive regulatory roles in this touch-responsive gene expression. Together, our results show that several regulatory systems can independently affect energy organelle function in response to stress, providing different means to exert operational control. PMID:27208304

The Gem GTP-binding protein promotes morphological differentiation in neuroblastoma.

PubMed

Leone, A; Mitsiades, N; Ward, Y; Spinelli, B; Poulaki, V; Tsokos, M; Kelly, K

2001-05-31

Gem is a small GTP-binding protein within the Ras superfamily whose function has not been determined. We report here that ectopic Gem expression is sufficient to stimulate cell flattening and neurite extension in N1E-115 and SH-SY5Y neuroblastoma cells, suggesting a role for Gem in cytoskeletal rearrangement and/or morphological differentiation of neurons. Consistent with this potential function, in clinical samples of neuroblastoma, Gem protein was most highly expressed within cells which had differentiated to express ganglionic morphology. Gem was also observed in developing trigeminal nerve ganglia in 12.5 day mouse embryos, demonstrating that Gem expression is a property of normal ganglionic development. Although Gem expression is rare in epithelial and hematopoietic cancer cell lines, constitutive Gem levels were detected in several neuroblastoma cell lines and could be further induced as much as 10-fold following treatment with PMA or the acetylcholine muscarinic agonist, carbachol.

Indian hedgehog contributes to human cartilage endplate degeneration.

PubMed

Wang, Shaowei; Yang, Kun; Chen, Shuai; Wang, Jiying; Du, Guoqing; Fan, Shunwu; Wei, Lei

2015-08-01

To determine the role of Indian hedgehog (Ihh) signaling in human cartilage endplate (CEP) degeneration. CEP-degenerated tissues from patients with Modic I or II changes (n = 9 and 45, respectively) and normal tissues from vertebral burst fracture patients (n = 17) were collected. Specimens were either cut into slices for organ culture ex vivo or digested to isolate chondrocytes for cell culture in vitro. Ihh expression and the effect of Ihh on cartilage degeneration were determined by investigating degeneration markers in this study. Ihh expression and cartilage degeneration markers significantly increased in the Modic I and II groups. The expression of cartilage degeneration markers was positively correlated with degeneration severity. Gain-of-function for Ihh promoted expression of cartilage degeneration markers in vitro, while loss-of-function for Ihh inhibited their expression both in vitro and ex vivo. These findings demonstrated that Ihh promotes CEP degeneration. Blocking Ihh pathway has potential clinical usage for attenuating CEP degeneration.

ER stress upregulated PGE2/IFNγ-induced IL-6 expression and down-regulated iNOS expression in glial cells

NASA Astrophysics Data System (ADS)

Hosoi, Toru; Honda, Miya; Oba, Tatsuya; Ozawa, Koichiro

2013-12-01

The disruption of endoplasmic reticulum (ER) function can lead to neurodegenerative disorders, in which inflammation has also been implicated. We investigated the possible correlation between ER stress and immune function using glial cells. We demonstrated that ER stress synergistically enhanced prostaglandin (PG) E2 + interferon (IFN) γ-induced interleukin (IL)-6 production. This effect was mediated through cAMP. Immune-activated glial cells produced inducible nitric oxide synthase (iNOS). Interestingly, ER stress inhibited PGE2 + IFNγ-induced iNOS expression. Similar results were obtained when cells were treated with dbcAMP + IFNγ. Thus, cAMP has a dual effect on immune reactions; cAMP up-regulated IL-6 expression, but down-regulated iNOS expression under ER stress. Therefore, our results suggest a link between ER stress and immune reactions in neurodegenerative diseases.

Joint sparse learning for 3-D facial expression generation.

PubMed

Song, Mingli; Tao, Dacheng; Sun, Shengpeng; Chen, Chun; Bu, Jiajun

2013-08-01

3-D facial expression generation, including synthesis and retargeting, has received intensive attentions in recent years, because it is important to produce realistic 3-D faces with specific expressions in modern film production and computer games. In this paper, we present joint sparse learning (JSL) to learn mapping functions and their respective inverses to model the relationship between the high-dimensional 3-D faces (of different expressions and identities) and their corresponding low-dimensional representations. Based on JSL, we can effectively and efficiently generate various expressions of a 3-D face by either synthesizing or retargeting. Furthermore, JSL is able to restore 3-D faces with holes by learning a mapping function between incomplete and intact data. Experimental results on a wide range of 3-D faces demonstrate the effectiveness of the proposed approach by comparing with representative ones in terms of quality, time cost, and robustness.

FoxA family members are crucial regulators of the hypertrophic chondrocyte differentiation program

PubMed Central

Ionescu, Andreia; Kozhemyakina, Elena; Nicolae, Claudia; Kaestner, Klaus H.; Olsen, Bjorn R.; Lassar, Andrew B.

2012-01-01

During endochondral ossification small immature chondrocytes enlarge to form hypertrophic chondrocytes, which express collagen X. In this work, we demonstrate that FoxA factors are induced during chondrogenesis, bind to conserved binding sites in the collagen X enhancer, and can promote the expression of a collagen X-luciferase reporter in both chondrocytes and fibroblasts. In addition, we demonstrate by both gain and loss of function analyses that FoxA factors play a crucial role driving the expression of both endogenous collagen X and other hypertrophic chondrocyte-specific genes. Mice engineered to lack expression of both FoxA2 and FoxA3 in their chondrocytes display defects in chondrocyte hypertrophy, alkaline phosphatase expression, and mineralization in their sternebrae and in addition exhibit postnatal dwarfism that is coupled to significantly decreased expression of both collagen X and MMP13 in their growth plates. Together, our findings indicate that FoxA family members are crucial regulators of the hypertrophic chondrocyte differentiation program. PMID:22595668

Functional characterisation of three members of the Vitis vinifera L. carotenoid cleavage dioxygenase gene family

PubMed Central

2013-01-01

Background In plants, carotenoids serve as the precursors to C13-norisoprenoids, a group of apocarotenoid compounds with diverse biological functions. Enzymatic cleavage of carotenoids catalysed by members of the carotenoid cleavage dioxygenase (CCD) family has been shown to produce a number of industrially important volatile flavour and aroma apocarotenoids including β-ionone, geranylacetone, pseudoionone, α-ionone and 3-hydroxy-β-ionone in a range of plant species. Apocarotenoids contribute to the floral and fruity attributes of many wine cultivars and are thereby, at least partly, responsible for the “varietal character”. Despite their importance in grapes and wine; carotenoid cleavage activity has only been described for VvCCD1 and the mechanism(s) and regulation of carotenoid catabolism remains largely unknown. Results Three grapevine-derived CCD-encoding genes have been isolated and shown to be functional with unique substrate cleavage capacities. Our results demonstrate that the VvCCD4a and VvCCD4b catalyse the cleavage of both linear and cyclic carotenoid substrates. The expression of VvCCD1, VvCCD4a and VvCCD4b was detected in leaf, flower and throughout berry development. VvCCD1 expression was constitutive, whereas VvCCD4a expression was predominant in leaves and VvCCD4b in berries. A transgenic population with a 12-fold range of VvCCD1 expression exhibited a lack of correlation between VvCCD1 expression and carotenoid substrates and/or apocarotenoid products in leaves, providing proof that the in planta function(s) of VvCCD1 in photosynthetically active tissue is distinct from the in vitro activities demonstrated. The isolation and functional characterisation of VvCCD4a and VvCCD4b identify two additional CCDs that are functional in grapevine. Conclusions Taken together, our results indicate that the three CCDs are under various levels of control that include gene expression (spatial and temporal), substrate specificity and compartmentalisation that act individually and/or co-ordinately to maintain carotenoid and volatile apocarotenoid levels in plants. Altering the expression of VvCCD1 in a transgenic grapevine population illustrated the divergence between the in vitro enzyme activity and the in planta activity of this enzyme, thereby contributing to the efforts to understand how enzymatic degradation of carotenoids involved in photosynthesis occurs. The identification and functional characterisation of VvCCD4a and VvCCD4b suggest that these enzymes are primarily responsible for catalysing the cleavage of plastidial carotenoids. PMID:24106789

Timed Knickkopf function is essential for wing cuticle formation in Drosophila melanogaster.

PubMed

Li, Kaixia; Zhang, Xubo; Zuo, Ying; Liu, Weimin; Zhang, Jianzhen; Moussian, Bernard

2017-10-01

The insect cuticle is an extracellular matrix that consists of the polysaccharide chitin, proteins, lipids and organic molecules that are arranged in distinct horizontal layers. In Drosophila melanogaster, these layers are not formed sequentially, but, at least partially, at the same time. Timing of the underlying molecular mechanisms is conceivably crucial for cuticle formation. To study this issue, we determined the time period during which the function of Knickkopf (Knk), a key factor of chitin organization, is required for wing cuticle differentiation in D. melanogaster. Although knk is expressed throughout metamorphosis, we demonstrate that its expression 30 h prior and 48 h after pupariation is essential for correct wing cuticle formation. In other words, expression beyond this period is futile. Importantly, manipulation of Knk expression during this time causes wing bending suggesting an effect of Knk amounts on the physical properties of the wing cuticle. Manipulation of Knk expression also interferes with the structure and function of the cuticle surface. First, we show that the shape of surface nano-structures depends on the expression levels of knk. Second, we find that cuticle impermeability is compromised in wings with reduced knk expression. In summary, despite the extended supply of Knk during metamorphosis, controlled amounts of Knk are important for correct wing cuticle differentiation and function in a concise period of time. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ectromelia virus encodes an anti-apoptotic protein that regulates cell death.

PubMed

Mehta, Ninad; Taylor, John; Quilty, Douglas; Barry, Michele

2015-01-15

Apoptosis serves as a powerful defense against damaged or pathogen-infected cells. Since apoptosis is an effective defense against viral infection, many viruses including poxviruses, encode proteins to prevent or delay apoptosis. Here we show that ectromelia virus, the causative agent of mousepox encodes an anti-apoptotic protein EVM025. Here we demonstrate that expression of functional EVM025 is crucial to prevent apoptosis triggered by virus infection and staurosporine. We demonstrate that the expression of EVM025 prevents the conformational activation of the pro-apoptotic proteins Bak and Bax, allowing the maintenance of mitochondrial membrane integrity upon infection with ECTV. Additionally, EVM025 interacted with intracellular Bak. We were able to demonstrate that EVM025 ability to inhibit Bax activation is a function of its ability to inhibit the activity of an upstream BH3 only protein Bim. Collectively, our data indicates that EVM025 inhibits apoptosis by sequestering Bak and inhibiting the activity of Bak and Bax. Copyright © 2014 Elsevier Inc. All rights reserved.

Functional linkage between NOXA and Bim in mitochondrial apoptotic events.

PubMed

Han, Jie; Goldstein, Leslie A; Hou, Wen; Rabinowich, Hannah

2007-06-01

NOXA is a BH3-only protein whose expression is induced by certain p53-depenent or independent apoptotic stimuli. Both NOXA and Bim are avid binders of Mcl-1, but a functional linkage between these BH3-only proteins has not yet been reported. In this study, we demonstrate that Mcl-1 binding of endogenously induced NOXA interferes with the ability of Mcl-1 to efficiently sequester endogenous Bim, as Bim is displaced from its complex with Mcl-1. Induced NOXA significantly enhances the UV sensitivity of cells, and the ensuing mitochondrial depolarization is entirely abrogated by Bim knockdown. These results demonstrate a Mcl-1-mediated cross-talk between endogenous NOXA and Bim that occurs upstream of the Bak/Bax-dependent execution of UV-induced mitochondrial depolarization. The current findings demonstrate that the mitochondrial response to an induced expression of NOXA is executed by endogenous Bim and suggest a plausible mechanism for the observed NOXA-Bim linkage.

Collateral Sensitivity of Multidrug-Resistant Cells to the Orphan Drug Tiopronin

PubMed Central

Goldsborough, Andrew S.; Handley, Misty D.; Dulcey, Andrés E.; Pluchino, Kristen M.; Kannan, Pavitra; Brimacombe, Kyle R.; Hall, Matthew D.; Griffiths, Gary; Gottesman, Michael M.

2011-01-01

A major challenge in the treatment of cancer is multidrug resistance (MDR) that develops during chemotherapy. Here we demonstrate that tiopronin (1), a thiol-substituted N-propanoylglycine derivative, was selectively toxic to a series of cell lines expressing the drug efflux pump P-glycoprotein (P-gp, ABCB1) and MRP1 (ABCC1). Treatment of MDR cells with 1 led to instability of the ABCB1 mRNA and consequently a reduction in P-gp protein, despite functional assays demonstrating that tiopronin does not interact with P-gp. Long-term exposure of P-gp-expressing cells to 1 sensitized them to doxorubicin and taxol, both P-gp substrates. Treatment of MRP1-overexpressing cells with tiopronin led to a significant reduction in MRP1 protein. Synthesis and screening of analogs of tiopronin demonstrated that the thiol functional group was essential for collateral sensitivity, while substitution of the amino acid backbone altered but did not destroy specificity, pointing to future development of targeted analogs. PMID:21657271

Dissecting the genetics of the human transcriptome identifies novel trait-related trans-eQTLs and corroborates the regulatory relevance of non-protein coding loci†

PubMed Central

Kirsten, Holger; Al-Hasani, Hoor; Holdt, Lesca; Gross, Arnd; Beutner, Frank; Krohn, Knut; Horn, Katrin; Ahnert, Peter; Burkhardt, Ralph; Reiche, Kristin; Hackermüller, Jörg; Löffler, Markus; Teupser, Daniel; Thiery, Joachim; Scholz, Markus

2015-01-01

Genetics of gene expression (eQTLs or expression QTLs) has proved an indispensable tool for understanding biological pathways and pathomechanisms of trait-associated SNPs. However, power of most genome-wide eQTL studies is still limited. We performed a large eQTL study in peripheral blood mononuclear cells of 2112 individuals increasing the power to detect trans-effects genome-wide. Going beyond univariate SNP-transcript associations, we analyse relations of eQTLs to biological pathways, polygenetic effects of expression regulation, trans-clusters and enrichment of co-localized functional elements. We found eQTLs for about 85% of analysed genes, and 18% of genes were trans-regulated. Local eSNPs were enriched up to a distance of 5 Mb to the transcript challenging typically implemented ranges of cis-regulations. Pathway enrichment within regulated genes of GWAS-related eSNPs supported functional relevance of identified eQTLs. We demonstrate that nearest genes of GWAS-SNPs might frequently be misleading functional candidates. We identified novel trans-clusters of potential functional relevance for GWAS-SNPs of several phenotypes including obesity-related traits, HDL-cholesterol levels and haematological phenotypes. We used chromatin immunoprecipitation data for demonstrating biological effects. Yet, we show for strongly heritable transcripts that still little trans-chromosomal heritability is explained by all identified trans-eSNPs; however, our data suggest that most cis-heritability of these transcripts seems explained. Dissection of co-localized functional elements indicated a prominent role of SNPs in loci of pseudogenes and non-coding RNAs for the regulation of coding genes. In summary, our study substantially increases the catalogue of human eQTLs and improves our understanding of the complex genetic regulation of gene expression, pathways and disease-related processes. PMID:26019233

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tong, Meng, E-mail: tong.59@osu.edu; Han, Byungdo B.; Holpuch, Andrew S.

The presence of the EMT (epithelial-mesenchymal transition), EndMT (endothelial-mesenchymal transition) and VM (vasculogenic mimicry) demonstrates the multidirectional extent of phenotypic plasticity in cancers. Previous findings demonstrating the crosstalk between head and neck squamous cell carcinoma (HNSCC) and vascular endothelial growth factor (VEGF) imply that HNSCC cells share some functional commonalities with endothelial cells. Our current results reveal that cultured HNSCC cells not only possess endothelial-specific markers, but also display endotheliod functional features including low density lipoprotein uptake, formation of tube-like structures on Matrigel and growth state responsiveness to VEGF and endostatin. HNSCC cell subpopulations are also highly responsive to transformingmore » growth factor-β1 and express its auxiliary receptor, endoglin. Furthermore, the endotheliod characteristics observed in vitro recapitulate phenotypic features observed in human HNSCC tumors. Conversely, cultured normal human oral keratinocytes and intact or ulcerated human oral epithelia do not express comparable endotheliod characteristics, which imply that assumption of endotheliod features is restricted to transformed keratinocytes. In addition, this phenotypic state reciprocity facilitates HNSCC progression by increasing production of factors that are concurrently pro-proliferative and pro-angiogenic, conserving cell energy stores by LDL internalization and enhancing cell mobility. Finally, recognition of this endotheliod phenotypic transition provides a solid rationale to evaluate the antitumorigenic potential of therapeutic agents formerly regarded as exclusively angiostatic in scope. - Highlights: ► HNSCC tumor cells express endothelial specific markers VE-cadherin, CD31 and vimentin. ► Similarly, cultured HNSCC cells retain expression of these markers. ► HNSCC cells demonstrate functional endotheliod characteristics i.e. AcLDL uptake. ► HNSCC cell subpopulations are highly responsive to TGF- β1, VEGF and endostatin. ► TGF-β1 facilitates cadherin switching and augments invasiveness of HNSCC subpopulations.« less

Clustered Regularly Interspaced Short Palindromic Repeat-Dependent, Biofilm-Specific Death of Pseudomonas aeruginosa Mediated by Increased Expression of Phage-Related Genes

PubMed Central

Heussler, Gary E.; Cady, Kyle C.; Koeppen, Katja; Bhuju, Sabin; Stanton, Bruce A.

2015-01-01

ABSTRACT The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (CRISPR/Cas) system is an adaptive immune system present in many archaea and bacteria. CRISPR/Cas systems are incredibly diverse, and there is increasing evidence of CRISPR/Cas systems playing a role in cellular functions distinct from phage immunity. Previously, our laboratory reported one such alternate function in which the type 1-F CRISPR/Cas system of the opportunistic pathogen Pseudomonas aeruginosa strain UCBPP-PA14 (abbreviated as P. aeruginosa PA14) inhibits both biofilm formation and swarming motility when the bacterium is lysogenized by the bacteriophage DMS3. In this study, we demonstrated that the presence of just the DMS3 protospacer and the protospacer-adjacent motif (PAM) on the P. aeruginosa genome is necessary and sufficient for this CRISPR-dependent loss of these group behaviors, with no requirement of additional DMS3 sequences. We also demonstrated that the interaction of the CRISPR system with the DMS3 protospacer induces expression of SOS-regulated phage-related genes, including the well-characterized pyocin operon, through the activity of the nuclease Cas3 and subsequent RecA activation. Furthermore, our data suggest that expression of the phage-related genes results in bacterial cell death on a surface due to the inability of the CRISPR-engaged strain to downregulate phage-related gene expression, while these phage-related genes have minimal impact on growth and viability under planktonic conditions. Deletion of the phage-related genes restores biofilm formation and swarming motility while still maintaining a functional CRISPR/Cas system, demonstrating that the loss of these group behaviors is an indirect effect of CRISPR self-targeting. PMID:25968642

Characterization of Rat Meibomian Gland Ion and Fluid Transport

PubMed Central

Yu, Dongfang; Davis, Richard M.; Aita, Megumi; Burns, Kimberlie A.; Clapp, Phillip W.; Gilmore, Rodney C.; Chua, Michael; O'Neal, Wanda K.; Schlegel, Richard; Randell, Scott H.; C. Boucher, Richard

2016-01-01

Purpose We establish novel primary rat meibomian gland (MG) cell culture systems and explore the ion transport activities of the rat MG. Methods Freshly excised rat MG tissues were characterized as follows: (1) mRNA expression of selected epithelial ion channels/transporters were measured by RT-PCR, (2) localization of epithelial sodium channel (ENaC) mRNAs was performed by in situ hybridization, and (3) protein expression and localization of βENaC, the Na+/K+/Cl− cotransporter (NKCC), and the Na+/K+ ATPase were evaluated by immunofluorescence. Primary isolated rat MG cells were cocultured with 3T3 feeder cells and a Rho-associated kinase (ROCK) inhibitor (Y-27632) for expansion. Passaged rat MG cells were cultured as planar sheets under air-liquid interface (ALI) conditions for gene expression and electrophysiologic studies. Passaged rat MG cells also were cultured in matrigel matrices to form spheroids, which were examined ultrastructurally by transmission electron microscopy (TEM) and functionally using swelling assays. Results Expression of multiple ion channel/transporter genes was detected in rat MG tissues. β-ENaC mRNA and protein were localized more to MG peripheral acinar cells than central acinar cells or ductular epithelial cells. Electrophysiologic studies of rat MG cell planar cultures demonstrated functional sodium, chloride, and potassium channels, and cotransporters activities. Transmission electron microscopic analyses of rat MG spheroids revealed highly differentiated MG cells with abundant lysosomal lamellar bodies. Rat MG spheroids culture-based measurements demonstrated active volume regulation by ion channels. Conclusions This study demonstrates the presence and function of ion channels and volume transport by rat MG. Two novel primary MG cell culture models that may be useful for MG research were established. PMID:27127933

Structural basis for decreased induction of class IB PI3-kinases expression by MIF inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Abhay Kumar; Pantouris, Georgios; Borosch, Sebastian

Macrophage migration inhibitory factor (MIF) is a master regulator of proinflammatory cytokines and plays pathological roles when not properly regulated in rheumatoid arthritis, lupus, atherosclerosis, asthma and cancer. Unlike canonical cytokines, MIF has vestigial keto-enol tautomerase activity. Most of the current MIF inhibitors were screened for the inhibition of this enzymatic activity. However, only some of the enzymatic inhibitors inhibit receptor-mediated biological functions of MIF, such as cell recruitment, through an unknown molecular mechanism. The goal of this study was to understand the molecular basis underlying the pharmacological inhibition of biological functions of MIF. Here, we demonstrate how the structuralmore » changes caused upon inhibitor binding translate into the alteration of MIF-induced downstream signalling. Macrophage migration inhibitory factor activates phosphoinositide 3-kinases (PI3Ks) that play a pivotal role in immune cell recruitment in health and disease. There are several different PI3K isoforms, but little is known about how they respond to MIF. We demonstrate that MIF up-regulates the expression of Class IB PI3Ks in leucocytes. We also demonstrate that MIF tautomerase active site inhibitors down-regulate the expression of Class IB PI3Ks as well as leucocyte recruitment in vitro and in vivo. Finally, based on our MIF:inhibitor complex crystal structures, we hypothesize that the reduction in Class IB PI3K expression occurs because of the displacement of Pro1 towards the second loop of MIF upon inhibitor binding, which results in increased flexibility of the loop 2 and sub-optimal MIF binding to its receptors. These results will provide molecular insights for fine-tuning the biological functions of MIF.« less

An interactive network of elastase, secretases, and PAR-2 protein regulates CXCR1 receptor surface expression on neutrophils.

PubMed

Bakele, Martina; Lotz-Havla, Amelie S; Jakowetz, Anja; Carevic, Melanie; Marcos, Veronica; Muntau, Ania C; Gersting, Soeren W; Hartl, Dominik

2014-07-25

CXCL8 (IL-8) recruits and activates neutrophils through the G protein-coupled chemokine receptor CXCR1. We showed previously that elastase cleaves CXCR1 and thereby impairs antibacterial host defense. However, the molecular intracellular machinery involved in this process remained undefined. Here we demonstrate by using flow cytometry, confocal microscopy, subcellular fractionation, co-immunoprecipitation, and bioluminescence resonance energy transfer that combined α- and γ-secretase activities are functionally involved in elastase-mediated regulation of CXCR1 surface expression on human neutrophils, whereas matrix metalloproteases are dispensable. We further demonstrate that PAR-2 is stored in mobilizable compartments in neutrophils. Bioluminescence resonance energy transfer and co-immunoprecipitation studies showed that secretases, PAR-2, and CXCR1 colocalize and physically interact in a novel protease/secretase-chemokine receptor network. PAR-2 blocking experiments provided evidence that elastase increased intracellular presenilin-1 expression through PAR-2 signaling. When viewed in combination, these studies establish a novel functional network of elastase, secretases, and PAR-2 that regulate CXCR1 expression on neutrophils. Interfering with this network could lead to novel therapeutic approaches in neutrophilic diseases, such as cystic fibrosis or rheumatoid arthritis.

An Interactive Network of Elastase, Secretases, and PAR-2 Protein Regulates CXCR1 Receptor Surface Expression on Neutrophils*

PubMed Central

Bakele, Martina; Lotz-Havla, Amelie S.; Jakowetz, Anja; Carevic, Melanie; Marcos, Veronica; Muntau, Ania C.; Gersting, Soeren W.; Hartl, Dominik

2014-01-01

CXCL8 (IL-8) recruits and activates neutrophils through the G protein-coupled chemokine receptor CXCR1. We showed previously that elastase cleaves CXCR1 and thereby impairs antibacterial host defense. However, the molecular intracellular machinery involved in this process remained undefined. Here we demonstrate by using flow cytometry, confocal microscopy, subcellular fractionation, co-immunoprecipitation, and bioluminescence resonance energy transfer that combined α- and γ-secretase activities are functionally involved in elastase-mediated regulation of CXCR1 surface expression on human neutrophils, whereas matrix metalloproteases are dispensable. We further demonstrate that PAR-2 is stored in mobilizable compartments in neutrophils. Bioluminescence resonance energy transfer and co-immunoprecipitation studies showed that secretases, PAR-2, and CXCR1 colocalize and physically interact in a novel protease/secretase-chemokine receptor network. PAR-2 blocking experiments provided evidence that elastase increased intracellular presenilin-1 expression through PAR-2 signaling. When viewed in combination, these studies establish a novel functional network of elastase, secretases, and PAR-2 that regulate CXCR1 expression on neutrophils. Interfering with this network could lead to novel therapeutic approaches in neutrophilic diseases, such as cystic fibrosis or rheumatoid arthritis. PMID:24914212

BAG3 promotes proliferation of ovarian cancer cells via post-transcriptional regulation of Skp2 expression.

PubMed

Yan, Jing; Liu, Chuan; Jiang, Jing-Yi; Liu, Hans; Li, Chao; Li, Xin-Yu; Yuan, Ye; Zong, Zhi-Hong; Wang, Hua-Qin

2017-10-01

Bcl-2 associated athanogene 3 (BAG3) contains a modular structure, through which BAG3 interacts with a wide range of proteins, thereby affording its capacity to regulate multifaceted biological processes. BAG3 is often highly expressed and functions as a pro-survival factor in many cancers. However, the oncogenic potential of BAG3 remains not fully understood. The cell cycle regulator, S-phase kinase associated protein 2 (Skp2) is increased in various cancers and plays an important role in tumorigenesis. The current study demonstrated that BAG3 promoted proliferation of ovarian cancer cells via upregulation of Skp2. BAG3 stabilized Skp2 mRNA via its 3'-untranslated region (UTR). The current study demonstrated that BAG3 interacted with Skp2 mRNA. In addition, miR-21-5p suppressed Skp2 expression, which was compromised by forced BAG3 expression. These results indicated that at least some oncogenic functions of BAG3 were mediated through posttranscriptional regulation of Skp2 via antagonizing suppressive action of miR-21-5p in ovarian cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.

The novel 19q13 KRAB zinc-finger tumour suppressor ZNF382 is frequently methylated in oesophageal squamous cell carcinoma and antagonises Wnt/β-catenin signalling.

PubMed

Zhang, Chong; Xiang, Tingxiu; Li, Shuman; Ye, Lin; Feng, Yixiao; Pei, Lijiao; Li, Lili; Wang, Xiangyu; Sun, Ran; Tao, Qian; Ren, Guosheng

2018-05-14

Zinc finger proteins (ZFPs) are the largest transcription factor family in mammals. About one-third of ZFPs are Krüppel-associated box domain (KRAB)-ZFPs and involved in the regulation of cell differentiation/proliferation/apoptosis and neoplastic transformation. We recently identified ZNF382 as a novel KRAB-ZFP epigenetically inactivated in multiple cancers due to frequent promoter CpG methylation. However, its epigenetic alterations, biological functions/mechanism and clinical significance in oesophageal squamous cell carcinoma (ESCC) are still unknown. Here, we demonstrate that ZNF382 expression was suppressed in ESCC due to aberrant promoter methylation, but highly expressed in normal oesophagus tissues. ZNF382 promoter methylation is correlated with ESCC differentiation levels. Restoration of ZNF382 expression in silenced ESCC cells suppressed tumour cell proliferation and metastasis through inducing cell apoptosis. Importantly, ZNF382 suppressed Wnt/β-catenin signalling and downstream target gene expression, likely through binding directly to FZD1 and DVL2 promoters. In summary, our findings demonstrate that ZNF382 functions as a bona fide tumour suppressor inhibiting ESCC pathogenesis through inhibiting the Wnt/β-catenin signalling pathway.

Distributed Function Mining for Gene Expression Programming Based on Fast Reduction.

PubMed

Deng, Song; Yue, Dong; Yang, Le-chan; Fu, Xiong; Feng, Ya-zhou

2016-01-01

For high-dimensional and massive data sets, traditional centralized gene expression programming (GEP) or improved algorithms lead to increased run-time and decreased prediction accuracy. To solve this problem, this paper proposes a new improved algorithm called distributed function mining for gene expression programming based on fast reduction (DFMGEP-FR). In DFMGEP-FR, fast attribution reduction in binary search algorithms (FAR-BSA) is proposed to quickly find the optimal attribution set, and the function consistency replacement algorithm is given to solve integration of the local function model. Thorough comparative experiments for DFMGEP-FR, centralized GEP and the parallel gene expression programming algorithm based on simulated annealing (parallel GEPSA) are included in this paper. For the waveform, mushroom, connect-4 and musk datasets, the comparative results show that the average time-consumption of DFMGEP-FR drops by 89.09%%, 88.85%, 85.79% and 93.06%, respectively, in contrast to centralized GEP and by 12.5%, 8.42%, 9.62% and 13.75%, respectively, compared with parallel GEPSA. Six well-studied UCI test data sets demonstrate the efficiency and capability of our proposed DFMGEP-FR algorithm for distributed function mining.

TEG-1 CD2BP2 controls miRNA levels by regulating miRISC stability in C. elegans and human cells

PubMed Central

Wang, Chris; Gupta, Pratyush; Fressigne, Lucile; Bossé, Gabriel D.; Wang, Xin; Simard, Martin J.

2017-01-01

Abstract MiRNAs post-transcriptionally regulate gene expression by recruiting the miRNA-induced silencing complex (miRISC) to target mRNAs. However, the mechanisms by which miRISC components are maintained at appropriate levels for proper function are largely unknown. Here, we demonstrate that Caenorhabditis elegans TEG-1 regulates the stability of two miRISC effectors, VIG-1 and ALG-1, which in turn affects the abundance of miRNAs in various families. We demonstrate that TEG-1 physically interacts with VIG-1, and complexes with mature let-7 miRNA. Also, loss of teg-1 in vivo phenocopies heterochronic defects observed in let-7 mutants, suggesting the association of TEG-1 with miRISC is necessary for let-7 to function properly during development. Loss of TEG-1 function also affects the abundance and function of other microRNAs, suggesting that TEG-1's role is not specific to let-7. We further demonstrate that the human orthologs of TEG-1, VIG-1 and ALG-1 (CD2BP2, SERBP1/PAI-RBP1 and AGO2) are found in a complex in HeLa cells, and knockdown of CD2BP2 results in reduced miRNA levels; therefore, TEG-1's role in affecting miRNA levels and function is likely conserved. Together, these data demonstrate that TEG-1 CD2BP2 stabilizes miRISC and mature miRNAs, maintaining them at levels necessary to properly regulate target gene expression. PMID:28180320

Chemokine Receptor CXCR4 Expression in Patients With Melanoma and Colorectal Cancer Liver Metastases and the Association With Disease Outcome

PubMed Central

Kim, Joseph; Mori, Takuji; Chen, Steven L.; Amersi, Farin F.; Martinez, Steve R.; Kuo, Christine; Turner, Roderick R.; Ye, Xing; Bilchik, Anton J.; Morton, Donald L.; Hoon, Dave S. B.

2006-01-01

Objective: To determine the role of chemokine receptor (CR) expression in patients with melanoma and colorectal cancer (CRC) liver metastases. Summary Background Data: Murine and in vitro models have identified CR as potential factors in organ-specific metastasis of multiple cancers. Chemokines via their respective receptors have been shown to promote cell migration to distant organs. Methods: Patients who underwent hepatic surgery for melanoma or CRC liver metastases were assessed. Screening cDNA microarrays of melanoma/CRC cell lines and tumor specimens were analyzed to identify CR. Microarray data were validated by quantitative real-time RT-PCR (qRT) in paraffin-embedded liver metastases. Migration assays and immunohistochemistry were performed to verify CR function and confirm CR expression, respectively. Results: Microarray analysis identified CXCR4 as the most common CR expressed by both cancers. qRT demonstrated CXCR4 expression in 24 of 27 (89%) melanoma and 28 of 29 (97%) CRC liver metastases. In vitro treatment of melanoma or CRC cells with CXCL12, the ligand for CXCR4, significantly increased cell migration (P < 0.001). Low versus high CXCR4 expression in CRC liver metastases correlated with a significant difference in overall survival (median 27 months vs. 10 months, respectively; P = 0.036). In melanoma, low versus high CXCR4 expression in liver metastases demonstrated no difference in overall survival (median 11 months vs. 8 months, respectively; P = not significant). Conclusions: CXCR4 is expressed and functional on melanoma and CRC cells. The ligand for CXCR4 is highly expressed in liver and may specifically attract melanoma and CRC CXCR4 (+) cells. Quantitative analysis of CXCR4 gene expression in patients with liver metastases has prognostic significance for disease outcome. PMID:16794396

Adjustable under-expression of yeast mating pathway proteins in Saccharomyces cerevisiae using a programmed ribosomal frameshift.

PubMed

Choi, Min-Yeon; Park, Sang-Hyun

2016-06-01

Experimental research in molecular biology frequently relies on the promotion or suppression of gene expression, an important tool in the study of its functions. Although yeast is among the most studied model systems with the ease of maintenance and manipulation, current experimental methods are mostly limited to gene deletion, suppression or overexpression of genes. Therefore, the ability to reduce protein expressions and then observing the effects would promote a better understanding of the exact functions and their interactions. Reducing protein expression is mainly limited by the difficulties associated with controlling the reduction level, and in some cases, the initial endogenous abundance is too low. For the under-expression to be useful as an experimental tool, repeatability and stability of reduced expression is important. We found that cis-elements in programmed -1 ribosomal frameshifting (-1RFS) of beet western yellow virus (BWYV) could be utilized to reduced protein expression in Saccharomyces cerevisiae. The two main advantages of using -1RFS are adjustable reduction rates and ease of use. To demonstrate the utility of this under-expression system, examples of reduced protein abundance were shown using yeast mating pathway components. The abundance of MAP kinase Fus3 was reduced to approximately 28-75 % of the wild-type value. Other MAP kinase mating pathway components, including Ste5, Ste11, and Ste7, were also under-expressed to verify that the -1RFS system works with different proteins. Furthermore, reduced Fus3 abundance altered the overall signal transduction outcome of the mating pathway, demonstrating the potential for further studies of signal transduction adjustment via under-expression.

Modulation of N-glycosylation by mesalamine facilitates membranous E-cadherin expression in colon epithelial cells☆

PubMed Central

Khare, Vineeta; Lang, Michaela; Dammann, Kyle; Campregher, Christoph; Lyakhovich, Alex; Gasche, Christoph

2014-01-01

Genome wide association studies have implicated intestinal barrier function genes in the pathogenesis of ulcerative colitis. One of such loci CDH1, encoding E-cadherin, a transmembrane glycoprotein with known tumor suppressor functions, is also linked to the susceptibility to colorectal cancer. Loss of membranous E-cadherin expression is common in both colitis and cancer. We have recently demonstrated that mesalamine (5-ASA); the anti-inflammatory drug used to treat ulcerative colitis, induces membranous expression of E-cadherin and increases intercellular adhesion. Using colorectal cancer epithelial cells with aberrant E-cadherin expression, we investigated the mechanism underlying such an effect of 5-ASA. Post-translational modification of E-cadherin glycosylation was analyzed by biotin/streptavidin detection of sialylated glycoproteins. GnT-III (N-acetylglucosaminyltransferase III) expression was assessed by qRT-PCR, Western blot and immunofluorescence. GnT-III activity was analyzed by reactivity with E-4/L-4-PHA. Expression, localization and interaction of E-cadherin and β-catenin were analyzed by Western blot, immunocytochemistry and RNA interference. 5-ASA activity modulated E-cadherin glycosylation and increased both mRNA and protein levels of GnT-III and its activity as detected by increased E4-lectin reactivity. Intestinal APCMin polyps in mice showed low expression of GnT-III and 5-ASA was effective in increasing its expression. The data demonstrated that remodeling of glycans by GnT-III mediated bisect glycosylation, contributes to the membranous retention of E-cadherin by 5-ASA; facilitating intercellular adhesion. Induction of membranous expression of E-cadherin by 5-ASA is a novel mechanism for mucosal healing in colitis that might impede tumor progression by modulation of GnT-III expression. PMID:24184502

MiR-21 is induced in endothelial cells by shear stress and modulates apoptosis and eNOS activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weber, Martina; Baker, Meredith B.; Moore, Jeffrey P.

Mechanical forces associated with blood flow play an important role in regulating vascular signaling and gene expression in endothelial cells (ECs). MicroRNAs (miRNAs) are a class of noncoding RNAs that posttranscriptionally regulate the expression of genes involved in diverse cell functions, including differentiation, growth, proliferation, and apoptosis. miRNAs are known to have an important role in modulating EC biology, but their expression and functions in cells subjected to shear stress conditions are unknown. We sought to determine the miRNA expression profile in human ECs subjected to unidirectional shear stress and define the role of miR-21 in shear stress-induced changes inmore » EC function. TLDA array and qRT-PCR analysis performed on HUVECs exposed to prolonged unidirectional shear stress (USS, 24 h, 15 dynes/cm{sup 2}) identified 13 miRNAs whose expression was significantly upregulated (p < 0.05). The miRNA with the greatest change was miR-21; it was increased 5.2-fold (p = 0.002) in USS-treated versus control cells. Western analysis demonstrated that PTEN, a known target of miR-21, was downregulated in HUVECs exposed to USS or transfected with pre-miR-21. Importantly, HUVECs overexpressing miR-21 had decreased apoptosis and increased eNOS phosphorylation and nitric oxide (NO{sup {center_dot}}) production. These data demonstrate that shear stress forces regulate the expression of miRNAs in ECs, and that miR-21 influences endothelial biology by decreasing apoptosis and activating the NO{sup {center_dot}} pathway. These studies advance our understanding of the mechanisms by which shear stress forces modulate vascular homeostasis.« less

Expression of an endoglucanase from Tribolium castaneum (TcEG1) in Saccharomyces cerevisiae.

PubMed

Shirley, Derek; Oppert, Cris; Reynolds, Todd B; Miracle, Bethany; Oppert, Brenda; Klingeman, William E; Jurat-Fuentes, Juan Luis

2014-10-01

Insects are a largely unexploited resource in prospecting for novel cellulolytic enzymes to improve the production of ethanol fuel from lignocellulosic biomass. The cost of lignocellulosic ethanol production is expected to decrease by the combination of cellulose degradation (saccharification) and fermentation of the resulting glucose to ethanol in a single process, catalyzed by the yeast Saccharomyces cerevisiae transformed to express efficient cellulases. While S. cerevisiae is an established heterologous expression system, there are no available data on the functional expression of insect cellulolytic enzymes for this species. To address this knowledge gap, S. cerevisiae was transformed to express the full-length cDNA encoding an endoglucanase from the red flour beetle, Tribolium castaneum (TcEG1), and evaluated the activity of the transgenic product (rTcEG1). Expression of the TcEG1 cDNA in S. cerevisiae was under control of the strong glyceraldehyde-3 phosphate dehydrogenase promoter. Cultured transformed yeast secreted rTcEG1 protein as a functional β-1,4-endoglucanase, which allowed transformants to survive on selective media containing cellulose as the only available carbon source. Evaluation of substrate specificity for secreted rTcEG1 demonstrated endoglucanase activity, although some activity was also detected against complex cellulose substrates. Potentially relevant to uses in biofuel production rTcEG1 activity increased with pH conditions, with the highest activity detected at pH 12. Our results demonstrate the potential for functional production of an insect cellulase in S. cerevisiae and confirm the stability of rTcEG1 activity in strong alkaline environments. © 2013 Institute of Zoology, Chinese Academy of Sciences.

Characterization of Wnt/β-catenin signaling in rhabdomyosarcoma.

PubMed

Annavarapu, Srinivas R; Cialfi, Samantha; Dominici, Carlo; Kokai, George K; Uccini, Stefania; Ceccarelli, Simona; McDowell, Heather P; Helliwell, Timothy R

2013-10-01

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and accounts for about 5% of all malignant paediatric tumours. β-Catenin, a multifunctional nuclear transcription factor in the canonical Wnt signaling pathway, is active in myogenesis and embryonal somite patterning. Dysregulation of Wnt signaling facilitates tumour invasion and metastasis. This study characterizes Wnt/β-catenin signaling and functional activity in paediatric embryonal and alveolar RMS. Immunohistochemical assessment of paraffin-embedded tissues from 44 RMS showed β-catenin expression in 26 cases with cytoplasmic/membranous expression in 9/14 cases of alveolar RMS, and 15/30 cases of embryonal RMS, whereas nuclear expression was only seen in 2 cases of embryonal RMS. The potential functional significance of β-catenin expression was tested in four RMS cell lines, two derived from embryonal (RD and RD18) RMS and two from alveolar (Rh4 and Rh30) RMS. Western blot analysis demonstrated the expression of Wnt-associated proteins including β-catenin, glycogen synthase kinase-3β, disheveled, axin-1, naked, LRP-6 and cadherins in all cell lines. Cell fractionation and immunofluorescence studies of the cell lines (after stimulation by human recombinant Wnt3a) showed reduced phosphorylation of β-catenin, stabilization of the active cytosolic form and nuclear translocation of β-catenin. Reporter gene assay demonstrated a T-cell factor/lymphoid-enhancing factor-mediated transactivation in these cells. In response to human recombinant Wnt3a, the alveolar RMS cells showed a significant decrease in proliferation rate and induction of myogenic differentiation (myogenin, MyoD1 and myf5). These data indicate that the central regulatory components of canonical Wnt/β-catenin signaling are expressed and that this pathway is functionally active in a significant subset of RMS tumours and might represent a novel therapeutic target.

Suppression of forkhead box Q1 by microRNA-506 represses the proliferation and epithelial-mesenchymal transition of cervical cancer cells.

PubMed

Zhang, Mingting; Xu, Qingli; Yan, Shufen; Li, Zhigang; Yan, Wei; Jia, Xiaojing

2016-05-01

MicroRNAs (miRNAs) play a pivotal role in cancer progression and development, representing novel therapeutic tools for cancer therapy. Forkhead box Q1 (FOXQ1) functions as an oncogene in various cancer types. However, the functional significance of FOXQ1 in cervical cancer remains unknown. In this study, we investigated the biological function of FOXQ1 in cervical cancer and tested whether or not FOXQ1 can be targeted and regulated by specific miRNAs. We found that FOXQ1 was highly expressed in cervical cancer cell lines. Knockdown of FOXQ1 by small interfering RNA (siRNA) significantly suppressed the proliferation and epithelial-mesenchymal transition (EMT) of cervical cancer cells. FOXQ1 was predicted as a target gene of microRNA-506 (miR-506), and this prediction was validated by dual-luciferase reporter assay. Quantitative real-time PCR and western blot analyses demonstrated that mRNA and protein expression was negatively regulated by miR-506. The expression of miR-506 was downregulated in cervical cancer tissues, and miR-506 expression was inversely correlated with FOXQ1 expression in cervical cancer. The overexpression of miR-506 dramatically suppressed the proliferation and EMT of cervical cancer cells that mimicked the suppression of FOXO1 siRNA. Furthermore, the restoration of FOXQ1 expression significantly reversed the inhibitory effect of miR-506. Overall, our study demonstrated that miR-506 inhibited the proliferation and EMT of cervical cancer cells by targeting FOXQ1 and provided evidence that the miR-506/FOXQ1 axis plays an important role in the pathogenesis of cervical cancer, representing potential molecular targets for the development of anticancer agents for cervical cancer treatment.

TALE factors use two distinct functional modes to control an essential zebrafish gene expression program.

PubMed

Ladam, Franck; Stanney, William; Donaldson, Ian J; Yildiz, Ozge; Bobola, Nicoletta; Sagerström, Charles G

2018-06-18

TALE factors are broadly expressed embryonically and known to function in complexes with transcription factors (TFs) like Hox proteins at gastrula/segmentation stages, but it is unclear if such generally expressed factors act by the same mechanism throughout embryogenesis. We identify a TALE-dependent gene regulatory network (GRN) required for anterior development and detect TALE occupancy associated with this GRN throughout embryogenesis. At blastula stages, we uncover a novel functional mode for TALE factors, where they occupy genomic DECA motifs with nearby NF-Y sites. We demonstrate that TALE and NF-Y form complexes and regulate chromatin state at genes of this GRN. At segmentation stages, GRN-associated TALE occupancy expands to include HEXA motifs near PBX:HOX sites. Hence, TALE factors control a key GRN, but utilize distinct DNA motifs and protein partners at different stages - a strategy that may also explain their oncogenic potential and may be employed by other broadly expressed TFs. © 2018, Ladam et al.

The Multifaceted Effects of Agmatine on Functional Recovery after Spinal Cord Injury through Modulations of BMP-2/4/7 Expressions in Neurons and Glial Cells

PubMed Central

Park, Yu Mi; Lee, Won Taek; Bokara, Kiran Kumar; Seo, Su Kyoung; Park, Seung Hwa; Kim, Jae Hwan; Yenari, Midori A.; Park, Kyung Ah; Lee, Jong Eun

2013-01-01

Presently, few treatments for spinal cord injury (SCI) are available and none have facilitated neural regeneration and/or significant functional improvement. Agmatine (Agm), a guanidinium compound formed from decarboxylation of L-arginine by arginine decarboxylase, is a neurotransmitter/neuromodulator and been reported to exert neuroprotective effects in central nervous system injury models including SCI. The purpose of this study was to demonstrate the multifaceted effects of Agm on functional recovery and remyelinating events following SCI. Compression SCI in mice was produced by placing a 15 g/mm2 weight for 1 min at thoracic vertebra (Th) 9 segment. Mice that received an intraperitoneal (i.p.) injection of Agm (100 mg/kg/day) within 1 hour after SCI until 35 days showed improvement in locomotor recovery and bladder function. Emphasis was made on the analysis of remyelination events, neuronal cell preservation and ablation of glial scar area following SCI. Agm treatment significantly inhibited the demyelination events, neuronal loss and glial scar around the lesion site. In light of recent findings that expressions of bone morphogenetic proteins (BMPs) are modulated in the neuronal and glial cell population after SCI, we hypothesized whether Agm could modulate BMP- 2/4/7 expressions in neurons, astrocytes, oligodendrocytes and play key role in promoting the neuronal and glial cell survival in the injured spinal cord. The results from computer assisted stereological toolbox analysis (CAST) demonstrate that Agm treatment dramatically increased BMP- 2/7 expressions in neurons and oligodendrocytes. On the other hand, BMP- 4 expressions were significantly decreased in astrocytes and oligodendrocytes around the lesion site. Together, our results reveal that Agm treatment improved neurological and histological outcomes, induced oligodendrogenesis, protected neurons, and decreased glial scar formation through modulating the BMP- 2/4/7 expressions following SCI. PMID:23349763

Tribbles 3 inhibits brown adipocyte differentiation and function by suppressing insulin signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jeong, Ha-Won; Choi, Ran Hee; McClellan, Jamie L.

Recent studies have demonstrated that adult humans have substantial amounts of functioning brown adipose tissue (BAT). Since BAT has been implicated as an anti-obese and anti-diabetic tissue, it is important to understand the signaling molecules that regulate BAT function. There has been a link between insulin signaling and BAT metabolism as deletion or pharmaceutical inhibition of insulin signaling impairs BAT differentiation and function. Tribbles 3 (TRB3) is a pseudo kinase that has been shown to regulate metabolism and insulin signaling in multiple tissues but the role of TRB3 in BAT has not been studied. In this study, we found thatmore » TRB3 expression was present in BAT and overexpression of TRB3 in brown preadipocytes impaired differentiation and decreased expression of BAT markers. Furthermore, TRB3 overexpression resulted in significantly lower oxygen consumption rates for basal and proton leakage, indicating decreased BAT activity. Based on previous studies showing that deletion or pharmaceutical inhibition of insulin signaling impairs BAT differentiation and function, we assessed insulin signaling in brown preadipocytes and BAT in vivo. Overexpression of TRB3 in cells impaired insulin-stimulated IRS1 and Akt phosphorylation, whereas TRB3KO mice displayed improved IRS1 and Akt phosphorylation. Finally, deletion of IRS1 abolished the function of TRB3 to regulate BAT differentiation and metabolism. These data demonstrate that TRB3 inhibits insulin signaling in BAT, resulting in impaired differentiation and function. - Highlights: • TRB3 is expressed in brown adipose tissue and its expression is increased during differentiation. • Overexpression of TRB3 inhibits differentiation and its activity. • Overexpression of TRB3 in brown preadipocytes inhibits insulin signaling. • TRB3KO mice displays improved insulin signaling in brown adipose tissue. • Insulin signaling is required for the effects of TRB3 to regulate brown adipose tissue differentiation and activity.« less

3D-printed gelatin scaffolds of differing pore geometry modulate hepatocyte function and gene expression.

PubMed

Lewis, Phillip L; Green, Richard M; Shah, Ramille N

2018-03-15

Three dimensional (3D) printing is highly amenable to the fabrication of tissue-engineered organs of a repetitive microstructure such as the liver. The creation of uniform and geometrically repetitive tissue scaffolds can also allow for the control over cellular aggregation and nutrient diffusion. However, the effect of differing geometries, while controlling for pore size, has yet to be investigated in the context of hepatocyte function. In this study, we show the ability to precisely control pore geometry of 3D-printed gelatin scaffolds. An undifferentiated hepatocyte cell line (HUH7) demonstrated high viability and proliferation when seeded on 3D-printed scaffolds of two different geometries. However, hepatocyte specific functions (albumin secretion, CYP activity, and bile transport) increases in more interconnected 3D-printed gelatin cultures compared to a less interconnected geometry and to 2D controls. Additionally, we also illustrate the disparity between gene expression and protein function in simple 2D culture modes, and that recreation of a physiologically mimetic 3D environment is necessary to induce both expression and function of cultured hepatocytes. Three dimensional (3D) printing provides tissue engineers the ability spatially pattern cells and materials in precise geometries, however the biological effects of scaffold geometry on soft tissues such as the liver have not been rigorously investigated. In this manuscript, we describe a method to 3D print gelatin into well-defined repetitive geometries that show clear differences in biological effects on seeded hepatocytes. We show that a relatively simple and widely used biomaterial, such as gelatin, can significantly modulate biological processes when fabricated into specific 3D geometries. Furthermore, this study expands upon past research into hepatocyte aggregation by demonstrating how it can be manipulated to enhance protein function, and how function and expression may not precisely correlate in 2D models. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

An essential cell-autonomous role for hepcidin in cardiac iron homeostasis

PubMed Central

Lakhal-Littleton, Samira; Wolna, Magda; Chung, Yu Jin; Christian, Helen C; Heather, Lisa C; Brescia, Marcella; Ball, Vicky; Diaz, Rebeca; Santos, Ana; Biggs, Daniel; Clarke, Kieran; Davies, Benjamin; Robbins, Peter A

2016-01-01

Hepcidin is the master regulator of systemic iron homeostasis. Derived primarily from the liver, it inhibits the iron exporter ferroportin in the gut and spleen, the sites of iron absorption and recycling respectively. Recently, we demonstrated that ferroportin is also found in cardiomyocytes, and that its cardiac-specific deletion leads to fatal cardiac iron overload. Hepcidin is also expressed in cardiomyocytes, where its function remains unknown. To define the function of cardiomyocyte hepcidin, we generated mice with cardiomyocyte-specific deletion of hepcidin, or knock-in of hepcidin-resistant ferroportin. We find that while both models maintain normal systemic iron homeostasis, they nonetheless develop fatal contractile and metabolic dysfunction as a consequence of cardiomyocyte iron deficiency. These findings are the first demonstration of a cell-autonomous role for hepcidin in iron homeostasis. They raise the possibility that such function may also be important in other tissues that express both hepcidin and ferroportin, such as the kidney and the brain. DOI: http://dx.doi.org/10.7554/eLife.19804.001 PMID:27897970

Characterization, expression patterns and functional analysis of the MAPK and MAPKK genes in watermelon (Citrullus lanatus).

PubMed

Song, Qiuming; Li, Dayong; Dai, Yi; Liu, Shixia; Huang, Lei; Hong, Yongbo; Zhang, Huijuan; Song, Fengming

2015-12-23

Mitogen-activated protein kinase (MAPK) cascades, which consist of three functionally associated protein kinases, namely MEKKs, MKKs and MPKs, are universal signaling modules in all eukaryotes and have been shown to play critical roles in many physiological and biochemical processes in plants. However, little or nothing is known about the MPK and MKK families in watermelon. In the present study, we performed a systematic characterization of the ClMPK and ClMKK families including the identification and nomenclature, chromosomal localization, phylogenetic relationships, ClMPK-ClMKK interactions, expression patterns in different tissues and in response to abiotic and biotic stress and transient expression-based functional analysis for their roles in disease resistance. Genome-wide survey identified fifteen ClMPK and six ClMKK genes in watermelon genome and phylogenetic analysis revealed that both of the ClMPK and ClMKK families can be classified into four distinct groups. Yeast two-hybrid assays demonstrated significant interactions between members of the ClMPK and ClMKK families, defining putative ClMKK2-1/ClMKK6-ClMPK4-1/ClMPK4-2/ClMPK13 and ClMKK5-ClMPK6 cascades. Most of the members in the ClMPK and ClMKK families showed differential expression patterns in different tissues and in response to abiotic (e.g. drought, salt, cold and heat treatments) and biotic (e.g. infection of Fusarium oxysporum f. sp. niveum) stresses. Transient expression of ClMPK1, ClMPK4-2 and ClMPK7 in Nicotiana benthamiana resulted in enhanced resistance to Botrytis cinerea and upregulated expression of defense genes while transient expression of ClMPK6 and ClMKK2-2 led to increased susceptibility to B. cinerea. Furthermore, transient expression of ClMPK7 also led to hypersensitive response (HR)-like cell death and significant accumulation of H2O2 in N. benthamiana. We identified fifteen ClMPK and six ClMKK genes from watermelon and analyzed their phylogenetic relationships, expression patterns and protein-protein interactions and functions in disease resistance. Our results demonstrate that ClMPK1, ClMPK4-2 and ClMPK7 positively but ClMPK6 and ClMKK2-2 negatively regulate the resistance to B. cinerea when transiently expressed in N. benthamiana and that ClMPK7 functions as a regulator of HR-like cell death through modulating the generation of H2O2.

Tissue Inhibitor of Metalloproteinases–3 Moderates the Proinflammatory Status of Macrophages

PubMed Central

Gharib, Sina A.; Bench, Eli M.; Sussman, Samuel W.; Wang, Roy T.; Rims, Cliff; Birkland, Timothy P.; Wang, Ying; Manicone, Anne M.; McGuire, John K.; Parks, William C.

2013-01-01

Tissue inhibitor of metalloproteinases–3 (TIMP-3) has emerged as a key mediator of inflammation. Recently, we reported that the resolution of inflammation is impaired in Timp3−/− mice after bleomycin-induced lung injury. Here, we demonstrate that after LPS instillation (another model of acute lung injury), Timp3−/− mice demonstrate enhanced and persistent neutrophilia, increased numbers of infiltrated macrophages, and delayed weight gain, compared with wild-type (WT) mice. Because macrophages possess broad immune functions and can differentiate into cells that either stimulate inflammation (M1 macrophages) or are immunosuppressive (M2 macrophages), we examined whether TIMP-3 influences macrophage polarization. Comparisons of the global gene expression of unstimulated or LPS-stimulated bone marrow–derived macrophages (BMDMs) from WT and Timp3−/− mice revealed that Timp3−/− BMDMs exhibited an increased expression of genes associated with proinflammatory (M1) macrophages, including Il6, Il12, Nos2, and Ccl2. Microarray analyses also revealed a baseline difference in gene expression between WT and Timp3−/− BMDMs, suggesting altered macrophage differentiation. Furthermore, the treatment of Timp3−/− BMDMs with recombinant TIMP-3 rescued this altered gene expression. We also examined macrophage function, and found that Timp3−/− M1 cells exhibit significantly more neutrophil chemotactic activity and significantly less soluble Fas ligand–induced caspase-3/7 activity, a marker of apoptosis, compared with WT M1 cells. Macrophage differentiation into immunosuppressive M2 cells is mediated by exposure to IL-4/IL-13, and we found that Timp3−/− M2 macrophages demonstrated a lower expression of genes associated with an anti-inflammatory phenotype, compared with WT M2 cells. Collectively, these findings indicate that TIMP-3 functions to moderate the differentiation of macrophages into proinflammatory (M1) cells. PMID:23742180

Carbonic anhydrase III regulates peroxisome proliferator-activated receptor-{gamma}2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mitterberger, Maria C.; Kim, Geumsoo; Rostek, Ursula

2012-05-01

Carbonic anhydrase III (CAIII) is an isoenzyme of the CA family. Because of its low specific anhydrase activity, physiological functions in addition to hydrating CO{sub 2} have been proposed. CAIII expression is highly induced in adipogenesis and CAIII is the most abundant protein in adipose tissues. The function of CAIII in both preadipocytes and adipocytes is however unknown. In the present study we demonstrate that adipogenesis is greatly increased in mouse embryonic fibroblasts (MEFs) from CAIII knockout (KO) mice, as demonstrated by a greater than 10-fold increase in the induction of fatty acid-binding protein-4 (FABP4) and increased triglyceride formation inmore » CAIII{sup -/-} MEFs compared with CAIII{sup +/+} cells. To address the underlying mechanism, we investigated the expression of the two adipogenic key regulators, peroxisome proliferator-activated receptor-{gamma}2 (PPAR{gamma}2) and CCAAT/enhancer binding protein-{alpha}. We found a considerable (approximately 1000-fold) increase in the PPAR{gamma}2 expression in the CAIII{sup -/-} MEFs. Furthermore, RNAi-mediated knockdown of endogenous CAIII in NIH 3T3-L1 preadipocytes resulted in a significant increase in the induction of PPAR{gamma}2 and FABP4. When both CAIII and PPAR{gamma}2 were knocked down, FABP4 was not induced. We conclude that down-regulation of CAIII in preadipocytes enhances adipogenesis and that CAIII is a regulator of adipogenic differentiation which acts at the level of PPAR{gamma}2 gene expression. -- Highlights: Black-Right-Pointing-Pointer We discover a novel function of Carbonic anhydrase III (CAIII). Black-Right-Pointing-Pointer We show that CAIII is a regulator of adipogenesis. Black-Right-Pointing-Pointer We demonstrate that CAIII acts at the level of PPAR{gamma}2 gene expression. Black-Right-Pointing-Pointer Our data contribute to a better understanding of the role of CAIII in fat tissue.« less

Expression of cyclophilin B is associated with malignant progression and regulation of genes implicated in the pathogenesis of breast cancer.

PubMed

Fang, Feng; Flegler, Ayanna J; Du, Pan; Lin, Simon; Clevenger, Charles V

2009-01-01

Cyclophilin B (CypB) is a 21-kDa protein with peptidyl-prolyl cis-trans isomerase activity that functions as a transcriptional inducer for Stat5 and as a ligand for CD147. To better understand the global function of CypB in breast cancer, T47D cells with a small interfering RNA-mediated knockdown of CypB were generated. Subsequent expression profiling analysis showed that 663 transcripts were regulated by CypB knockdown, and that many of these gene products contributed to cell proliferation, cell motility, and tumorigenesis. Real-time PCR confirmed that STMN3, S100A4, S100A6, c-Myb, estrogen receptor alpha, growth hormone receptor, and progesterone receptor were all down-regulated in si-CypB cells. A linkage analysis of these array data to protein networks resulted in the identification of 27 different protein networks that were impacted by CypB knockdown. Functional assays demonstrated that CypB knockdown also decreased cell growth, proliferation, and motility. Immunohistochemical and immunofluorescent analyses of a matched breast cancer progression tissue microarray that was labeled with an anti-CypB antibody demonstrated a highly significant increase in CypB protein levels as a function of breast cancer progression. Taken together, these results suggest that the enhanced expression of CypB in malignant breast epithelium may contribute to the pathogenesis of this disease through its regulation of the expression of hormone receptors and gene products that are involved in cell proliferation and motility.

Expression of Cyclophilin B is Associated with Malignant Progression and Regulation of Genes Implicated in the Pathogenesis of Breast Cancer

PubMed Central

Fang, Feng; Flegler, Ayanna J.; Du, Pan; Lin, Simon; Clevenger, Charles V.

2009-01-01

Cyclophilin B (CypB) is a 21-kDa protein with peptidyl-prolyl cis-trans isomerase activity that functions as a transcriptional inducer for Stat5 and as a ligand for CD147. To better understand the global function of CypB in breast cancer, T47D cells with a small interfering RNA-mediated knockdown of CypB were generated. Subsequent expression profiling analysis showed that 663 transcripts were regulated by CypB knockdown, and that many of these gene products contributed to cell proliferation, cell motility, and tumorigenesis. Real-time PCR confirmed that STMN3, S100A4, S100A6, c-Myb, estrogen receptor α, growth hormone receptor, and progesterone receptor were all down-regulated in si-CypB cells. A linkage analysis of these array data to protein networks resulted in the identification of 27 different protein networks that were impacted by CypB knockdown. Functional assays demonstrated that CypB knockdown also decreased cell growth, proliferation, and motility. Immunohistochemical and immunofluorescent analyses of a matched breast cancer progression tissue microarray that was labeled with an anti-CypB antibody demonstrated a highly significant increase in CypB protein levels as a function of breast cancer progression. Taken together, these results suggest that the enhanced expression of CypB in malignant breast epithelium may contribute to the pathogenesis of this disease through its regulation of the expression of hormone receptors and gene products that are involved in cell proliferation and motility. PMID:19056847

Knockdown of long non-coding RNA HOTAIR increases miR-454-3p by targeting Stat3 and Atg12 to inhibit chondrosarcoma growth

PubMed Central

Bao, Xing; Ren, Tingting; Huang, Yi; Sun, Kunkun; Wang, Shidong; Liu, Kuisheng; Zheng, Bingxin; Guo, Wei

2017-01-01

Current practices for the therapy of chondrosarcoma, including wide-margin surgical resection and chemotherapy, are less than satisfactory. Recently, emerging evidence has demonstrated that long non-coding RNAs (lncRNAs) have an essential role in the initiation and progression of tumors. As a typical lncRNA, HOTAIR is significantly overexpressed in various tumors. However, the function and potential biological mechanisms of HOTAIR in human chondrosarcoma remain unknown. Quantitative RT-PCR demonstrated that HOTAIR expression was upregulated in chondrosarcoma tissues and cell lines. High HOTAIR expression is correlated with tumor stage and poor prognosis. Functional experiments reveal that HOTAIR knockdown leads to growth inhibition of human chondrosarcoma cells in vitro and in vivo. In addition to cycle arrest and apoptosis, knockdown of HOTAIR inhibits autophagy, which favors cell death. Mechanistically, we demonstrated that HOTAIR induced DNA methylation of miR-454-3p by recruiting EZH2 and DNMT1 to the miR-454-3p promoter regions, which markedly silences miR-454-3p expression. Further analysis revealed that STAT3 and ATG12 are targets of miR-454-3p, initiate HOTAIR deficiency-induced apoptosis and reduce autophagy. Collectively, our data reveal the roles and functional mechanisms of HOTAIR in human chondrosarcoma and suggest that HOTAIR may act as a prognostic biomarker and potential therapeutic target for chondrosarcoma. PMID:28182000

Microarray profiling of gene expression in human adipocytes in response to anthocyanins.

PubMed

Tsuda, Takanori; Ueno, Yuki; Yoshikawa, Toshikazu; Kojo, Hitoshi; Osawa, Toshihiko

2006-04-14

Adipocyte dysfunction is strongly associated with the development of obesity and insulin resistance. It is accepted that the regulation of adipocytokine secretion or the adipocyte specific gene expression is one of the most important targets for the prevention of obesity and amelioration of insulin sensitivity. Recently, we demonstrated that anthocyanins, which are pigments widespread in the plant kingdom, have the potency of anti-obesity in mice and the enhancement adipocytokine secretion and its gene expression in adipocytes. In this study, we have shown the gene expression profile in human adipocytes treated with anthocyanins (cyanidin 3-glucoside; C3G or cyanidin; Cy). The human adipocytes were treated with 100 microM C3G, Cy or vehicle for 24 h. The total RNA from the adipocytes was isolated and carried out GeneChip microarray analysis. Based on the gene expression profile, we demonstrated the significant changes of adipocytokine expression (up-regulation of adiponectin and down-regulation of plasminogen activator inhibitor-1 and interleukin-6). Some of lipid metabolism related genes (uncoupling protein2, acylCoA oxidase1 and perilipin) also significantly induced in both common the C3G or Cy treatment groups. These studies have provided an overview of the gene expression profiles in human adipocytes treated with anthocyanins and demonstrated that anthocyanins can regulate adipocytokine gene expression to ameliorate adipocyte function related with obesity and diabetes that merit further investigation.

ICAM-1 (CD54) expression on B lymphocytes is associated with their costimulatory function and can be increased by coactivation with IL-1 and IL-7.

PubMed

Dennig, D; Lacerda, J; Yan, Y; Gasparetto, C; O'Reilly, R J

1994-07-01

Recent studies have demonstrated that acute lymphoblastic leukemia-derived pre-B cell lines are deficient in their costimulatory function for T cell proliferation in response to the mitogen Con A and the superantigens TSST-1 and SEB. Stimulation of these pre-B cells with IL-7 increased their costimulatory function which involved the B7/CD28 pathway. In the present study, we stimulated T cells with Con A, TSST-1, and SEB in the presence of peripheral blood B lineage cells that do not constitutively express B7/BB1 on their surface and investigated whether their costimulatory function could also be enhanced by IL-7. We found that, in the presence of IL-1, stimulation with IL-7 increased the costimulatory function of B cells and their surface expression level of ICAM-1 (CD54). We then investigated whether costimulatory B cell function could be inhibited by blocking the ICAM-1/LFA-1 pathway. Addition of anti-ICAM-1 mAb to the coculture of T and B cells inhibited T cell proliferation by approximately 20%. In contrast, addition of anti-LFA-1 beta (CD18) mAb, directed against the T cell ligand of ICAM-1, inhibited T cell proliferation almost completely. To determine the role of ICAM-1 in costimulatory B cell function, we sorted B cells into ICAM-1low-and ICAM-1high-expressing populations. We found that B cells expressing high levels of surface ICAM-1 elicited significantly higher T cell responses than those with low levels, suggesting that the expression level of ICAM-1 on peripheral blood B cells correlates with their costimulatory function.

Drug screens based on the newly found role of dystroglycan proteolysis and restoration of dystroglycan function thereof

DOEpatents

Bissell, Mina J.; Muschler, John L.

2010-02-23

The present invention provides methods and compositions for the diagnosis and treatment of cells lacking normal growth arresting characteristic. The present invention demonstrates that many tumor cells lack normal cell surface .alpha.-dystroglycan and thereby lack dystroglycan function. Dystroglycan can be lost from the cell surface by proteolytic shedding of a fragment of .alpha.-dystroglycan into the surrounding medium. Upon restoration of dystroglycan function and over-expression of the dystroglycan gene, the once tumorigenic cells revert to non-tumorigenic cells which polarize and arrest cell growth in the presence of basement membrane proteins, demonstrating that dystroglycan functions as a tumor marker and suppressor.

A portable expression resource for engineering cross-species genetic circuits and pathways

PubMed Central

Kushwaha, Manish; Salis, Howard M.

2015-01-01

Genetic circuits and metabolic pathways can be reengineered to allow organisms to process signals and manufacture useful chemicals. However, their functions currently rely on organism-specific regulatory parts, fragmenting synthetic biology and metabolic engineering into host-specific domains. To unify efforts, here we have engineered a cross-species expression resource that enables circuits and pathways to reuse the same genetic parts, while functioning similarly across diverse organisms. Our engineered system combines mixed feedback control loops and cross-species translation signals to autonomously self-regulate expression of an orthogonal polymerase without host-specific promoters, achieving nontoxic and tuneable gene expression in diverse Gram-positive and Gram-negative bacteria. Combining 50 characterized system variants with mechanistic modelling, we show how the cross-species expression resource's dynamics, capacity and toxicity are controlled by the control loops' architecture and feedback strengths. We also demonstrate one application of the resource by reusing the same genetic parts to express a biosynthesis pathway in both model and non-model hosts. PMID:26184393

Long noncoding RNA AB074169 inhibits cell proliferation via modulation of KHSRP-mediated p21 expression in papillary thyroid carcinoma.

PubMed

Gou, Qiheng; Gao, Linbo; Nie, Xinwen; Pu, Wenchen; Zhu, Jingqiang; Wang, Yichao; Liu, Xuesha; Tan, Shuangyan; Zhou, Jian-Kang; Gong, Yanqiu; He, Juan; Wu, Ke; Xie, Yuxin; Zhao, Wanjun; Dai, Lunzhi; Liu, Lunxu; Xiang, Rong; Wei, Yu-Quan; Zhang, Lin; Peng, Yong

2018-05-07

Long noncoding RNAs (lncRNAs) are emerging as a novel class of regulators in gene expression associated with tumorigenesis. However, the role of lncRNAs in papillary thyroid carcinoma (PTC) is poorly understood. Here we conducted global lncRNA profiling and identified lncRNA AB074169 (lncAB) as significantly downregulated in PTC. Decreased expression of lncAB in PTC was caused by CpG hypermethylation within its gene promoter. Functional studies showed that lncAB overexpression led to cell cycle arrest and tumor growth inhibition in vitro and in vivo, whereas lncAB knockdown promoted cell proliferation. Mechanistic analyses revealed that lncAB bound KH-type splicing regulatory protein (KHSRP) and also decreased expression of KHSRP, thus increasing CDKN1a (p21) expression and decreasing CDK2 expression to repress cell proliferation. Taken together, these findings demonstrate that lncAB functions as a tumor suppressor during PTC tumorigenesis. Copyright ©2018, American Association for Cancer Research.

The lineage-specific gene ponzr1 is essential for zebrafish pronephric and pharyngeal arch development.

PubMed

Bedell, Victoria M; Person, Anthony D; Larson, Jon D; McLoon, Anna; Balciunas, Darius; Clark, Karl J; Neff, Kevin I; Nelson, Katie E; Bill, Brent R; Schimmenti, Lisa A; Beiraghi, Soraya; Ekker, Stephen C

2012-02-01

The Homeobox (Hox) and Paired box (Pax) gene families are key determinants of animal body plans and organ structure. In particular, they function within regulatory networks that control organogenesis. How these conserved genes elicit differences in organ form and function in response to evolutionary pressures is incompletely understood. We molecularly and functionally characterized one member of an evolutionarily dynamic gene family, plac8 onzin related protein 1 (ponzr1), in the zebrafish. ponzr1 mRNA is expressed early in the developing kidney and pharyngeal arches. Using ponzr1-targeting morpholinos, we show that ponzr1 is required for formation of the glomerulus. Loss of ponzr1 results in a nonfunctional glomerulus but retention of a functional pronephros, an arrangement similar to the aglomerular kidneys found in a subset of marine fish. ponzr1 is integrated into the pax2a pathway, with ponzr1 expression requiring pax2a gene function, and proper pax2a expression requiring normal ponzr1 expression. In addition to pronephric function, ponzr1 is required for pharyngeal arch formation. We functionally demonstrate that ponzr1 can act as a transcription factor or co-factor, providing the first molecular mode of action for this newly described gene family. Together, this work provides experimental evidence of an additional mechanism that incorporates evolutionarily dynamic, lineage-specific gene families into conserved regulatory gene networks to create functional organ diversity.

Heterologous Expression of the Clostridium carboxidivorans CO Dehydrogenase Alone or Together with the Acetyl Coenzyme A Synthase Enables both Reduction of CO2 and Oxidation of CO by Clostridium acetobutylicum

PubMed Central

Carlson, Ellinor D.

2017-01-01

ABSTRACT With recent advances in synthetic biology, CO2 could be utilized as a carbon feedstock by native or engineered organisms, assuming the availability of electrons. Two key enzymes used in autotrophic CO2 fixation are the CO dehydrogenase (CODH) and acetyl coenzyme A (acetyl-CoA) synthase (ACS), which form a bifunctional heterotetrameric complex. The CODH/ACS complex can reversibly catalyze CO2 to CO, effectively enabling a biological water-gas shift reaction at ambient temperatures and pressures. The CODH/ACS complex is part of the Wood-Ljungdahl pathway (WLP) used by acetogens to fix CO2, and it has been well characterized in native hosts. So far, only a few recombinant CODH/ACS complexes have been expressed in heterologous hosts, none of which demonstrated in vivo CO2 reduction. Here, functional expression of the Clostridium carboxidivorans CODH/ACS complex is demonstrated in the solventogen Clostridium acetobutylicum, which was engineered to express CODH alone or together with the ACS. Both strains exhibited CO2 reduction and CO oxidation activities. The CODH reactions were interrogated using isotopic labeling, thus verifying that CO was a direct product of CO2 reduction, and vice versa. CODH apparently uses a native C. acetobutylicum ferredoxin as an electron carrier for CO2 reduction. Heterologous CODH activity depended on actively growing cells and required the addition of nickel, which is inserted into CODH without the need to express the native Ni insertase protein. Increasing CO concentrations in the gas phase inhibited CODH activity and altered the metabolite profile of the CODH-expressing cells. This work provides the foundation for engineering a complete and functional WLP in nonnative host organisms. IMPORTANCE Functional expression of CO dehydrogenase (CODH) from Clostridium carboxidivorans was demonstrated in C. acetobutylicum, which is natively incapable of CO2 fixation. The expression of CODH, alone or together with the C. carboxidivorans acetyl-CoA synthase (ACS), enabled C. acetobutylicum to catalyze both CO2 reduction and CO oxidation. Importantly, CODH exhibited activity in both the presence and absence of ACS. 13C-tracer studies confirmed that the engineered C. acetobutylicum strains can reduce CO2 to CO and oxidize CO during growth on glucose. PMID:28625981

Heterologous Expression of the Clostridium carboxidivorans CO Dehydrogenase Alone or Together with the Acetyl Coenzyme A Synthase Enables both Reduction of CO2 and Oxidation of CO by Clostridium acetobutylicum.

PubMed

Carlson, Ellinor D; Papoutsakis, Eleftherios T

2017-08-15

With recent advances in synthetic biology, CO 2 could be utilized as a carbon feedstock by native or engineered organisms, assuming the availability of electrons. Two key enzymes used in autotrophic CO 2 fixation are the CO dehydrogenase (CODH) and acetyl coenzyme A (acetyl-CoA) synthase (ACS), which form a bifunctional heterotetrameric complex. The CODH/ACS complex can reversibly catalyze CO 2 to CO, effectively enabling a biological water-gas shift reaction at ambient temperatures and pressures. The CODH/ACS complex is part of the Wood-Ljungdahl pathway (WLP) used by acetogens to fix CO 2 , and it has been well characterized in native hosts. So far, only a few recombinant CODH/ACS complexes have been expressed in heterologous hosts, none of which demonstrated in vivo CO 2 reduction. Here, functional expression of the Clostridium carboxidivorans CODH/ACS complex is demonstrated in the solventogen Clostridium acetobutylicum , which was engineered to express CODH alone or together with the ACS. Both strains exhibited CO 2 reduction and CO oxidation activities. The CODH reactions were interrogated using isotopic labeling, thus verifying that CO was a direct product of CO 2 reduction, and vice versa. CODH apparently uses a native C. acetobutylicum ferredoxin as an electron carrier for CO 2 reduction. Heterologous CODH activity depended on actively growing cells and required the addition of nickel, which is inserted into CODH without the need to express the native Ni insertase protein. Increasing CO concentrations in the gas phase inhibited CODH activity and altered the metabolite profile of the CODH-expressing cells. This work provides the foundation for engineering a complete and functional WLP in nonnative host organisms. IMPORTANCE Functional expression of CO dehydrogenase (CODH) from Clostridium carboxidivorans was demonstrated in C. acetobutylicum , which is natively incapable of CO 2 fixation. The expression of CODH, alone or together with the C. carboxidivorans acetyl-CoA synthase (ACS), enabled C. acetobutylicum to catalyze both CO 2 reduction and CO oxidation. Importantly, CODH exhibited activity in both the presence and absence of ACS. 13 C-tracer studies confirmed that the engineered C. acetobutylicum strains can reduce CO 2 to CO and oxidize CO during growth on glucose. Copyright © 2017 American Society for Microbiology.

Genes Responsive to Low-Intensity Pulsed Ultrasound in MC3T3-E1 Preosteoblast Cells

PubMed Central

Tabuchi, Yoshiaki; Sugahara, Yuuki; Ikegame, Mika; Suzuki, Nobuo; Kitamura, Kei-ichiro; Kondo, Takashi

2013-01-01

Although low-intensity pulsed ultrasound (LIPUS) has been shown to enhance bone fracture healing, the underlying mechanism of LIPUS remains to be fully elucidated. Here, to better understand the molecular mechanism underlying cellular responses to LIPUS, we investigated gene expression profiles in mouse MC3T3-E1 preosteoblast cells exposed to LIPUS using high-density oligonucleotide microarrays and computational gene expression analysis tools. Although treatment of the cells with a single 20-min LIPUS (1.5 MHz, 30 mW/cm2) did not affect the cell growth or alkaline phosphatase activity, the treatment significantly increased the mRNA level of Bglap. Microarray analysis demonstrated that 38 genes were upregulated and 37 genes were downregulated by 1.5-fold or more in the cells at 24-h post-treatment. Ingenuity pathway analysis demonstrated that the gene network U (up) contained many upregulated genes that were mainly associated with bone morphology in the category of biological functions of skeletal and muscular system development and function. Moreover, the biological function of the gene network D (down), which contained downregulated genes, was associated with gene expression, the cell cycle and connective tissue development and function. These results should help to further clarify the molecular basis of the mechanisms of the LIPUS response in osteoblast cells. PMID:24252911

WNT10B Functional Dualism: β-Catenin/Tcf-dependent Growth Promotion or Independent Suppression with Deregulated Expression in Cancer

PubMed Central

Yoshikawa, Hirohide; Matsubara, Kenichi; Zhou, Xiaoling; Okamura, Shu; Kubo, Takahiko; Murase, Yaeko; Shikauchi, Yuko; Esteller, Manel; Herman, James G.; Wei Wang, Xin

2007-01-01

We found aberrant DNA methylation of the WNT10B promoter region in 46% of primary hepatocellular carcinoma (HCC) and 15% of colon cancer samples. Three of 10 HCC and one of two colon cancer cell lines demonstrated low or no expression, and 5-aza-2′deoxycytidine reactivated WNT10B expression with the induction of demethylation, indicating that WNT10B is silenced by DNA methylation in some cancers, whereas WNT10B expression is up-regulated in seven of the 10 HCC cell lines and a colon cancer cell line. These results indicate that WNT10B can be deregulated by either overexpression or silencing in cancer. We found that WNT10B up-regulated β-catenin/Tcf activity. However, WNT10B-overexpressing cells demonstrated a reduced growth rate and anchorage-independent growth that is independent of the β-catenin/Tcf activation, because mutant β-catenin–transduced cells did not suppress growth, and dominant-negative hTcf-4 failed to alleviate the growth suppression by WNT10B. Although WNT10B expression alone inhibits cell growth, it acts synergistically with the fibroblast growth factor (FGF) to stimulate cell growth. WNT10B is bifunctional, one function of which is involved in β-catenin/Tcf activation, and the other function is related to the down-regulation of cell growth through a different mechanism. We suggest that FGF switches WNT10B from a negative to a positive cell growth regulator. PMID:17761539

Effector Regulatory T Cell Differentiation and Immune Homeostasis Depend on the Transcription Factor Myb.

PubMed

Dias, Sheila; D'Amico, Angela; Cretney, Erika; Liao, Yang; Tellier, Julie; Bruggeman, Christine; Almeida, Francisca F; Leahy, Jamie; Belz, Gabrielle T; Smyth, Gordon K; Shi, Wei; Nutt, Stephen L

2017-01-17

FoxP3-expressing regulatory T (Treg) cells are essential for maintaining immune homeostasis. Activated Treg cells undergo further differentiation into an effector state that highly expresses genes critical for Treg cell function, although how this process is coordinated on a transcriptional level is poorly understood. Here, we demonstrate that mice lacking the transcription factor Myb in Treg cells succumbed to a multi-organ inflammatory disease. Myb was specifically expressed in, and required for the differentiation of, thymus-derived effector Treg cells. The combination of transcriptome and genomic footprint analyses revealed that Myb directly regulated a large proportion of the gene expression specific to effector Treg cells, identifying Myb as a critical component of the gene regulatory network controlling effector Treg cell differentiation and function. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression of checkpoint molecules on myeloid-derived suppressor cells.

PubMed

Ballbach, Marlene; Dannert, Angelika; Singh, Anurag; Siegmund, Darina M; Handgretinger, Rupert; Piali, Luca; Rieber, Nikolaus; Hartl, Dominik

2017-12-01

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous cell population expanded in cancer, infection and autoimmunity capable of suppressing T-cell functions. Checkpoint inhibitors have emerged as a key therapeutic strategy in immune-oncology. While checkpoint molecules were initially associated with T cell functions, recent evidence suggests a broader expression and function in innate myeloid cells. Previous studies provided first evidence for a potential role for checkpoints on MDSCs, yet the human relevance remained poorly understood. Therefore, we investigated the expression and functional relevance of checkpoint molecules in human MDSC-T-cell interactions. Our studies demonstrate that programmed death-ligand 1 (PD-L1) is expressed on granulocytic MDSCs upon co-culture with T cells. Transwell experiments showed that cell-to-cell contact was required for MDSC-T-cell interactions and antibody blocking studies showed that targeting PD-L1 partially impaired MDSC-mediated T-cell suppression. Collectively, these studies suggest a role for PD-L1 in human MDSC function and thereby expand the functionality of this checkpoint beyond T cells, which could pave the way for further understanding and therapeutic targeting of PD-1/PD-L1 in innate immune-mediated diseases. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

The nestin-expressing and non-expressing neurons in rat basal forebrain display different electrophysiological properties and project to hippocampus

PubMed Central

2011-01-01

Background Nestin-immunoreactive (nestin-ir) neurons have been identified in the medial septal/diagonal band complex (MS/DBB) of adult rat and human, but the significance of nestin expression in functional neurons is not clear. This study investigated electrophysiological properties and neurochemical phenotypes of nestin-expressing (nestin+) neurons using whole-cell recording combined with single-cell RT-PCR to explore the significance of nestin expression in functional MS/DBB neurons. The retrograde labelling and immunofluorescence were used to investigate the nestin+ neuron related circuit in the septo-hippocampal pathway. Results The results of single-cell RT-PCR showed that 87.5% (35/40) of nestin+ cells expressed choline acetyltransferase mRNA (ChAT+), only 44.3% (35/79) of ChAT+ cells expressed nestin mRNA. Furthermore, none of the nestin+ cells expressed glutamic acid decarboxylases 67 (GAD67) or vesicular glutamate transporters (VGLUT) mRNA. All of the recorded nestin+ cells were excitable and demonstrated slow-firing properties, which were distinctive from those of GAD67 or VGLUT mRNA-positive neurons. These results show that the MS/DBB cholinergic neurons could be divided into nestin-expressing cholinergic neurons (NEChs) and nestin non-expressing cholinergic neurons (NNChs). Interestingly, NEChs had higher excitability and received stronger spontaneous excitatory synaptic inputs than NNChs. Retrograde labelling combined with choline acetyltransferase and nestin immunofluorescence showed that both of the NEChs and NNChs projected to hippocampus. Conclusions These results suggest that there are two parallel cholinergic septo-hippocampal pathways that may have different functions. The significance of nestin expressing in functional neurons has been discussed. PMID:22185478

Neurochemical phenotype of cytoglobin-expressing neurons in the rat hippocampus.

PubMed

Hundahl, Christian Ansgar; Fahrenkrug, Jan; Hannibal, Jens

2014-09-01

Cytoglobin (Cygb), a novel oxygen-binding protein, is expressed in the majority of tissues and has been proposed to function in nitric oxide (NO) metabolism in the vasculature and to have cytoprotective properties. However, the overall functions of Cygb remain elusive. Cygb is also expressed in a subpopulation of brain neurons. Recently, it has been shown that stress upregulates Cygb expression in the brain and the majority of neuronal nitric oxide synthase (nNOS)-positive neurons, an enzyme that produces NO, co-express Cygb. However, there are more neurons expressing Cygb than nNOS, thus a large number of Cygb neurons remain uncharacterized by the neurochemical content. The aim of the present study was to provide an additional and more detailed neurochemical phenotype of Cygb-expressing neurons in the rat hippocampus. The rat hippocampus was chosen due to the abundance of Cygb, as well as this limbic structure being an important target in a number of neurodegenerative diseases. Using triple immunohistochemistry, it was demonstrated that nearly all the parvalbumin- and heme oxygenase 1-positive neurons co-express Cygb and to a large extent, these neuron populations are distinct from the population of Cygb neurons co-expressing nNOS. Furthermore, it was shown that the majority of neurons expressing somastostatin and vasoactive intestinal peptide also co-express Cygb and nNOS. Detailed information regarding the neurochemical phenotype of Cygb neurons in the hippocampus can be a valuable tool in determining the function of Cygb in the brain.

Nppa and Nppb act redundantly during zebrafish cardiac development to confine AVC marker expression and reduce cardiac jelly volume.

PubMed

Grassini, Daniela R; Lagendijk, Anne K; De Angelis, Jessica E; Da Silva, Jason; Jeanes, Angela; Zettler, Nicole; Bower, Neil I; Hogan, Benjamin M; Smith, Kelly A

2018-05-11

Atrial natriuretic peptide ( nppa/anf ) and brain natriuretic peptide ( nppb/bnp ) form a gene cluster with expression in the chambers of the developing heart. Despite restricted expression, a function in cardiac development has not been demonstrated by mutant analysis. This is attributed to functional redundancy however their genomic location in cis has impeded formal analysis. Using genome-editing, we generated mutants for nppa and nppb and found single mutants indistinguishable from wildtype whereas nppa / nppb double mutants display heart morphogenesis defects and pericardial oedema. Analysis of atrioventricular canal (AVC) markers show expansion of bmp4 , tbx2b, has2 and versican expression into the atrium of double mutants. This expanded expression correlates with increased extracellular matrix in the atrium. Using a biosensor for Hyaluronic acid to measure the cardiac jelly (cardiac extracellular matrix), we confirm cardiac jelly expansion in nppa / nppb double mutants. Finally, bmp4 knockdown rescues the expansion of has2 expression and cardiac jelly in double mutants. This definitively shows that nppa and nppb function redundantly during cardiac development to restrict gene expression to the AVC, preventing excessive cardiac jelly synthesis in the atrial chamber. © 2018. Published by The Company of Biologists Ltd.

A Sharp Cadherin-6 Gene Expression Boundary in the Developing Mouse Cortical Plate Demarcates the Future Functional Areal Border

PubMed Central

Terakawa, Youhei W.; Inoue, Yukiko U.; Asami, Junko; Hoshino, Mikio; Inoue, Takayoshi

2013-01-01

The mammalian cerebral cortex can be tangentially subdivided into tens of functional areas with distinct cyto-architectures and neural circuitries; however, it remains elusive how these areal borders are genetically elaborated during development. Here we establish original bacterial artificial chromosome transgenic mouse lines that specifically recapitulate cadherin-6 (Cdh6) mRNA expression profiles in the layer IV of the somatosensory cortex and by detailing their cortical development, we show that a sharp Cdh6 gene expression boundary is formed at a mediolateral coordinate along the cortical layer IV as early as the postnatal day 5 (P5). By further applying mouse genetics that allows rigid cell fate tracing with CreERT2 expression, it is demonstrated that the Cdh6 gene expression boundary set at around P4 eventually demarcates the areal border between the somatosensory barrel and limb field at P20. In the P6 cortical cell pellet culture system, neurons with Cdh6 expression preferentially form aggregates in a manner dependent on Ca2+ and electroporation-based Cdh6 overexpression limited to the postnatal stages perturbs area-specific cell organization in the barrel field. These results suggest that Cdh6 expression in the nascent cortical plate may serve solidification of the protomap for cortical functional areas. PMID:22875867

Roles of GATA6 during Gonadal Development in Japanese Flounder: Gonadogenesis, Regulation of Gender-Related Genes, Estrogen Formation and Gonadal Function Maintenance.

PubMed

Li, Zan; Liu, Xiumei; Sun, Yan; Liu, Jinxiang; Liu, Yuezhong; Wang, Mengxun; Zhang, Quanqi; Wang, Xubo

2017-01-16

GATA-binding protein 6 (GATA6), a highly-conserved transcription factor of the GATA family plays an important role in gonadal cell proliferation, differentiation and endoderm development. In this study, the full-length cDNA of GATA6 of Paralichthys olivaceus (Japanese flounder) was obtained. Phylogenetic, gene structure and synteny analyses demonstrated that GATA6 of P. olivaceus is homologous to that of teleosts and tetrapods. The P. olivaceus GATA6 transcript showed higher expression in testis than in ovary, demonstrating a sexually dimorphic gene expression. During embryonic development, the expression of P. olivaceus GATA6 increased at the blastula stage, demonstrating that GATA6 is involved in morphogenesis. Results of in situ hybridization showed that GATA6 signals were detected in Sertoli cells, oogonia and oocytes. Moreover, 17α methyl testosterone, a male hormone, could moderately upregulate P. olivaceus GATA6 and downregulate P. olivaceus aromatase CYP19A1 in testis cells. These results suggest that GATA6 may play an important role in gonadal development in P. olivaceus . This study provides valuable information on the function of P. olivaceus GATA6, laying the foundation for further development of breeding techniques in this species.

Roles of GATA6 during Gonadal Development in Japanese Flounder: Gonadogenesis, Regulation of Gender-Related Genes, Estrogen Formation and Gonadal Function Maintenance

PubMed Central

Li, Zan; Liu, Xiumei; Sun, Yan; Liu, Jinxiang; Liu, Yuezhong; Wang, Mengxun; Zhang, Quanqi; Wang, Xubo

2017-01-01

GATA-binding protein 6 (GATA6), a highly-conserved transcription factor of the GATA family plays an important role in gonadal cell proliferation, differentiation and endoderm development. In this study, the full-length cDNA of GATA6 of Paralichthys olivaceus (Japanese flounder) was obtained. Phylogenetic, gene structure and synteny analyses demonstrated that GATA6 of P. olivaceus is homologous to that of teleosts and tetrapods. The P. olivaceus GATA6 transcript showed higher expression in testis than in ovary, demonstrating a sexually dimorphic gene expression. During embryonic development, the expression of P. olivaceus GATA6 increased at the blastula stage, demonstrating that GATA6 is involved in morphogenesis. Results of in situ hybridization showed that GATA6 signals were detected in Sertoli cells, oogonia and oocytes. Moreover, 17α methyl testosterone, a male hormone, could moderately upregulate P. olivaceus GATA6 and downregulate P. olivaceus aromatase CYP19A1 in testis cells. These results suggest that GATA6 may play an important role in gonadal development in P. olivaceus. This study provides valuable information on the function of P. olivaceus GATA6, laying the foundation for further development of breeding techniques in this species. PMID:28275215

TSH Receptor Function Is Required for Normal Thyroid Differentiation in Zebrafish

PubMed Central

Opitz, Robert; Maquet, Emilie; Zoenen, Maxime; Dadhich, Rajesh

2011-01-01

TSH is the primary physiological regulator of thyroid gland function. The effects of TSH on thyroid cells are mediated via activation of its membrane receptor [TSH receptor (TSHR)]. In this study, we examined functional thyroid differentiation in zebrafish and characterized the role of TSHR signaling during thyroid organogenesis. Cloning of a cDNA encoding zebrafish Tshr showed conservation of primary structure and functional properties between zebrafish and mammalian TSHR. In situ hybridization confirmed that the thyroid is the major site of tshr expression during zebrafish development. In addition, we identified tpo, iyd, duox, and duoxa as novel thyroid differentiation markers in zebrafish. Temporal analyses of differentiation marker expression demonstrated the induction of an early thyroid differentiation program along with thyroid budding, followed by a delayed onset of duox and duoxa expression coincident with thyroid hormone synthesis. Furthermore, comparative analyses in mouse and zebrafish revealed for the first time a thyroid-enriched expression of cell death regulators of the B-cell lymphoma 2 family during early thyroid morphogenesis. Knockdown of tshr function by morpholino microinjection into embryos did not affect early thyroid morphogenesis but caused defects in later functional differentiation. The thyroid phenotype observed in tshr morphants at later stages comprised a reduction in number and size of functional follicles, down-regulation of differentiation markers, as well as reduced thyroid transcription factor expression. A comparison of our results with phenotypes observed in mouse models of defective TSHR and cAMP signaling highlights the value of zebrafish as a model to enhance the understanding of functional differentiation in the vertebrate thyroid. PMID:21737742

ΔNp63α induces the expression of FAT2 and Slug to promote tumor invasion

PubMed Central

Dang, Tuyen T.; Westcott, Jill M.; Maine, Erin A.; Kanchwala, Mohammed; Xing, Chao; Pearson, Gray W.

2016-01-01

Tumor invasion can be induced by changes in gene expression that alter cell phenotype. The transcription factor ΔNp63α promotes basal-like breast cancer (BLBC) migration by inducing the expression of the mesenchymal genes Slug and Axl, which confers cells with a hybrid epithelial/mesenchymal state. However, the extent of the ΔNp63α regulated genes that support invasive behavior is not known. Here, using gene expression analysis, ChIP-seq, and functional testing, we find that ΔNp63α promotes BLBC motility by inducing the expression of the atypical cadherin FAT2, the vesicular binding protein SNCA, the carbonic anhydrase CA12, the lipid binding protein CPNE8 and the kinase NEK1, along with Slug and Axl. Notably, lung squamous cell carcinoma migration also required ΔNp63α dependent FAT2 and Slug expression, demonstrating that ΔNp63α promotes migration in multiple tumor types by inducing mesenchymal and non-mesenchymal genes. ΔNp63α activation of FAT2 and Slug influenced E-cadherin localization to cell-cell contacts, which can restrict spontaneous cell movement. Moreover, live-imaging of spheroids in organotypic culture demonstrated that ΔNp63α, FAT2 and Slug were essential for the extension of cellular protrusions that initiate collective invasion. Importantly, ΔNp63α is co-expressed with FAT2 and Slug in patient tumors and the elevated expression of ΔNp63α, FAT2 and Slug correlated with poor patient outcome. Together, these results reveal how ΔNp63α promotes cell migration by directly inducing the expression of a cohort of genes with distinct cellular functions and suggest that FAT2 is a new regulator of collective invasion that may influence patient outcome. PMID:27081041

Gene Expression Architecture of Mouse Dorsal and Tail Skin Reveals Functional Differences in Inflammation and Cancer | Office of Cancer Genomics

Cancer.gov

Inherited germline polymorphisms can cause gene expression levels in normal tissues to differ substantially between individuals. We present an analysis of the genetic architecture of normal adult skin from 470 genetically unique mice, demonstrating the effect of germline variants, skin tissue location, and perturbation by exogenous inflammation or tumorigenesis on gene signaling pathways.

HIV-1-encoded antisense RNA suppresses viral replication for a prolonged period

PubMed Central

2012-01-01

Background Recent evidence proposes a novel concept that mammalian natural antisense RNAs play important roles in cellular homeostasis by regulating the expression of several genes. Identification and characterization of retroviral antisense RNA would provide new insights into mechanisms of replication and pathogenesis. HIV-1 encoded-antisense RNAs have been reported, although their structures and functions remain to be studied. We have tried to identify and characterize antisense RNAs of HIV-1 and their function in viral infection. Results Characterization of transcripts of HEK293T cells that were transiently transfected with an expression plasmid with HIV-1NL4–3 DNA in the antisense orientation showed that various antisense transcripts can be expressed. By screening and characterizing antisense RNAs in HIV-1NL4–3-infected cells, we defined the primary structure of a major form of HIV-1 antisense RNAs, which corresponds to a variant of previously reported ASP mRNA. This 2.6 kb RNA was transcribed from the U3 region of the 3′ LTR and terminated at the env region in acutely or chronically infected cell lines and acutely infected human peripheral blood mononuclear cells. Reporter assays clearly demonstrated that the HIV-1 LTR harbours promoter activity in the reverse orientation. Mutation analyses suggested the involvement of NF-κΒ binding sites in the regulation of antisense transcription. The antisense RNA was localized in the nuclei of the infected cells. The expression of this antisense RNA suppressed HIV-1 replication for more than one month. Furthermore, the specific knockdown of this antisense RNA enhanced HIV-1 gene expression and replication. Conclusions The results of the present study identified an accurate structure of the major form of antisense RNAs expressed from the HIV-1NL4–3 provirus and demonstrated its nuclear localization. Functional studies collectively demonstrated a new role of the antisense RNA in viral replication. Thus, we suggest a novel viral mechanism that self-limits HIV-1 replication and provides new insight into the viral life cycle. PMID:22569184

The Cytoplasmic Zinc Finger Protein ZPR1 Accumulates in the Nucleolus of Proliferating Cells

PubMed Central

Galcheva-Gargova, Zoya; Gangwani, Laxman; Konstantinov, Konstantin N.; Mikrut, Monique; Theroux, Steven J.; Enoch, Tamar; Davis, Roger J.

1998-01-01

The zinc finger protein ZPR1 translocates from the cytoplasm to the nucleus after treatment of cells with mitogens. The function of nuclear ZPR1 has not been defined. Here we demonstrate that ZPR1 accumulates in the nucleolus of proliferating cells. The role of ZPR1 was examined using a gene disruption strategy. Cells lacking ZPR1 are not viable. Biochemical analysis demonstrated that the loss of ZPR1 caused disruption of nucleolar function, including preribosomal RNA expression. These data establish ZPR1 as an essential protein that is required for normal nucleolar function in proliferating cells. PMID:9763455

Engineering biosynthetic excitable tissues from unexcitable cells for electrophysiological and cell therapy studies.

PubMed

Kirkton, Robert D; Bursac, Nenad

2011-01-01

Patch-clamp recordings in single-cell expression systems have been traditionally used to study the function of ion channels. However, this experimental setting does not enable assessment of tissue-level function such as action potential (AP) conduction. Here we introduce a biosynthetic system that permits studies of both channel activity in single cells and electrical conduction in multicellular networks. We convert unexcitable somatic cells into an autonomous source of electrically excitable and conducting cells by stably expressing only three membrane channels. The specific roles that these expressed channels have on AP shape and conduction are revealed by different pharmacological and pacing protocols. Furthermore, we demonstrate that biosynthetic excitable cells and tissues can repair large conduction defects within primary 2- and 3-dimensional cardiac cell cultures. This approach enables novel studies of ion channel function in a reproducible tissue-level setting and may stimulate the development of new cell-based therapies for excitable tissue repair.

Human Prostatic Acid Phosphatase: Structure, Function and Regulation

PubMed Central

Muniyan, Sakthivel; Chaturvedi, Nagendra K.; Dwyer, Jennifer G.; LaGrange, Chad A.; Chaney, William G.; Lin, Ming-Fong

2013-01-01

Human prostatic acid phosphatase (PAcP) is a 100 kDa glycoprotein composed of two subunits. Recent advances demonstrate that cellular PAcP (cPAcP) functions as a protein tyrosine phosphatase by dephosphorylating ErbB-2/Neu/HER-2 at the phosphotyrosine residues in prostate cancer (PCa) cells, which results in reduced tumorigenicity. Further, the interaction of cPAcP and ErbB-2 regulates androgen sensitivity of PCa cells. Knockdown of cPAcP expression allows androgen-sensitive PCa cells to develop the castration-resistant phenotype, where cells proliferate under an androgen-reduced condition. Thus, cPAcP has a significant influence on PCa cell growth. Interestingly, promoter analysis suggests that PAcP expression can be regulated by NF-κB, via a novel binding sequence in an androgen-independent manner. Further understanding of PAcP function and regulation of expression will have a significant impact on understanding PCa progression and therapy. PMID:23698773

Germline MLH1 Mutations Are Frequently Identified in Lynch Syndrome Patients With Colorectal and Endometrial Carcinoma Demonstrating Isolated Loss of PMS2 Immunohistochemical Expression.

PubMed

Dudley, Beth; Brand, Randall E; Thull, Darcy; Bahary, Nathan; Nikiforova, Marina N; Pai, Reetesh K

2015-08-01

Current guidelines on germline mutation testing for patients suspected of having Lynch syndrome are not entirely clear in patients with tumors demonstrating isolated loss of PMS2 immunohistochemical expression. We analyzed the clinical and pathologic features of patients with tumors demonstrating isolated loss of PMS2 expression in an attempt to (1) determine the frequency of germline MLH1 and PMS2 mutations and (2) correlate mismatch-repair protein immunohistochemistry and tumor histology with germline mutation results. A total of 3213 consecutive colorectal carcinomas and 215 consecutive endometrial carcinomas were prospectively analyzed for DNA mismatch-repair protein expression by immunohistochemistry. In total, 32 tumors from 31 patients demonstrated isolated loss of PMS2 immunohistochemical expression, including 16 colorectal carcinomas and 16 endometrial carcinomas. Microsatellite instability (MSI) polymerase chain reaction was performed in 29 tumors from 28 patients with the following results: 28 tumors demonstrated high-level MSI, and 1 tumor demonstrated low-level MSI. Twenty of 31 (65%) patients in the study group had tumors demonstrating histopathology associated with high-level MSI. Seventeen patients underwent germline mutation analysis with the following results: 24% with MLH1 mutations, 35% with PMS2 mutations, 12% with PMS2 variants of undetermined significance, and 29% with no mutations in either MLH1 or PMS2. Three of the 4 patients with MLH1 germline mutations had a mutation that results in decreased stability and quantity of the MLH1 protein that compromises the MLH1-PMS2 protein complex, helping to explain the presence of immunogenic but functionally inactive MLH1 protein within the tumor. The high frequency of MLH1 germline mutations identified in our study has important implications for testing strategies in patients suspected of having Lynch syndrome and indicates that patients with tumors demonstrating isolated loss of PMS2 expression without a germline PMS2 mutation must have MLH1 mutation analysis performed.

Complementary Information Derived from CRISPR Cas9 Mediated Gene Deletion and Suppression. | Office of Cancer Genomics

Cancer.gov

CRISPR-Cas9 provides the means to perform genome editing and facilitates loss-of-function screens. However, we and others demonstrated that expression of the Cas9 endonuclease induces a gene-independent response that correlates with the number of target sequences in the genome. An alternative approach to suppressing gene expression is to block transcription using a catalytically inactive Cas9 (dCas9). Here we directly compare genome editing by CRISPR-Cas9 (cutting, CRISPRc) and gene suppression using KRAB-dCas9 (CRISPRi) in loss-of-function screens to identify cell essential genes.

GP96 is a GARP chaperone and controls regulatory T cell functions.

PubMed

Zhang, Yongliang; Wu, Bill X; Metelli, Alessandra; Thaxton, Jessica E; Hong, Feng; Rachidi, Saleh; Ansa-Addo, Ephraim; Sun, Shaoli; Vasu, Chenthamarakshan; Yang, Yi; Liu, Bei; Li, Zihai

2015-02-01

Molecular chaperones control a multitude of cellular functions via folding chaperone-specific client proteins. CD4+FOXP3+ Tregs play key roles in maintaining peripheral tolerance, which is subject to regulation by multiple molecular switches, including mTOR and hypoxia-inducible factor. It is not clear whether GP96 (also known as GRP94), which is a master TLR and integrin chaperone, controls Treg function. Using murine genetic models, we demonstrated that GP96 is required for Treg maintenance and function, as loss of GP96 resulted in instability of the Treg lineage and impairment of suppressive functions in vivo. In the absence of GP96, Tregs were unable to maintain FOXP3 expression levels, resulting in systemic accumulation of pathogenic IFN-γ-producing and IL-17-producing T cells. We determined that GP96 serves as an essential chaperone for the cell-surface protein glycoprotein A repetitions predominant (GARP), which is a docking receptor for latent membrane-associated TGF-β (mLTGF-β). The loss of both GARP and integrins on GP96-deficient Tregs prevented expression of mLTGF-β and resulted in inefficient production of active TGF-β. Our work demonstrates that GP96 regulates multiple facets of Treg biology, thereby placing Treg stability and immunosuppressive functions strategically under the control of a major stress chaperone.

Discovery of functional non-coding conserved regions in the α-synuclein gene locus

PubMed Central

Sterling, Lori; Walter, Michael; Ting, Dennis; Schüle, Birgitt

2014-01-01

Several single nucleotide polymorphisms (SNPs) and the Rep-1 microsatellite marker of the α-synuclein ( SNCA) gene have consistently been shown to be associated with Parkinson’s disease, but the functional relevance is unclear. Based on these findings we hypothesized that conserved cis-regulatory elements in the SNCA genomic region regulate expression of SNCA, and that SNPs in these regions could be functionally modulating the expression of SNCA, thus contributing to neuronal demise and predisposing to Parkinson’s disease. In a pair-wise comparison of a 206kb genomic region encompassing the SNCA gene, we revealed 34 evolutionary conserved DNA sequences between human and mouse. All elements were cloned into reporter vectors and assessed for expression modulation in dual luciferase reporter assays.  We found that 12 out of 34 elements exhibited either an enhancement or reduction of the expression of the reporter gene. Three elements upstream of the SNCA gene displayed an approximately 1.5 fold (p<0.009) increase in expression. Of the intronic regions, three showed a 1.5 fold increase and two others indicated a 2 and 2.5 fold increase in expression (p<0.002). Three elements downstream of the SNCA gene showed 1.5 fold and 2.5 fold increase (p<0.0009). One element downstream of SNCA had a reduced expression of the reporter gene of 0.35 fold (p<0.0009) of normal activity. Our results demonstrate that the SNCA gene contains cis-regulatory regions that might regulate the transcription and expression of SNCA. Further studies in disease-relevant tissue types will be important to understand the functional impact of regulatory regions and specific Parkinson’s disease-associated SNPs and its function in the disease process. PMID:25566351

INHIBITION OF ERN1 SIGNALING ENZYME AFFECTS HYPOXIC REGULATION OF THE EXPRESSION OF E2F8, EPAS1, HOXC6, ATF3, TBX3 AND FOXF1 GENES IN U87 GLIOMA CELLS.

PubMed

Minchenko, O H; Tsymbal, D O; Minchenko, D O; Kovalevska, O V; Karbovskyi, L L; Bikfalvi, A

2015-01-01

Hypoxia as well as the endoplasmic reticulum stress are important factors of malignant tumor growth and control of the expression of genes, which regulate numerous metabolic processes and cell proliferation. Furthermore, blockade of ERN1 (endoplasmic reticulum to nucleus 1) suppresses cell proliferation and tumor growth. We studied the effect of hypoxia on the expression of genes encoding the transcription factors such as E2F8 (E2F transcription factor 8), EPAS1 (endothelial PAS domain protein 1), TBX3 (T-box 3), ATF3 (activating transcription factor 3), FOXF1 (forkhead box F), and HOXC6 (homeobox C6) in U87 glioma cells with and without ERN1 signaling enzyme function. We have established that hypoxia enhances the expression of HOXC6, E2F8, ATF3, and EPAS1 genes but does not change TBX3 and FOXF1 gene expression in glioma cells with ERNI function. At the same time, the expression level of all studied genes is strongly decreased, except for TBX3 gene, in glioma cells without ERN1 function. Moreover, the inhibition of ERN1 signaling enzyme function significantly modifies the effect of hypoxia on the expression of these transcription factor genes. removes or introduces this regulation as well as changes a direction or magnitude of hypoxic regulation. Present study demonstrates that fine-tuning of the expression of proliferation related genes depends upon hypoxia and ERN1-mediated endoplasmic reticulum stress signaling and correlates with slower proliferation rate of glioma cells without ERN1 function.

Voltage-gated Na+ Channel Activity Increases Colon Cancer Transcriptional Activity and Invasion Via Persistent MAPK Signaling

NASA Astrophysics Data System (ADS)

House, Carrie D.; Wang, Bi-Dar; Ceniccola, Kristin; Williams, Russell; Simaan, May; Olender, Jacqueline; Patel, Vyomesh; Baptista-Hon, Daniel T.; Annunziata, Christina M.; Silvio Gutkind, J.; Hales, Tim G.; Lee, Norman H.

2015-06-01

Functional expression of voltage-gated Na+ channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes.

Expression of Notch1 and Numb in small cell lung cancer.

PubMed

Kikuchi, Hajime; Sakakibara-Konishi, Jun; Furuta, Megumi; Yokouchi, Hiroshi; Nishihara, Hiroshi; Yamazaki, Shigeo; Uramoto, Hidetaka; Tanaka, Fumihiro; Harada, Masao; Akie, Kenji; Sugaya, Fumiko; Fujita, Yuka; Takamura, Kei; Kojima, Tetsuya; Harada, Toshiyuki; Higuchi, Mitsunori; Honjo, Osamu; Minami, Yoshinori; Watanabe, Naomi; Oizumi, Satoshi; Suzuki, Hiroyuki; Ishida, Takashi; Dosaka-Akita, Hirotoshi; Isobe, Hiroshi; Munakata, Mitsuru; Nishimura, Masaharu

2017-02-07

Notch signaling in tumorigenesis functions as an oncogene or tumor suppressor according to the type of malignancy. Numb represses intracellular Notch signaling. Previous studies have demonstrated that Notch signaling suppresses the proliferation of small cell lung cancer (SCLC) cell lines. However, in SCLC, the association between Notch1 and Numb expression and clinicopathological factors or prognosis has remained unclear. In this study, we evaluated the expression of Notch1 and Numb in SCLC. We immunohistochemically assessed 125 SCLCs that were surgically resected at 16 institutions participating in either the Hokkaido Lung Cancer Clinical Study Group Trial (HOT) or the Fukushima Investigative Group for Healing Thoracic Malignancy (FIGHT) between 2003 and 2013. Correlations between Notch1 or Numb expression and various clinicopathological features were evaluated. Notch1 expression was associated with ECOG performance status. Numb expression was associated with age, sex, and pathological histology (SCLC or Combined SCLC). Analysis of cellular biological expression did not demonstrate a significant correlation between the expression of Notch1 and of Numb. Multivariate Cox regression analysis showed that high Notch1 expression was an independent favorable prognostic factor for SCLC(hazard ratio = 0.503, P = 0.023). High Notch1 expression, but not Numb expression, is associated with favorable prognosis in SCLC.

Functional characterization of an oxytocin receptor gene variant (rs2268498) previously associated with social cognition by expression analysis in vitro and in human brain biopsy.

PubMed

Reuter, Martin; Montag, Christian; Altmann, Steffen; Bendlow, Fabian; Elger, Christian; Kirsch, Peter; Becker, Albert; Schoch-McGovern, Susanne; Simon, Matthias; Weber, Bernd; Felten, Andrea

2017-10-01

The oxytocin system plays a prominent role in social behavior across species, and numerous genetic studies in humans have reported associations between polymorphisms on the oxytocin receptor (OXTR) gene and phenotypes related to social cognition, affiliation, perspective taking, and sociability in healthy subjects and in patients with atypical social behavior, such as in autism spectrum disorders (ASD). Recently, the first study demonstrating altered agonist-induced OXTR internalization and recycling for the exonic variant rs35062132 emerged. Beside this, there has been no further demonstration of the functionality of the OXTR variants especially there does not exist any for the regulatory units. To address this gap in the literature, we tested the functionality of the promoter flanking single nucleotide polymorphism (SNP) rs2268498, which has proven an interesting candidate for predicting social behavior in recent association studies. Results of genetic expression analyses in human hippocampal tissue showed a twofold difference in messenger RNA transcription, dependent on the presence or absence of the C-allele. This finding was corroborated by cloning, i.e., in vitro reporter gene expression analysis after transfection of OXTR promoter plasmids into HEK-293 cells. Our results underline the importance of OXTR rs2268498 for genetic research in social behavior and ASD.

Regulated proteolysis of Trop2 drives epithelial hyperplasia and stem cell self-renewal via β-catenin signaling.

PubMed

Stoyanova, Tanya; Goldstein, Andrew S; Cai, Houjian; Drake, Justin M; Huang, Jiaoti; Witte, Owen N

2012-10-15

The cell surface protein Trop2 is expressed on immature stem/progenitor-like cells and is overexpressed in many epithelial cancers. However the biological function of Trop2 in tissue maintenance and tumorigenesis remains unclear. In this study, we demonstrate that Trop2 is a regulator of self-renewal, proliferation, and transformation. Trop2 controls these processes through a mechanism of regulated intramembrane proteolysis that leads to cleavage of Trop2, creating two products: the extracellular domain and the intracellular domain. The intracellular domain of Trop2 is released from the membrane and accumulates in the nucleus. Heightened expression of the Trop2 intracellular domain promotes stem/progenitor self-renewal through signaling via β-catenin and is sufficient to initiate precursor lesions to prostate cancer in vivo. Importantly, we demonstrate that loss of β-catenin or Trop2 loss-of-function cleavage mutants abrogates Trop2-driven self-renewal and hyperplasia in the prostate. These findings suggest that heightened expression of Trop2 is selected for in epithelial cancers to enhance the stem-like properties of self-renewal and proliferation. Defining the mechanism of Trop2 function in self-renewal and transformation is essential to identify new therapeutic strategies to block Trop2 activation in cancer.

Regulated proteolysis of Trop2 drives epithelial hyperplasia and stem cell self-renewal via β-catenin signaling

PubMed Central

Stoyanova, Tanya; Goldstein, Andrew S.; Cai, Houjian; Drake, Justin M.; Huang, Jiaoti; Witte, Owen N.

2012-01-01

The cell surface protein Trop2 is expressed on immature stem/progenitor-like cells and is overexpressed in many epithelial cancers. However the biological function of Trop2 in tissue maintenance and tumorigenesis remains unclear. In this study, we demonstrate that Trop2 is a regulator of self-renewal, proliferation, and transformation. Trop2 controls these processes through a mechanism of regulated intramembrane proteolysis that leads to cleavage of Trop2, creating two products: the extracellular domain and the intracellular domain. The intracellular domain of Trop2 is released from the membrane and accumulates in the nucleus. Heightened expression of the Trop2 intracellular domain promotes stem/progenitor self-renewal through signaling via β-catenin and is sufficient to initiate precursor lesions to prostate cancer in vivo. Importantly, we demonstrate that loss of β-catenin or Trop2 loss-of-function cleavage mutants abrogates Trop2-driven self-renewal and hyperplasia in the prostate. These findings suggest that heightened expression of Trop2 is selected for in epithelial cancers to enhance the stem-like properties of self-renewal and proliferation. Defining the mechanism of Trop2 function in self-renewal and transformation is essential to identify new therapeutic strategies to block Trop2 activation in cancer. PMID:23070813

p39, the Primary Activator for Cyclin-dependent Kinase 5 (Cdk5) in Oligodendroglia, Is Essential for Oligodendroglia Differentiation and Myelin Repair*

PubMed Central

Bankston, Andrew N.; Li, Wenqi; Zhang, Hui; Ku, Li; Liu, Guanglu; Papa, Filomena; Zhao, Lixia; Bibb, James A.; Cambi, Franca; Tiwari-Woodruff, Seema K.; Feng, Yue

2013-01-01

Cyclin-dependent kinase 5 (Cdk5) plays key roles in normal brain development and function. Dysregulation of Cdk5 may cause neurodegeneration and cognitive impairment. Besides the well demonstrated role of Cdk5 in neurons, emerging evidence suggests the functional requirement of Cdk5 in oligodendroglia (OL) and CNS myelin development. However, whether neurons and OLs employ similar or distinct mechanisms to regulate Cdk5 activity remains elusive. We report here that in contrast to neurons that harbor high levels of two Cdk5 activators, p35 and p39, OLs express abundant p39 but negligible p35. In addition, p39 is selectively up-regulated in OLs during differentiation along with elevated Cdk5 activity, whereas p35 expression remains unaltered. Specific knockdown of p39 by siRNA significantly attenuates Cdk5 activity and OL differentiation without affecting p35. Finally, expression of p39, but not p35, is increased during myelin repair, and remyelination is impaired in p39−/− mice. Together, these results reveal that neurons and OLs harbor distinct preference of Cdk5 activators and demonstrate important functions of p39-dependent Cdk5 activation in OL differentiation during de novo myelin development and myelin repair. PMID:23645679

Related-to-receptor tyrosine kinase receptor regulates hematopoietic stem and progenitor sensitivity to myelosuppressive injury in mice.

PubMed

Povinelli, Benjamin J; Srivastava, Pragya; Nemeth, Michael J

2015-03-01

Maintaining a careful balance between quiescence and proliferation of hematopoietic stem and progenitor cells (HSPCs) is necessary for lifelong blood formation. Previously, we demonstrated that the Wnt5a ligand inhibits HSPC proliferation through a functional interaction with a noncanonical Wnt ligand receptor termed 'related-to-receptor tyrosine kinase' (Ryk). Expression of Ryk on HSPCs in vivo is associated with a lower rate of proliferation, and, following treatment with fluorouracil (5-FU), the percentage of Ryk(+/high) HSPCs increased and the percentage of Ryk(-/low) HSPCs decreased. Based on these data, we hypothesized that one function of the Ryk receptor is to protect HSPCs from the effects of myeloablative agents. We found that Ryk expression on HSPCs is associated with lower rates of apoptosis following 5-FU and radiation. Transient inhibition of Ryk signaling in vivo resulted in increased hematopoietic-stem-cell proliferation and decreased hematopoietic-stem-cell function in bone marrow transplant assays. Furthermore, inhibition of Ryk signaling sensitized HSPCs to 5-FU treatment in association with increased levels of reactive oxygen species. Together, these results demonstrated an association between Ryk expression and survival of HSPCs following suppressive injury. Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Tumor Suppressor WWOX inhibits osteosarcoma metastasis by modulating RUNX2 function.

PubMed

Del Mare, Sara; Aqeilan, Rami I

2015-08-10

Osteosarcoma (OS) is among the most frequently occurring primary bone tumors, primarily affecting adolescents and young adults. This malignant osteoid forming tumor is characterized by its metastatic potential, mainly to lungs. We recently demonstrated that WW domain-containing oxidoreductase (WWOX) is frequently inactivated in human OS and that WWOX restoration in WWOX-negative OS cells suppresses tumorigenicity. Of note, WWOX levels are reduced in paired OS samples of post-treatment metastastectomies as compared to pre-treatment biopsies suggesting that decreased WWOX levels are associated with a more aggressive phenotype at the metastatic site. Nevertheless, little is known about WWOX function in OS metastasis. Here, we investigated the role of tumor suppressor WWOX in suppressing pulmonary OS metastasis both in vitro and in vivo. We demonstrated that ectopic expression of WWOX in OS cells, HOS and LM-7, inhibits OS invasion and cell migration in vitro. Furthermore, WWOX expression reduced tumor burden in vivo and inhibited metastases' seeding and colonization. Mechanistically, WWOX function is associated with reduced levels of RUNX2 metastatic target genes implicated in adhesion and motility. Our results suggest that WWOX plays a critical role in determining the aggressive phenotype of OS, and its expression could be an attractive therapeutic target to combat this devastating adolescent disease.

Tumor Suppressor WWOX inhibits osteosarcoma metastasis by modulating RUNX2 function

PubMed Central

Del Mare, Sara; Aqeilan, Rami I.

2015-01-01

Osteosarcoma (OS) is among the most frequently occurring primary bone tumors, primarily affecting adolescents and young adults. This malignant osteoid forming tumor is characterized by its metastatic potential, mainly to lungs. We recently demonstrated that WW domain-containing oxidoreductase (WWOX) is frequently inactivated in human OS and that WWOX restoration in WWOX-negative OS cells suppresses tumorigenicity. Of note, WWOX levels are reduced in paired OS samples of post-treatment metastastectomies as compared to pre-treatment biopsies suggesting that decreased WWOX levels are associated with a more aggressive phenotype at the metastatic site. Nevertheless, little is known about WWOX function in OS metastasis. Here, we investigated the role of tumor suppressor WWOX in suppressing pulmonary OS metastasis both in vitro and in vivo. We demonstrated that ectopic expression of WWOX in OS cells, HOS and LM-7, inhibits OS invasion and cell migration in vitro. Furthermore, WWOX expression reduced tumor burden in vivo and inhibited metastases’ seeding and colonization. Mechanistically, WWOX function is associated with reduced levels of RUNX2 metastatic target genes implicated in adhesion and motility. Our results suggest that WWOX plays a critical role in determining the aggressive phenotype of OS, and its expression could be an attractive therapeutic target to combat this devastating adolescent disease. PMID:26256646

Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system

PubMed Central

Lee, Young Je; Hoynes-O'Connor, Allison; Leong, Matthew C.; Moon, Tae Seok

2016-01-01

A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA–asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions. PMID:26837577

In-vivo extravasation induces the expression of interleukin 1 receptor type 1 in human neutrophils

PubMed Central

Paulsson, J M; Moshfegh, A; Dadfar, E; Held, C; Jacobson, S H; Lundahl, J

2012-01-01

In order to address neutrophil activation during inflammation we assessed the expression of interleukin 1 receptor type 1 (IL-1R1) following in-vivo extravasation. Extravasated neutrophils were collected from 11 healthy study subjects by a skin chamber technique and compared to neutrophils in peripheral blood. Expression of IL-1R1 was assessed by microarray, quantitative polymerase chain reaction (qPCR), Western blot, flow cytometry, enzyme linked immunosorbent assay (ELISA) and immunoelectron microscopy (iEM). IL-1R1 was induced following extravasation, demonstrated by both gene array and qPCR. Western blot demonstrated an increased expression of IL-1R1 in extravasated leucocytes. This was confirmed further in neutrophils by flow cytometry and iEM that also demonstrated an increased intracellular pool of IL-1R1 that could be mobilized by N-formyl-methionine-leucine-phenylalanine (fMLP). Stimulation of peripheral neutrophils with IL-1 resulted in transcription of NFκB and a number of downstream chemokines and the corresponding chemokines were also induced following in-vivo extravasation. The present results demonstrate that IL-1R1 is induced following extravasation and exists on the neutrophil surface, as well as in a mobile intracellular pool. Furthermore, neutrophils express functional IL-1R1 as demonstrated by the induction of chemokines following IL-1 stimulation. The results indicate a potential role for IL-1 in the activation of neutrophils at inflammatory sites. PMID:22385245

Resistin-like molecule β regulates innate colonic function: Barrier integrity and inflammation susceptibility

PubMed Central

Hogan, Simon P.; Seidu, Luqman; Blanchard, Carine; Groschwitz, Katherine; Mishra, Anil; Karow, Margaret L.; Ahrens, Richard; Artis, David; Murphy, Andrew J.; Valenzuela, David M.; Yancopoulos, George D.; Rothenberg, Marc E.

2007-01-01

Background: Resistin-like molecule (RELM) β is a cysteine-rich cytokine expressed in the gastrointestinal tract and implicated in insulin resistance and gastrointestinal nematode immunity; however, its function primarily remains an enigma. Objective: We sought to elucidate the function of RELM-β in the gastrointestinal tract. Methods: We generated RELM-β gene-targeted mice and examined colonic epithelial barrier function, gene expression profiles, and susceptibility to acute colonic inflammation. Results: We show that RELM-β is constitutively expressed in the colon by goblet cells and enterocytes and has a role in homeostasis, as assessed by alterations in colon mRNA transcripts and epithelial barrier function in the absence of RELM-β. Using acute colonic inflammatory models, we demonstrate that RELM-β has a central role in the regulation of susceptibility to colonic inflammation. Mechanistic studies identify that RELM-β regulates expression of type III regenerating gene (REG) (REG3β and γ), molecules known to influence nuclear factor κB signaling. Conclusions: These data define a critical role for RELM-β in the maintenance of colonic barrier function and gastrointestinal innate immunity. Clinical implications: These findings identify RELM-β as an important molecule in homeostatic gastrointestinal function and colonic inflammation, and as such, these results have implications for a variety of human inflammatory gastrointestinal conditions, including allergic gastroenteropathies. PMID:16815164

PROS-1/Prospero Is a Major Regulator of the Glia-Specific Secretome Controlling Sensory-Neuron Shape and Function in C. elegans.

PubMed

Wallace, Sean W; Singhvi, Aakanksha; Liang, Yupu; Lu, Yun; Shaham, Shai

2016-04-19

Sensory neurons are an animal's gateway to the world, and their receptive endings, the sites of sensory signal transduction, are often associated with glia. Although glia are known to promote sensory-neuron functions, the molecular bases of these interactions are poorly explored. Here, we describe a post-developmental glial role for the PROS-1/Prospero/PROX1 homeodomain protein in sensory-neuron function in C. elegans. Using glia expression profiling, we demonstrate that, unlike previously characterized cell fate roles, PROS-1 functions post-embryonically to control sense-organ glia-specific secretome expression. PROS-1 functions cell autonomously to regulate glial secretion and membrane structure, and non-cell autonomously to control the shape and function of the receptive endings of sensory neurons. Known glial genes controlling sensory-neuron function are PROS-1 targets, and we identify additional PROS-1-dependent genes required for neuron attributes. Drosophila Prospero and vertebrate PROX1 are expressed in post-mitotic sense-organ glia and astrocytes, suggesting conserved roles for this class of transcription factors. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Transcriptional Control by PARP-1: Chromatin Modulation, Enhancer-binding, Coregulation, and Insulation

PubMed Central

Kraus, W. Lee

2008-01-01

Summary The regulation of gene expression requires a wide array of protein factors that can modulate chromatin structure, act at enhancers, function as transcriptional coregulators, or regulate insulator function. Poly(ADP-ribose) polymerase-1 (PARP-1), an abundant and ubiquitous nuclear enzyme that catalyzes the NAD+-dependent addition of ADP-ribose polymers on a variety of nuclear proteins, has been implicated in all of these functions. Recent biochemical, genomic, proteomic, and cell-based studies have highlighted the role of PARP-1 in each of these processes and provided new insights about the molecular mechanisms governing PARP-1-dependent regulation of gene expression. In addition, these studies have demonstrated how PARP-1 functions as an integral part of cellular signaling pathways that culminate in gene regulatory outcomes. PMID:18450439

Analysis of Msx1 and Msx2 transactivation function in the context of the heat shock 70 (Hspa1b) gene promoter.

PubMed

Zhuang, Fengfeng; Nguyen, Manuel P; Shuler, Charles; Liu, Yi-Hsin

2009-04-03

Previous studies have shown that Msx proteins control gene transcription predominantly through repression mechanisms. However, gene expression studies using either the gain-of-function or the loss-of-function mutants revealed many gene targets whose expression require functional Msx proteins. To date, investigations into the mechanisms of Msx-dependent transactivation have been hindered by the lack of a responsive promoter. Here, we demonstrated the usefulness of the mouse Hspa1b promoter in probing Msx-dependent mechanisms of gene activation. We showed that Msx protein activates Hspa1b promoter via its C-terminal domain. The activation absolutely depends on the HSEs and physical interactions between Msx proteins and heat shock factors may play a contributing role.

Anyalysis of Msx1 and Msx2 Transactivation Function in the Context of the Heat Shock 70 (Hspa1b) Gene Promoter

PubMed Central

Zhuang, Fengfeng; Nguyen, Manuel P.; Shuler, Charles; Liu, Yi-Hsin

2009-01-01

Previous studies have shown that Msx proteins control gene transcription predominantly through repression mechanisms. However, gene expression studies using either the gain-of-function or the loss-of-function mutants revealed many gene targets whose expression require functional Msx proteins. To date, investigations into the mechanisms of Msx-dependent trans-activation have been hindered by the lack of a responsive promoter. Here, we demonstrated the usefulness of the mouse Hspa1b promoter in probing Msx-dependent mechanisms of gene activation. We showed that Msx protein activates Hspa1b promoter via its C-terminal domain. The activation absolutely depends on the HSEs and physical interactions between Msx proteins and Heat shock factors may play a contributing role. PMID:19338779

Regulation of myeloid leukemia factor-1 interacting protein (MLF1IP) expression in glioblastoma.

PubMed

Hanissian, Silva H; Teng, Bin; Akbar, Umar; Janjetovic, Zorica; Zhou, Qihong; Duntsch, Christopher; Robertson, Jon H

2005-06-14

The myelodysplasia/myeloid leukemia factor 1-interacting protein MLF1IP is a novel gene which encodes for a putative transcriptional repressor. It is localized to human chromosome 4q35.1 and is expressed in both the nuclei and cytoplasm of cells. Northern and Western blot analyses have revealed MLF1IP to be present at very low amounts in normal brain tissues, whereas a number of human and rat glioblastoma (GBM) cell lines demonstrated a high level expression of the MLF1IP protein. Immunohistochemical analysis of rat F98 and C6 GBM tumor models showed that MLF1IP was highly expressed in the tumor core where it was co-localized with MLF1 and nestin. Moreover, MLF1IP expression was elevated in the contralateral brain where no tumor cells were detected. These observations, together with previous data demonstrating a role for MLF1IP in erythroleukemias, suggest a possible function for this protein in glioma pathogenesis and potentially in other types of malignancies.

Gene expression analysis uncovers novel Hedgehog interacting protein (HHIP) effects in human bronchial epithelial cells

PubMed Central

Zhou, Xiaobo; Qiu, Weiliang; Sathirapongsasuti, J. Fah.; Cho, Michael H.; Mancini, John D.; Lao, Taotao; Thibault, Derek M.; Litonjua, Gus; Bakke, Per S.; Gulsvik, Amund; Lomas, David A.; Beaty, Terri H.; Hersh, Craig P.; Anderson, Christopher; Geigenmuller, Ute; Raby, Benjamin A.; Rennard, Stephen I.; Perrella, Mark A.; Choi, Augustine M.K.; Quackenbush, John; Silverman, Edwin K.

2013-01-01

Hedgehog Interacting Protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis. PMID:23459001

Allelic Expression of Deleterious Protein-Coding Variants across Human Tissues

PubMed Central

Kukurba, Kimberly R.; Zhang, Rui; Li, Xin; Smith, Kevin S.; Knowles, David A.; How Tan, Meng; Piskol, Robert; Lek, Monkol; Snyder, Michael; MacArthur, Daniel G.; Li, Jin Billy; Montgomery, Stephen B.

2014-01-01

Personal exome and genome sequencing provides access to loss-of-function and rare deleterious alleles whose interpretation is expected to provide insight into individual disease burden. However, for each allele, accurate interpretation of its effect will depend on both its penetrance and the trait's expressivity. In this regard, an important factor that can modify the effect of a pathogenic coding allele is its level of expression; a factor which itself characteristically changes across tissues. To better inform the degree to which pathogenic alleles can be modified by expression level across multiple tissues, we have conducted exome, RNA and deep, targeted allele-specific expression (ASE) sequencing in ten tissues obtained from a single individual. By combining such data, we report the impact of rare and common loss-of-function variants on allelic expression exposing stronger allelic bias for rare stop-gain variants and informing the extent to which rare deleterious coding alleles are consistently expressed across tissues. This study demonstrates the potential importance of transcriptome data to the interpretation of pathogenic protein-coding variants. PMID:24786518

Expression of TNF-alpha and immunohistochemical distribution of hepatic macrophage surface markers in carbon tetrachloride-induced chronic liver injury in rats.

PubMed

Orfila, C; Lepert, J C; Alric, L; Carrera, G; Beraud, M; Vinel, J P; Pipy, B

1999-10-01

In liver injury induced by carbon tetrachloride, secondary hepatic injury occurs from inflammatory processes originating from products released by activated Kupffer cells, which play a central role in hepatic inflammation. The purpose of our study was to demonstrate, in rats, the relationships between a function of the hepatic macrophages, TNF-alpha production and the state of activation of these cells, characterized by their phenotype, in the different phases of the process and development of fibrosis in a carbon tetrachloride-induced cirrhosis model. The immunohistochemical localization of proinflammatory cytokine TNF-alpha and surface surface makers (ED1 and ED2) was studied in hepatitis and cirrhosis in response to 3 and 9 weeks ingestion of carbon tetrachloride. After carbon tetrachloride ingestion, accompanying the increased necrosis, immunohistochemical analysis of liver tissue sections demonstrated the significantly increased number of cells expressing ED1, ED2 and TNF-alpha, compared to normal. The number of cells expressing the surface phenotypic markers of liver macrophages increased and this change was concomitantly associated with an increased cellular expression of TNF-alpha. Local macrophage proliferation and influx of newly recruited blood monocytes resulted in an increase of the macrophage population. The populational changes involved difference in functional activity and enhanced TNF-alpha expression. This cytokine expressed in the carbon tetrachloride-induced inflammatory process is associated with the development of fibrosis and may contribute to disease severity.

Analysis of the Nicotiana tabacum Stigma/Style Transcriptome Reveals Gene Expression Differences between Wet and Dry Stigma Species1[W][OA

PubMed Central

Quiapim, Andréa C.; Brito, Michael S.; Bernardes, Luciano A.S.; daSilva, Idalete; Malavazi, Iran; DePaoli, Henrique C.; Molfetta-Machado, Jeanne B.; Giuliatti, Silvana; Goldman, Gustavo H.; Goldman, Maria Helena S.

2009-01-01

The success of plant reproduction depends on pollen-pistil interactions occurring at the stigma/style. These interactions vary depending on the stigma type: wet or dry. Tobacco (Nicotiana tabacum) represents a model of wet stigma, and its stigmas/styles express genes to accomplish the appropriate functions. For a large-scale study of gene expression during tobacco pistil development and preparation for pollination, we generated 11,216 high-quality expressed sequence tags (ESTs) from stigmas/styles and created the TOBEST database. These ESTs were assembled in 6,177 clusters, from which 52.1% are pistil transcripts/genes of unknown function. The 21 clusters with the highest number of ESTs (putative higher expression levels) correspond to genes associated with defense mechanisms or pollen-pistil interactions. The database analysis unraveled tobacco sequences homologous to the Arabidopsis (Arabidopsis thaliana) genes involved in specifying pistil identity or determining normal pistil morphology and function. Additionally, 782 independent clusters were examined by macroarray, revealing 46 stigma/style preferentially expressed genes. Real-time reverse transcription-polymerase chain reaction experiments validated the pistil-preferential expression for nine out of 10 genes tested. A search for these 46 genes in the Arabidopsis pistil data sets demonstrated that only 11 sequences, with putative equivalent molecular functions, are expressed in this dry stigma species. The reverse search for the Arabidopsis pistil genes in the TOBEST exposed a partial overlap between these dry and wet stigma transcriptomes. The TOBEST represents the most extensive survey of gene expression in the stigmas/styles of wet stigma plants, and our results indicate that wet and dry stigmas/styles express common as well as distinct genes in preparation for the pollination process. PMID:19052150

Extending bicluster analysis to annotate unclassified ORFs and predict novel functional modules using expression data

PubMed Central

Bryan, Kenneth; Cunningham, Pádraig

2008-01-01

Background Microarrays have the capacity to measure the expressions of thousands of genes in parallel over many experimental samples. The unsupervised classification technique of bicluster analysis has been employed previously to uncover gene expression correlations over subsets of samples with the aim of providing a more accurate model of the natural gene functional classes. This approach also has the potential to aid functional annotation of unclassified open reading frames (ORFs). Until now this aspect of biclustering has been under-explored. In this work we illustrate how bicluster analysis may be extended into a 'semi-supervised' ORF annotation approach referred to as BALBOA. Results The efficacy of the BALBOA ORF classification technique is first assessed via cross validation and compared to a multi-class k-Nearest Neighbour (kNN) benchmark across three independent gene expression datasets. BALBOA is then used to assign putative functional annotations to unclassified yeast ORFs. These predictions are evaluated using existing experimental and protein sequence information. Lastly, we employ a related semi-supervised method to predict the presence of novel functional modules within yeast. Conclusion In this paper we demonstrate how unsupervised classification methods, such as bicluster analysis, may be extended using of available annotations to form semi-supervised approaches within the gene expression analysis domain. We show that such methods have the potential to improve upon supervised approaches and shed new light on the functions of unclassified ORFs and their co-regulation. PMID:18831786

Functional Characterization of a Novel Class of Morantel-Sensitive Acetylcholine Receptors in Nematodes

PubMed Central

Courtot, Elise; Charvet, Claude L.; Beech, Robin N.; Harmache, Abdallah; Wolstenholme, Adrian J.; Holden-Dye, Lindy; O’Connor, Vincent; Peineau, Nicolas; Woods, Debra J.; Neveu, Cedric

2015-01-01

Acetylcholine receptors are pentameric ligand–gated channels involved in excitatory neuro-transmission in both vertebrates and invertebrates. In nematodes, they represent major targets for cholinergic agonist or antagonist anthelmintic drugs. Despite the large diversity of acetylcholine-receptor subunit genes present in nematodes, only a few receptor subtypes have been characterized so far. Interestingly, parasitic nematodes affecting human or animal health possess two closely related members of this gene family, acr-26 and acr-27 that are essentially absent in free-living or plant parasitic species. Using the pathogenic parasitic nematode of ruminants, Haemonchus contortus, as a model, we found that Hco-ACR-26 and Hco-ACR-27 are co-expressed in body muscle cells. We demonstrated that co-expression of Hco-ACR-26 and Hco-ACR-27 in Xenopus laevis oocytes led to the functional expression of an acetylcholine-receptor highly sensitive to the anthelmintics morantel and pyrantel. Importantly we also reported that ACR-26 and ACR-27, from the distantly related parasitic nematode of horses, Parascaris equorum, also formed a functional acetylcholine-receptor highly sensitive to these two drugs. In Caenorhabditis elegans, a free-living model nematode, we demonstrated that heterologous expression of the H. contortus and P. equorum receptors drastically increased its sensitivity to morantel and pyrantel, mirroring the pharmacological properties observed in Xenopus oocytes. Our results are the first to describe significant molecular determinants of a novel class of nematode body wall muscle AChR. PMID:26625142

Utilization of the Tango beta-arrestin recruitment technology for cell-based EDG receptor assay development and interrogation.

PubMed

Wetter, Justin A; Revankar, Chetana; Hanson, Bonnie J

2009-10-01

Cellular assay development for the endothelial differentiation gene (EDG) family of G-protein-coupled receptors (GPCRs) and related lysophospholipid (LP) receptors is complicated by endogenous receptor expression and divergent receptor signaling. Endogenously expressed LP receptors exist in most tissue culture cell lines. These LP receptors, along with other endogenously expressed GPCRs, contribute to off-target signaling that can complicate interpretation of second-messenger-based cellular assay results. These receptors also activate a diverse and divergent set of cellular signaling pathways, necessitating the use of a variety of assay formats with mismatched procedures and functional readouts. This complicates examination and comparison of these receptors across the entire family. The Tango technology uses the conserved beta-arrestin-dependent receptor deactivation process to allow interrogation of the EDG and related receptors with a single functional assay. This method also isolates the target receptor signal, allowing the use of tissue culture cell lines regardless of their endogenous receptor expression. The authors describe the use of this technique to build cell-based receptor-specific assays for all 8 members of the EDG receptor family as well as the related LPA receptors GPR23, GPR92, and GPR87. In addition, they demonstrate the value of this technology for identification and investigation of functionally selective receptor compounds as demonstrated by the immunosuppressive compound FtY720-P and its action at the EDG(1) and EDG(3) receptors.

SCF/C-Kit/JNK/AP-1 Signaling Pathway Promotes Claudin-3 Expression in Colonic Epithelium and Colorectal Carcinoma.

PubMed

Wang, Yaxi; Sun, Tingyi; Sun, Haimei; Yang, Shu; Li, Dandan; Zhou, Deshan

2017-04-06

Claudin-3 is a major protein of tight junctions (TJs) in the intestinal epithelium and is critical for maintaining cell-cell adhesion, barrier function, and epithelium polarity. Recent studies have shown high claudin-3 levels in several solid tumors, but the regulation mechanism of claudin-3 expression remains poorly understood. In the present study, colorectal cancer (CRC) tissues, HT-29 and DLD-1 CRC cell lines, CRC murine model (C57BL/6 mice) and c-kit loss-of-function mutant mice were used. We demonstrated that elevated claudin-3 levels were positively correlated with highly expressed c-kit in CRC tissues based upon analysis of protein expression. In vitro, claudin-3 expression was clearly increased in CRC cells by overexpressed c-kit or stimulated by exogenous recombinant human stem cell factor (rhSCF), while significantly decreased by the treatment with c-kit or c-Jun N-terminal kinase (JNK) inhibitors. Chromatin immunoprecipitation (ChIP) and luciferase reporter assay showed that SCF/c-kit signaling significantly promoted activator protein-1 (AP-1) binding with CLDN-3 promoter and enhanced its transcription activity. Furthermore, decreased expression of claudin-3 was obtained in the colonic epithelium from the c-Kit loss-of-function mutant mice. In conclusion, SCF/c-kit-JNK/AP-1 signaling pathway significantly promoted claudin-3 expression in colonic epithelium and CRC, which could contribute to epithelial barrier function maintenance and to CRC development.

Activation of the mitogen-activated protein kinase pathway by bone sialoprotein regulates osteoblast differentiation.

PubMed

Gordon, Jonathan A R; Hunter, Graeme K; Goldberg, Harvey A

2009-01-01

Bone sialoprotein (BSP) is an abundant protein in the extracellular matrix of bone that has been suggested to have several different physiological functions, including the nucleation of hydroxyapatite (HA), promotion of cell attachment and binding of collagen. Studies in our lab have demonstrated that increased expression of BSP in osteoblast cells can increase expression of the osteoblast-related genes Runx2 and Osx as well as alkaline phosphatase and osteocalcin and increase matrix mineralization. To determine the molecular mechanisms responsible for the BSP-mediated increase in osteoblastic differentiation, several functional domain mutants of BSP were expressed in primary rat bone osteoblastic cells, including the contiguous glutamic acid sequences (polyGlu) and the arginine-glycine-aspartic acid (RGD) motif. Markers of osteoblast differentiation, including matrix mineralization and alkaline phosphatase staining, were increased in cells expressing BSP mutants of the polyGlu sequences but not in cells expressing RGD-mutated BSP. We also determined the dependence on integrin-associated pathways in promoting BSP-mediated differentiation responses in osteoblasts by demonstrating the activation of focal adhesion kinase, MAP kinase-associated proteins ERK1/2, ribosomal s6 kinase 2 and the AP-1 protein cFos. Thus, the mechanism regulating osteoblast differentiation by BSP was determined to be dependent on integrin-mediated intracellular signaling pathways. Copyright 2008 S. Karger AG, Basel.

The Tregs' world according to GARP.

PubMed

Battaglia, Manuela; Roncarolo, Maria Grazia

2009-12-01

Naturally occurring CD4+CD25(high) regulatory T cells (nTreg) are essential for maintaining tolerance. FOXP3 has been established as a molecular marker of nTreg; however, FOXP3 cannot be used as a reliable marker for bona fide human nTreg since effector T cells also up-regulate FOXP3 expression upon activation. Despite the important function of nTreg, the underlying molecular mechanisms of nTreg-mediated suppression are far from defined. Previous studies have demonstrated that the TGF-beta latency-associated peptide (LAP) is expressed on the surface of nTreg, and that immunosuppression can be mediated by membrane TGF-beta; however, it remains unknown how LAP is bound to nTreg and what is the functional significance of its selective expression on activated nTreg. The nTreg's world may now change according to GARP, an orphan toll-like receptor composed of leucine-rich repeats. In this issue of the European Journal of Immunology, a study provides further demonstration that GARP is selectively expressed only in activated human nTreg and nTreg cell clones but not in activated effector T cells, confirming GARP as a bona fide nTreg marker. In addition, GARP binds directly to LAP; yet, GARP over-expression is insufficient to induce modification of latent TGF-beta into active TGF-beta further clarifying its role in nTreg-mediated suppression.

MCT Expression and Lactate Influx/Efflux in Tanycytes Involved in Glia-Neuron Metabolic Interaction

PubMed Central

Cortés-Campos, Christian; Elizondo, Roberto; Llanos, Paula; Uranga, Romina María; Nualart, Francisco; García, María Angeles

2011-01-01

Metabolic interaction via lactate between glial cells and neurons has been proposed as one of the mechanisms involved in hypothalamic glucosensing. We have postulated that hypothalamic glial cells, also known as tanycytes, produce lactate by glycolytic metabolism of glucose. Transfer of lactate to neighboring neurons stimulates ATP synthesis and thus contributes to their activation. Because destruction of third ventricle (III-V) tanycytes is sufficient to alter blood glucose levels and food intake in rats, it is hypothesized that tanycytes are involved in the hypothalamic glucose sensing mechanism. Here, we demonstrate the presence and function of monocarboxylate transporters (MCTs) in tanycytes. Specifically, MCT1 and MCT4 expression as well as their distribution were analyzed in Sprague Dawley rat brain, and we demonstrate that both transporters are expressed in tanycytes. Using primary tanycyte cultures, kinetic analyses and sensitivity to inhibitors were undertaken to confirm that MCT1 and MCT4 were functional for lactate influx. Additionally, physiological concentrations of glucose induced lactate efflux in cultured tanycytes, which was inhibited by classical MCT inhibitors. Because the expression of both MCT1 and MCT4 has been linked to lactate efflux, we propose that tanycytes participate in glucose sensing based on a metabolic interaction with neurons of the arcuate nucleus, which are stimulated by lactate released from MCT1 and MCT4-expressing tanycytes. PMID:21297988

Circadian Behaviour in Neuroglobin Deficient Mice

PubMed Central

Hundahl, Christian A.; Fahrenkrug, Jan; Hay-Schmidt, Anders; Georg, Birgitte; Faltoft, Birgitte; Hannibal, Jens

2012-01-01

Neuroglobin (Ngb), a neuron-specific oxygen-binding globin with an unknown function, has been proposed to play a key role in neuronal survival. We have previously shown Ngb to be highly expressed in the rat suprachiasmatic nucleus (SCN). The present study addresses the effect of Ngb deficiency on circadian behavior. Ngb-deficient and wild-type (wt) mice were placed in running wheels and their activity rhythms, endogenous period and response to light stimuli were investigated. The effect of Ngb deficiency on the expression of Period1 (Per1) and the immediate early gene Fos was determined after light stimulation at night and the neurochemical phenotype of Ngb expressing neurons in wt mice was characterized. Loss of Ngb function had no effect on overall circadian entrainment, but resulted in a significantly larger phase delay of circadian rhythm upon light stimulation at early night. A light-induced increase in Per1, but not Fos, gene expression was observed in Ngb-deficient mice. Ngb expressing neurons which co-stored Gastrin Releasing Peptide (GRP) and were innervated from the eye and the geniculo-hypothalamic tract expressed FOS after light stimulation. No PER1 expression was observed in Ngb-positive neurons. The present study demonstrates for the first time that the genetic elimination of Ngb does not affect core clock function but evokes an increased behavioural response to light concomitant with increased Per1 gene expression in the SCN at early night. PMID:22496809

Circadian behaviour in neuroglobin deficient mice.

PubMed

Hundahl, Christian A; Fahrenkrug, Jan; Hay-Schmidt, Anders; Georg, Birgitte; Faltoft, Birgitte; Hannibal, Jens

2012-01-01

Neuroglobin (Ngb), a neuron-specific oxygen-binding globin with an unknown function, has been proposed to play a key role in neuronal survival. We have previously shown Ngb to be highly expressed in the rat suprachiasmatic nucleus (SCN). The present study addresses the effect of Ngb deficiency on circadian behavior. Ngb-deficient and wild-type (wt) mice were placed in running wheels and their activity rhythms, endogenous period and response to light stimuli were investigated. The effect of Ngb deficiency on the expression of Period1 (Per1) and the immediate early gene Fos was determined after light stimulation at night and the neurochemical phenotype of Ngb expressing neurons in wt mice was characterized. Loss of Ngb function had no effect on overall circadian entrainment, but resulted in a significantly larger phase delay of circadian rhythm upon light stimulation at early night. A light-induced increase in Per1, but not Fos, gene expression was observed in Ngb-deficient mice. Ngb expressing neurons which co-stored Gastrin Releasing Peptide (GRP) and were innervated from the eye and the geniculo-hypothalamic tract expressed FOS after light stimulation. No PER1 expression was observed in Ngb-positive neurons. The present study demonstrates for the first time that the genetic elimination of Ngb does not affect core clock function but evokes an increased behavioural response to light concomitant with increased Per1 gene expression in the SCN at early night.

Molecular mechanism of G1 arrest and cellular senescence induced by LEE011, a novel CDK4/CDK6 inhibitor, in leukemia cells.

PubMed

Tao, Yan-Fang; Wang, Na-Na; Xu, Li-Xiao; Li, Zhi-Heng; Li, Xiao-Lu; Xu, Yun-Yun; Fang, Fang; Li, Mei; Qian, Guang-Hui; Li, Yan-Hong; Li, Yi-Ping; Wu, Yi; Ren, Jun-Li; Du, Wei-Wei; Lu, Jun; Feng, Xing; Wang, Jian; He, Wei-Qi; Hu, Shao-Yan; Pan, Jian

2017-01-01

Overexpression of cyclin D1 dependent kinases 4 and 6 (CDK4/6) is a common feature of many human cancers including leukemia. LEE011 is a novel inhibitor of both CDK4 and 6. To date, the molecular function of LEE011 in leukemia remains unclear. Leukemia cell growth and apoptosis following LEE011 treatment was assessed through CCK-8 and annexin V/propidium iodide staining assays. Cell senescence was assessed by β-galactosidase staining and p16 INK4a expression analysis. Gene expression profiles of LEE011 treated HL-60 cells were investigated using an Arraystar Human LncRNA array. Gene ontology and KEGG pathway analysis were then used to analyze the differentially expressed genes from the cluster analysis. Our studies demonstrated that LEE011 inhibited proliferation of leukemia cells and could induce apoptosis. Hoechst 33,342 staining analysis showed DNA fragmentation and distortion of nuclear structures following LEE011 treatment. Cell cycle analysis showed LEE011 significantly induced cell cycle G 1 arrest in seven of eight acute leukemia cells lines, the exception being THP-1 cells. β-Galactosidase staining analysis and p16 INK4a expression analysis showed that LEE011 treatment can induce cell senescence of leukemia cells. LncRNA microarray analysis showed 2083 differentially expressed mRNAs and 3224 differentially expressed lncRNAs in LEE011-treated HL-60 cells compared with controls. Molecular function analysis showed that LEE011 induced senescence in leukemia cells partially through downregulation of the transcriptional expression of MYBL2. We demonstrate for the first time that LEE011 treatment results in inhibition of cell proliferation and induction of G 1 arrest and cellular senescence in leukemia cells. LncRNA microarray analysis showed differentially expressed mRNAs and lncRNAs in LEE011-treated HL-60 cells and we demonstrated that LEE011 induces cellular senescence partially through downregulation of the expression of MYBL2. These results may open new lines of investigation regarding the molecular mechanism of LEE011 induced cellular senescence.

miRNAs in brain development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Petri, Rebecca; Malmevik, Josephine; Fasching, Liana

2014-02-01

MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. In the brain, a large number of miRNAs are expressed and there is a growing body of evidence demonstrating that miRNAs are essential for brain development and neuronal function. Conditional knockout studies of the core components in the miRNA biogenesis pathway, such as Dicer and DGCR8, have demonstrated a crucial role for miRNAs during the development of the central nervous system. Furthermore, mice deleted for specific miRNAs and miRNA-clusters demonstrate diverse functional roles for different miRNAs during the development of different brain structures. miRNAs havemore » been proposed to regulate cellular functions such as differentiation, proliferation and fate-determination of neural progenitors. In this review we summarise the findings from recent studies that highlight the importance of miRNAs in brain development with a focus on the mouse model. We also discuss the technical limitations of current miRNA studies that still limit our understanding of this family of non-coding RNAs and propose the use of novel and refined technologies that are needed in order to fully determine the impact of specific miRNAs in brain development. - Highlights: • miRNAs are essential for brain development and neuronal function. • KO of Dicer is embryonically lethal. • Conditional Dicer KO results in defective proliferation or increased apoptosis. • KO of individual miRNAs or miRNA families is necessary to determine function.« less

Functional Heterologous Expression of an Engineered Full Length CipA from Clostridium thermocellum in Thermoanaerobacterium saccharolyticum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Currie, Devin; Herring, Christopher; Guss, Adam M

BACKGROUND: Cellulose is highly recalcitrant and thus requires a specialized suite of enzymes to solubilize it into fermentable sugars. In C. thermocellum, these extracellular enzymes are present as a highly active multi-component system known as the cellulosome. This study explores the expression of a critical C. thermocellum cellulosomal component in T. saccharolyticum as a step toward creating a thermophilic bacterium capable of consolidated bioprocessing by employing heterologously expressed cellulosomes. RESULTS: We developed an inducible promoter system based on the native T. saccharolyticum xynA promoter, which was shown to be induced by xylan and xylose. The promoter was used to expressmore » the cellulosomal component cipA*, an engineered form of the wild-type cipA from C. thermocellum. Expression and localization to the supernatant were both verified for CipA*. When a cipA mutant C. thermocellum strain was cultured with a CipA*-expressing T. saccharolyticum strain, hydrolysis and fermentation of 10 grams per liter SigmaCell 101, a highly crystalline cellulose, were observed. This trans-species complementation of a cipA deletion demonstrated the ability for CipA* to assemble a functional cellulosome. CONCLUSION: This study is the first example of an engineered thermophile heterologously expressing a structural component of a cellulosome. To achieve this goal we developed and tested an inducible promoter for controlled expression in T. saccharolyticum as well as a synthetic cipA. In addition, we demonstrate a high degree of hydrolysis (up to 93%) on microcrystalline cellulose.« less

miR-135A Regulates Preimplantation Embryo Development through Down-Regulation of E3 Ubiquitin Ligase Seven in Absentia Homolog 1A (SIAH1A) Expression

PubMed Central

Leung, Carmen O. N.; Ye, Tian-Min; Kwan, Peter C. K.; Lee, Kai-Fai; Yeung, William S. B.

2011-01-01

Background MicroRNAs (miRNAs) are small non-coding RNA molecules capable of regulating transcription and translation. Previously, a cluster of miRNAs that are specifically expressed in mouse zygotes but not in oocytes or other preimplantation stages embryos are identified by multiplex real-time polymerase chain reaction-based miRNA profiling. The functional role of one of these zygote-specific miRNAs, miR-135a, in preimplantation embryo development was investigated. Methodology/Principal Findings Microinjection of miR-135a inhibitor suppressed first cell cleavage in more than 30% of the zygotes. Bioinformatics analysis identified E3 Ubiquitin Ligase Seven In Absentia Homolog 1A (Siah1a) as a predicted target of miR-135a. Western blotting and 3′UTR luciferase functional assays demonstrated that miR-135a down-regulated the expression of Siah1 in HeLa cells and in mouse zygotes. Siah1a was expressed in preimplantation embryos and its expression pattern negatively correlated with that of miR-135a. Co-injection of Siah1a-specific antibody with miR-135a inhibitor partially nullified the effect of miR-135a inhibition. Proteasome inhibition by MG-132 revealed that miR-135a regulated proteasomal degradation and potentially controlled the expression of chemokinesin DNA binding protein (Kid). Conclusions/Significance The present study demonstrated for the first time that zygotic specific miRNA modulates the first cell cleavage through regulating expression of Siah1a. PMID:22132158

An RNAi based screen in Drosophila larvae identifies fascin as a regulator of myoblast fusion and myotendinous junction structure.

PubMed

Camuglia, Jaclyn M; Mandigo, Torrey R; Moschella, Richard; Mark, Jenna; Hudson, Christine H; Sheen, Derek; Folker, Eric S

2018-04-06

A strength of Drosophila as a model system is its utility as a tool to screen for novel regulators of various functional and developmental processes. However, the utility of Drosophila as a screening tool is dependent on the speed and simplicity of the assay used. Here, we use larval locomotion as an assay to identify novel regulators of skeletal muscle function. We combined this assay with muscle-specific depletion of 82 genes to identify genes that impact muscle function by their expression in muscle cells. The data from the screen were supported with characterization of the muscle pattern in embryos and larvae that had disrupted expression of the strongest hit from the screen. With this assay, we showed that 12/82 tested genes regulate muscle function. Intriguingly, the disruption of five genes caused an increase in muscle function, illustrating that mechanisms that reduce muscle function exist and that the larval locomotion assay is sufficiently quantitative to identify conditions that both increase and decrease muscle function. We extended the data from this screen and tested the mechanism by which the strongest hit, fascin, impacted muscle function. Compared to controls, animals in which fascin expression was disrupted with either a mutant allele or muscle-specific expression of RNAi had fewer muscles, smaller muscles, muscles with fewer nuclei, and muscles with disrupted myotendinous junctions. However, expression of RNAi against fascin only after the muscle had finished embryonic development did not recapitulate any of these phenotypes. These data suggest that muscle function is reduced due to impaired myoblast fusion, muscle growth, and muscle attachment. Together, these data demonstrate the utility of Drosophila larval locomotion as an assay for the identification of novel regulators of muscle development and implicate fascin as necessary for embryonic muscle development.

A constitutively active dioxin/aryl hydrocarbon receptor induces stomach tumors

NASA Astrophysics Data System (ADS)

Andersson, Patrik; McGuire, Jacqueline; Rubio, Carlos; Gradin, Katarina; Whitelaw, Murray L.; Pettersson, Sven; Hanberg, Annika; Poellinger, Lorenz

2002-07-01

The dioxin/aryl hydrocarbon receptor (AhR) functions as a ligand-activated transcription factor regulating transcription of a battery of genes encoding xenobiotic metabolizing enzymes. Known receptor ligands are environmental pollutants including polycyclic aromatic hydrocarbons and polychlorinated dioxins. Loss-of-function (gene-disruption) studies in mice have demonstrated that the AhR is involved in toxic effects of dioxins but have not yielded unequivocal results concerning the physiological function of the receptor. Gain-of-function studies therefore were performed to unravel the biological functions of the AhR. A constitutively active AhR expressed in transgenic mice reduced the life span of the mice and induced tumors in the glandular part of the stomach, demonstrating the oncogenic potential of the AhR and implicating the receptor in regulation of cell proliferation.

Multiple chimeric antigen receptors successfully target chondroitin sulfate proteoglycan 4 in several different cancer histologies and cancer stem cells

PubMed Central

2014-01-01

Background The development of immunotherapy has led to significant progress in the treatment of metastatic cancer, including the development of genetic engineering technologies that redirect lymphocytes to recognize and target a wide variety of tumor antigens. Chimeric antigen receptors (CARs) are hybrid proteins combining antibody recognition domains linked to T cell signaling elements. Clinical trials of CAR-transduced peripheral blood lymphocytes (PBL) have induced remission of both solid organ and hematologic malignancies. Chondroitin sulfate proteoglycan 4 (CSPG4) is a promising target antigen that is overexpressed in multiple cancer histologies including melanoma, triple-negative breast cancer, glioblastoma, mesothelioma and sarcoma. Methods CSPG4 expression in cancer cell lines was assayed using flow cytometry (FACS) and reverse-transcription PCR (RT-PCR). Immunohistochemistry was utilized to assay resected melanomas and normal human tissues (n = 30) for CSPG4 expression and a reverse-phase protein array comprising 94 normal tissue samples was also interrogated for CSPG4 expression. CARs were successfully constructed from multiple murine antibodies (225.28S, TP41.2, 149.53) using second generation (CD28.CD3ζ) signaling domains. CAR sequences were cloned into a gamma-retroviral vector with subsequent successful production of retroviral supernatant and PBL transduction. CAR efficacy was assayed by cytokine release and cytolysis following coculture with target cell lines. Additionally, glioblastoma stem cells were generated from resected human tumors, and CSPG4 expression was determined by RT-PCR and FACS. Results Immunohistochemistry demonstrated prominent CSPG4 expression in melanoma tumors, but failed to demonstrate expression in any of the 30 normal human tissues studied. Two of 94 normal tissue protein lysates were positive by protein array. CAR constructs demonstrated cytokine secretion and cytolytic function after co-culture with tumor cell lines from multiple different histologies, including melanoma, breast cancer, mesothelioma, glioblastoma and osteosarcoma. Furthermore, we report for the first time that CSPG4 is expressed on glioblastoma cancer stem cells (GSC) and demonstrate that anti-CSPG4 CAR-transduced T cells recognize and kill these GSC. Conclusions The functionality of multiple different CARs, with the widespread expression of CSPG4 on multiple malignancies, suggests that CSPG4 may be an attractive candidate tumor antigen for CAR-based immunotherapies using appropriate technology to limit possible off-tumor toxicity. PMID:25197555

Effect of iron deficiency on the expression of insulin-like growth factor-II and its receptor in neuronal and glial cells.

PubMed

Morales González, E; Contreras, I; Estrada, J A

2014-09-01

Many studies have demonstrated that iron deficiency modifies the normal function of the central nervous system and alters cognitive abilities. When cellular damage occurs in the central nervous system, neuroprotective mechanisms, such as the production of neurotrophic factors, are essential in order for nervous tissue to function correctly. Insulin-like growth factor II (IGF- II) is a neurotrophic factor that was recently shown to be involved in the normal functioning of cognitive processes in animal models. However, the impact of iron deficiency on the expression and function of this molecule has not yet been clarified. Mixed primary cell cultures from the central nervous system were collected to simulate iron deficiency using deferoxamine. The expression of IGF-I, IGF-II, IGF-IR, and IGF-IIR was determined with the western blot test. We observed increased expression of IGF-II, along with a corresponding decrease in the expression of IGF-IIR, in iron-deficient mixed primary cell cultures. We did not observe alterations in the expression of these proteins in isolated microglia or neuronal cultures under the same conditions. We did not detect differences in the expression of IGF-I and IGF-IR in iron-deficient cultures. In vitro iron deficiency increases the expression of IGF-II in mixed glial cell cultures, which may have a beneficial effect on brain tissue homeostasis in a situation in which iron availability is decreased. Copyright © 2013 Sociedad Española de Neurología. Published by Elsevier Espana. All rights reserved.

Fungal Gene Expression on Demand: an Inducible, Tunable, and Metabolism-Independent Expression System for Aspergillus niger▿†

PubMed Central

Meyer, Vera; Wanka, Franziska; van Gent, Janneke; Arentshorst, Mark; van den Hondel, Cees A. M. J. J.; Ram, Arthur F. J.

2011-01-01

Filamentous fungi are the cause of serious human and plant diseases but are also exploited in biotechnology as production platforms. Comparative genomics has documented their genetic diversity, and functional genomics and systems biology approaches are under way to understand the functions and interaction of fungal genes and proteins. In these approaches, gene functions are usually inferred from deletion or overexpression mutants. However, studies at these extreme points give only limited information. Moreover, many overexpression studies use metabolism-dependent promoters, often causing pleiotropic effects and thus limitations in their significance. We therefore established and systematically evaluated a tunable expression system for Aspergillus niger that is independent of carbon and nitrogen metabolism and silent under noninduced conditions. The system consists of two expression modules jointly targeted to a defined genomic locus. One module ensures constitutive expression of the tetracycline-dependent transactivator rtTA2S-M2, and one module harbors the rtTA2S-M2-dependent promoter that controls expression of the gene of interest (the Tet-on system). We show here that the system is tight, responds within minutes after inducer addition, and allows fine-tuning based on the inducer concentration or gene copy number up to expression levels higher than the expression levels of the gpdA promoter. We also validate the Tet-on system for the generation of conditional overexpression mutants and demonstrate its power when combined with a gene deletion approach. Finally, we show that the system is especially suitable when the functions of essential genes must be examined. PMID:21378046

Src-like adaptor protein down-regulates T cell receptor (TCR)-CD3 expression by targeting TCRzeta for degradation.

PubMed

Myers, Margaret D; Dragone, Leonard L; Weiss, Arthur

2005-07-18

Src-like adaptor protein (SLAP) down-regulates expression of the T cell receptor (TCR)-CD3 complex during a specific stage of thymocyte development when the TCR repertoire is selected. Consequently, SLAP-/- thymocytes display alterations in thymocyte development. Here, we have studied the mechanism of SLAP function. We demonstrate that SLAP-deficient thymocytes have increased TCRzeta chain expression as a result of a defect in TCRzeta degradation. Failure to degrade TCRzeta leads to an increased pool of fully assembled TCR-CD3 complexes that are capable of recycling back to the cell surface. We also provide evidence that SLAP functions in a pathway that requires the phosphorylated TCRzeta chain and the Src family kinase Lck, but not ZAP-70 (zeta-associated protein of 70 kD). These studies reveal a unique mechanism by which SLAP contributes to the regulation of TCR expression during a distinct stage of thymocyte development.

Altered RECQL5 expression in urothelial bladder carcinoma increases cellular proliferation and makes RECQL5 helicase activity a novel target for chemotherapy

PubMed Central

Patterson, Karl; Arya, Lovleen; Bottomley, Sarah; Morgan, Susan; Cox, Angela; Catto, James; Bryant, Helen E.

2016-01-01

RECQ helicases are a family of enzymes with both over lapping and unique functions. Functional autosomal recessive loss of three members of the family BLM, WRN and RECQL4, results in hereditary human syndromes characterized by cancer predisposition and premature aging, but despite the finding that RECQL5 deficient mice are cancer prone, no such link has been made to human RECQL5. Here we demonstrate that human urothelial carcinoma of the bladder (UCC) has increased expression of RECQL5 compared to normal bladder tissue and that increasing RECQL5 expression can drive proliferation of normal bladder cells and is associated with poor prognosis. Further, by expressing a helicase dead RECQL5 and by depleting bladder cancer cells of RECQL5 we show that inhibition of RECQL5 activity has potential as a new target for treatment of UCC. PMID:27764811

CD54+ rabbit adipose-derived stem cells overexpressing HIF-1α facilitate vascularized fat flap regeneration

PubMed Central

Liang, Zhi-Jie; Huang, Min-Hong; Peng, Qi-Liu; Zou, Dong-Hua; Gu, Rong-He; Xu, Fang-Tian; Gao, Hui; Chen, Zhen-Dong; Chi, Guang-Yi; Wei, Zhong-Heng; Chen, Li; Li, Hong-Mian

2017-01-01

Fat flap transplantation is frequently performed in patients suffering from soft tissue defects resulting from disease or trauma. This study explored the feasibility of constructing vascularized fat flaps using rabbit adipose-derived stem cells (rASCs) and collagen scaffolds in a rabbit model. We evaluated rASCs proliferation, paracrine function, adipogenesis, vascularization, and CD54 expression, with or without HIF-1α transfection in vitro and in vivo. We observed that adipogenic differentiation potential was greater in rASCs with high CD54 expression (CD54+rASCs) than in those with low expression (CD54–rASCs), both in vitro and in vivo. HIF-1α overexpression not only augmented this effect, but also enhanced cell proliferation and paracrine function in vitro. We also demonstrated that HIF-1α-transfected CD54+rASCs showed enhanced paracrine function and adipogenic capacity, and that paracrine function increases expression of angiogenesis-related markers. Thus, CD54+rASCs overexpressing HIF-1α enhanced large volume vascularized fat flap regeneration in rabbits, suggesting CD54 may be an ideal candidate marker for ASCs adipogenic differentiation. PMID:28423354

Insulin-like growth factor-I regulates GPER expression and function in cancer cells.

PubMed

De Marco, P; Bartella, V; Vivacqua, A; Lappano, R; Santolla, M F; Morcavallo, A; Pezzi, V; Belfiore, A; Maggiolini, M

2013-02-07

Functional cross talk between insulin-like growth factor-I (IGF-I) system and estrogen signaling has been largely reported, although the underlying molecular mechanisms remain to be fully elucidated. As GPR30/GPER mediates rapid cell responses to estrogens, we evaluated the potential of IGF-I to regulate GPER expression and function in estrogen receptor (ER)α-positive breast (MCF-7) and endometrial (Ishikawa) cancer cells. We found that IGF-I transactivates the GPER promoter sequence and upregulates GPER mRNA and protein levels in both cells types. Similar data were found, at least in part, in carcinoma-associated fibroblasts. The upregulation of GPER expression by IGF-I involved the IGF-IR/PKCδ/ERK/c-fos/AP1 transduction pathway and required ERα, as ascertained by specific pharmacological inhibitors and gene-silencing. In both MCF-7 and Ishikawa cancer cells, the IGF-I-dependent cell migration required GPER and its main target gene CTGF, whereas the IGF-I-induced proliferation required both GPER and cyclin D1. Our data demonstrate that the IGF-I system regulates GPER expression and function, triggering the activation of a signaling network that leads to the migration and proliferation of cancer cells.

Vibrio cholerae ToxR downregulates virulence factor production in response to cyclo(Phe-Pro).

PubMed

Bina, X Renee; Taylor, Dawn L; Vikram, Amit; Ante, Vanessa M; Bina, James E

2013-08-27

Vibrio cholerae is an aquatic organism that causes the severe acute diarrheal disease cholera. The ability of V. cholerae to cause disease is dependent upon the production of two critical virulence determinants, cholera toxin (CT) and the toxin-coregulated pilus (TCP). The expression of the genes that encode for CT and TCP production is under the control of a hierarchical regulatory system called the ToxR regulon, which functions to activate virulence gene expression in response to in vivo stimuli. Cyclic dipeptides have been found to be produced by numerous bacteria, yet their biological function remains unknown. V. cholerae has been shown to produce cyclo(Phe-Pro). Previous studies in our laboratory demonstrated that cyclo(Phe-Pro) inhibited V. cholerae virulence factor production. For this study, we report on the mechanism by which cyclo(Phe-Pro) inhibited virulence factor production. We have demonstrated that exogenous cyclo(Phe-Pro) activated the expression of leuO, a LysR-family regulator that had not been previously associated with V. cholerae virulence. Increased leuO expression repressed aphA transcription, which resulted in downregulation of the ToxR regulon and attenuated CT and TCP production. The cyclo(Phe-Pro)-dependent induction of leuO expression was found to be dependent upon the virulence regulator ToxR. Cyclo(Phe-Pro) did not affect toxR transcription or ToxR protein levels but appeared to enhance the ToxR-dependent transcription of leuO. These results have identified leuO as a new component of the ToxR regulon and demonstrate for the first time that ToxR is capable of downregulating virulence gene expression in response to an environmental cue. The ToxR regulon has been a focus of cholera research for more than three decades. During this time, a model has emerged wherein ToxR functions to activate the expression of Vibrio cholerae virulence factors upon host entry. V. cholerae and other enteric bacteria produce cyclo(Phe-Pro), a cyclic dipeptide that we identified as an inhibitor of V. cholerae virulence factor production. This finding suggested that cyclo(Phe-Pro) was a negative effector of virulence factor production and represented a molecule that could potentially be exploited for therapeutic development. In this work, we investigated the mechanism by which cyclo(Phe-Pro) inhibited virulence factor production. We found that cyclo(Phe-Pro) signaled through ToxR to activate the expression of leuO, a new virulence regulator that functioned to repress virulence factor production. Our results have identified a new arm of the ToxR regulon and suggest that ToxR may play a broader role in pathogenesis than previously known.

In silico design of context-responsive mammalian promoters with user-defined functionality

PubMed Central

Gibson, Suzanne J.; Hatton, Diane

2017-01-01

Abstract Comprehensive de novo-design of complex mammalian promoters is restricted by unpredictable combinatorial interactions between constituent transcription factor regulatory elements (TFREs). In this study, we show that modular binding sites that do not function cooperatively can be identified by analyzing host cell transcription factor expression profiles, and subsequently testing cognate TFRE activities in varying homotypic and heterotypic promoter architectures. TFREs that displayed position-insensitive, additive function within a specific expression context could be rationally combined together in silico to create promoters with highly predictable activities. As TFRE order and spacing did not affect the performance of these TFRE-combinations, compositions could be specifically arranged to preclude the formation of undesirable sequence features. This facilitated simple in silico-design of promoters with context-required, user-defined functionalities. To demonstrate this, we de novo-created promoters for biopharmaceutical production in CHO cells that exhibited precisely designed activity dynamics and long-term expression-stability, without causing observable retroactive effects on cellular performance. The design process described can be utilized for applications requiring context-responsive, customizable promoter function, particularly where co-expression of synthetic TFs is not suitable. Although the synthetic promoter structure utilized does not closely resemble native mammalian architectures, our findings also provide additional support for a flexible billboard model of promoter regulation. PMID:28977454

Global Landscape of a Co-Expressed Gene Network in Barley and its Application to Gene Discovery in Triticeae Crops

PubMed Central

Mochida, Keiichi; Uehara-Yamaguchi, Yukiko; Yoshida, Takuhiro; Sakurai, Tetsuya; Shinozaki, Kazuo

2011-01-01

Accumulated transcriptome data can be used to investigate regulatory networks of genes involved in various biological systems. Co-expression analysis data sets generated from comprehensively collected transcriptome data sets now represent efficient resources that are capable of facilitating the discovery of genes with closely correlated expression patterns. In order to construct a co-expression network for barley, we analyzed 45 publicly available experimental series, which are composed of 1,347 sets of GeneChip data for barley. On the basis of a gene-to-gene weighted correlation coefficient, we constructed a global barley co-expression network and classified it into clusters of subnetwork modules. The resulting clusters are candidates for functional regulatory modules in the barley transcriptome. To annotate each of the modules, we performed comparative annotation using genes in Arabidopsis and Brachypodium distachyon. On the basis of a comparative analysis between barley and two model species, we investigated functional properties from the representative distributions of the gene ontology (GO) terms. Modules putatively involved in drought stress response and cellulose biogenesis have been identified. These modules are discussed to demonstrate the effectiveness of the co-expression analysis. Furthermore, we applied the data set of co-expressed genes coupled with comparative analysis in attempts to discover potentially Triticeae-specific network modules. These results demonstrate that analysis of the co-expression network of the barley transcriptome together with comparative analysis should promote the process of gene discovery in barley. Furthermore, the insights obtained should be transferable to investigations of Triticeae plants. The associated data set generated in this analysis is publicly accessible at http://coexpression.psc.riken.jp/barley/. PMID:21441235

The Epidermal Growth Factor Receptor Critically Regulates Endometrial Function during Early Pregnancy

PubMed Central

Large, Michael J.; Wetendorf, Margeaux; Lanz, Rainer B.; Hartig, Sean M.; Creighton, Chad J.; Mancini, Michael A.; Kovanci, Ertug; Lee, Kuo-Fen; Threadgill, David W.; Lydon, John P.; Jeong, Jae-Wook; DeMayo, Francesco J.

2014-01-01

Infertility and adverse gynecological outcomes such as preeclampsia and miscarriage represent significant female reproductive health concerns. The spatiotemporal expression of growth factors indicates that they play an important role in pregnancy. The goal of this study is to define the role of the ERBB family of growth factor receptors in endometrial function. Using conditional ablation in mice and siRNA in primary human endometrial stromal cells, we identified the epidermal growth factor receptor (Egfr) to be critical for endometrial function during early pregnancy. While ablation of Her2 or Erbb3 led to only a modest reduction in litter size, mice lacking Egfr expression are severely subfertile. Pregnancy demise occurred shortly after blastocyst implantation due to defects in decidualization including decreased proliferation, cell survival, differentiation and target gene expression. To place Egfr in a genetic regulatory hierarchy, transcriptome analyses was used to compare the gene signatures from mice with conditional ablation of Egfr, wingless-related MMTV integration site 4 (Wnt4) or boneless morphogenic protein 2 (Bmp2); revealing that not only are Bmp2 and Wnt4 key downstream effectors of Egfr, but they also regulate distinct physiological functions. In primary human endometrial stromal cells, marker gene expression, a novel high content image-based approach and phosphokinase array analysis were used to demonstrate that EGFR is a critical regulator of human decidualization. Furthermore, inhibition of EGFR signaling intermediaries WNK1 and AKT1S1, members identified in the kinase array and previously unreported to play a role in the endometrium, also attenuate decidualization. These results demonstrate that EGFR plays an integral role in establishing the cellular context necessary for successful pregnancy via the activation of intricate signaling and transcriptional networks, thereby providing valuable insight into potential therapeutic targets. PMID:24945252

Probiotic Bifidobacterium species stimulate human SLC26A3 gene function and expression in intestinal epithelial cells

PubMed Central

Kumar, Anoop; Hecht, Cameron; Priyamvada, Shubha; Anbazhagan, Arivarasu N.; Alakkam, Anas; Borthakur, Alip; Alrefai, Waddah A.; Gill, Ravinder K.

2014-01-01

SLC26A3, or downregulated in adenoma (DRA), plays a major role in mediating Cl− absorption in the mammalian intestine. Disturbances in DRA function and expression have been implicated in intestinal disorders such as congenital Cl− diarrhea and gut inflammation. We previously showed that an increase in DRA function and expression by Lactobacillus acidophilus and its culture supernatant (CS) might underlie antidiarrheal effects of this probiotic strain. However, the effects of Bifidobacterium species, important inhabitants of the human colon, on intestinal Cl−/HCO3− exchange activity are not known. Our current results demonstrate that CS derived from Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium bifidum increased anion exchange activity in Caco-2 cells (∼1.8- to 2.4-fold). Consistent with the increase in DRA function, CS also increased the protein, as well as the mRNA, level of DRA (but not putative anion transporter 1). CS of all three Bifidobacterium sp. increased DRA promoter activity (−1,183/+114 bp) in Caco-2 cells (1.5- to 1.8-fold). Furthermore, the increase in DRA mRNA expression by CS of B. breve and B. infantis was blocked in the presence of the transcription inhibitor actinomycin D (5 μM) and the ERK1/2 MAPK pathway inhibitor U0126 (10 μM). Administration of live B. breve, B. infantis, and B. bifidum by oral gavage to mice for 24 h increased DRA mRNA and protein levels in the colon. These data demonstrate an upregulation of DRA via activation of the ERK1/2 pathway that may underlie potential antidiarrheal effects of Bifidobacterium sp. PMID:25143346

FOXO3 Modulates Endothelial Gene Expression and Function by Classical and Alternative Mechanisms*

PubMed Central

Czymai, Tobias; Viemann, Dorothee; Sticht, Carsten; Molema, Grietje; Goebeler, Matthias; Schmidt, Marc

2010-01-01

FOXO transcription factors represent targets of the phosphatidylinositol 3-kinase/protein kinase B survival pathway controlling important biological processes, such as cell cycle progression, apoptosis, vascular remodeling, stress responses, and metabolism. Recent studies suggested the existence of alternative mechanisms of FOXO-dependent gene expression beyond classical binding to a FOXO-responsive DNA-binding element (FRE). Here we analyzed the relative contribution of those mechanisms to vascular function by comparing the transcriptional and cellular responses to conditional activation of FOXO3 and a corresponding FRE-binding mutant in human primary endothelial cells. We demonstrate that FOXO3 controls expression of vascular remodeling genes in an FRE-dependent manner. In contrast, FOXO3-induced cell cycle arrest and apoptosis occurs independently of FRE binding, albeit FRE-dependent gene expression augments the proapoptotic response. These findings are supported by bioinformatical analysis, which revealed a statistical overrepresentation of cell cycle regulators and apoptosis-related genes in the group of co-regulated genes. Molecular analysis of FOXO3-induced endothelial apoptosis excluded modulators of the extrinsic death receptor pathway and demonstrated important roles for the BCL-2 family members BIM and NOXA in this process. Although NOXA essentially contributed to FRE-dependent apoptosis, BIM was effectively induced in the absence of FRE-binding, and small interfering RNA-mediated BIM depletion could rescue apoptosis induced by both FOXO3 mutants. These data suggest BIM as a critical cell type-specific mediator of FOXO3-induced endothelial apoptosis, whereas NOXA functions as an amplifying factor. Our study provides the first comprehensive analysis of alternatively regulated FOXO3 targets in relevant primary cells and underscores the importance of such genes for endothelial function and integrity. PMID:20123982

Eucalyptus hairy roots, a fast, efficient and versatile tool to explore function and expression of genes involved in wood formation.

PubMed

Plasencia, Anna; Soler, Marçal; Dupas, Annabelle; Ladouce, Nathalie; Silva-Martins, Guilherme; Martinez, Yves; Lapierre, Catherine; Franche, Claudine; Truchet, Isabelle; Grima-Pettenati, Jacqueline

2016-06-01

Eucalyptus are of tremendous economic importance being the most planted hardwoods worldwide for pulp and paper, timber and bioenergy. The recent release of the Eucalyptus grandis genome sequence pointed out many new candidate genes potentially involved in secondary growth, wood formation or lineage-specific biosynthetic pathways. Their functional characterization is, however, hindered by the tedious, time-consuming and inefficient transformation systems available hitherto for eucalypts. To overcome this limitation, we developed a fast, reliable and efficient protocol to obtain and easily detect co-transformed E. grandis hairy roots using fluorescent markers, with an average efficiency of 62%. We set up conditions both to cultivate excised roots in vitro and to harden composite plants and verified that hairy root morphology and vascular system anatomy were similar to wild-type ones. We further demonstrated that co-transformed hairy roots are suitable for medium-throughput functional studies enabling, for instance, protein subcellular localization, gene expression patterns through RT-qPCR and promoter expression, as well as the modulation of endogenous gene expression. Down-regulation of the Eucalyptus cinnamoyl-CoA reductase1 (EgCCR1) gene, encoding a key enzyme in lignin biosynthesis, led to transgenic roots with reduced lignin levels and thinner cell walls. This gene was used as a proof of concept to demonstrate that the function of genes involved in secondary cell wall biosynthesis and wood formation can be elucidated in transgenic hairy roots using histochemical, transcriptomic and biochemical approaches. The method described here is timely because it will accelerate gene mining of the genome for both basic research and industry purposes. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Temperature regulates methane production through the function centralization of microbial community in anaerobic digestion.

PubMed

Lin, Qiang; De Vrieze, Jo; He, Guihua; Li, Xiangzhen; Li, Jiabao

2016-09-01

Temperature is crucial for the performance of anaerobic digestion process. In this study of anaerobic digestion of swine manure, the relationship between the microbial gene expression and methane production at different temperatures (25-55°C) was revealed through metatranscriptomic analysis. Daily methane production and total biogas production increased with temperature up to 50°C, but decreased at 55°C. The functional gene expression showed great variation at different temperatures. The function centralization (opposite to alpha-diversity), assessed by the least proportions of functional pathways contributing for at least 50% of total reads positively correlated to methane production. Temperature regulated methane production probably through reducing the diversity of functional pathways, but enhancing central functional pathways, so that most of cellular activities and resource were invested in methanogenesis and related pathways, enhancing the efficiency of conversion of substrates to methane. This research demonstrated the importance of function centralization for efficient system functioning. Copyright © 2016 Elsevier Ltd. All rights reserved.

FAS/FASL are dysregulated in chordoma and their loss-of-function impairs zebrafish notochord formation.

PubMed

Ferrari, Luca; Pistocchi, Anna; Libera, Laura; Boari, Nicola; Mortini, Pietro; Bellipanni, Gianfranco; Giordano, Antonio; Cotelli, Franco; Riva, Paola

2014-07-30

Chordoma is a rare malignant tumor that recapitulates the notochord phenotype and is thought to derive from notochord remnants not correctly regressed during development. Apoptosis is necessary for the proper notochord development in vertebrates, and the apoptotic pathway mediated by Fas and Fasl has been demonstrated to be involved in notochord cells regression. This study was conducted to investigate the expression of FAS/FASL pathway in a cohort of skull base chordomas and to analyze the role of fas/fasl homologs in zebrafish notochord formation. FAS/FASL expression was found to be dysregulated in chordoma leading to inactivation of the downstream Caspases in the samples analyzed. Both fas and fasl were specifically expressed in zebrafish notochord sorted cells. fas and fasl loss-of-function mainly resulted in larvae with notochord multi-cell-layer jumps organization, larger vacuolated notochord cells, defects in the peri-notochordal sheath structure and in vertebral mineralization. Interestingly, we observed the persistent expression of ntla and col2a1a, the zebrafish homologs of the human T gene and COL2A1 respectively, which are specifically up-regulated in chordoma. These results demonstrate for the first time the dysregulation of FAS/FASL in chordoma and their role in notochord formation in the zebrafish model, suggesting their possible implication in chordoma onset.

PD-1 expression on dendritic cells suppresses CD8+ T cell function and antitumor immunity.

PubMed

Lim, Tong Seng; Chew, Valerie; Sieow, Je Lin; Goh, Siting; Yeong, Joe Poh-Sheng; Soon, Ai Ling; Ricciardi-Castagnoli, Paola

2016-03-01

Programmed death one (PD-1) is a well-established co-inhibitory regulator that suppresses proliferation and cytokine production of T cells. Despite remarkable progress in delineating the functional roles of PD-1 on T lymphocytes, little is known about the regulatory role of PD-1 expressed on myeloid cells such as dendritic cells (DCs). Here, we show that CD8 + T cells can be more potently activated to secrete IL-2 and IFNγ by PD-1-deficient DCs compared to wild-type DCs. Adoptive transfer of PD-1-deficient DCs demonstrated their superior capabilities in inducing antigen-specific CD8 + T cell proliferation in vivo . In addition, we provide first evidence demonstrating the existence of peripheral blood DCs and CD11c + tumor-infiltrating myeloid cells that co-express PD-1 in patients with hepatocellular carcinoma (HCC). The existence of PD-1-expressing HCC-infiltrating DCs (HIDCs) was further supported in a mouse model of HCC. Intratumoral transfer of PD-1-deficient DCs rendered recipient mice resistant to the growth of HCC by promoting tumor-infiltrating CD8 + effector T cells to secrete perforin and granzyme B. This novel finding provides a deeper understanding of the role of PD-1 in immune regulation and has significant implications for cancer immunotherapies targeting PD-1.

PD-1 expression on dendritic cells suppresses CD8+ T cell function and antitumor immunity

PubMed Central

Lim, Tong Seng; Chew, Valerie; Sieow, Je Lin; Goh, Siting; Yeong, Joe Poh-Sheng; Soon, Ai Ling; Ricciardi-Castagnoli, Paola

2016-01-01

ABSTRACT Programmed death one (PD-1) is a well-established co-inhibitory regulator that suppresses proliferation and cytokine production of T cells. Despite remarkable progress in delineating the functional roles of PD-1 on T lymphocytes, little is known about the regulatory role of PD-1 expressed on myeloid cells such as dendritic cells (DCs). Here, we show that CD8+ T cells can be more potently activated to secrete IL-2 and IFNγ by PD-1-deficient DCs compared to wild-type DCs. Adoptive transfer of PD-1-deficient DCs demonstrated their superior capabilities in inducing antigen-specific CD8+ T cell proliferation in vivo. In addition, we provide first evidence demonstrating the existence of peripheral blood DCs and CD11c+ tumor-infiltrating myeloid cells that co-express PD-1 in patients with hepatocellular carcinoma (HCC). The existence of PD-1-expressing HCC-infiltrating DCs (HIDCs) was further supported in a mouse model of HCC. Intratumoral transfer of PD-1-deficient DCs rendered recipient mice resistant to the growth of HCC by promoting tumor-infiltrating CD8+ effector T cells to secrete perforin and granzyme B. This novel finding provides a deeper understanding of the role of PD-1 in immune regulation and has significant implications for cancer immunotherapies targeting PD-1. PMID:27141339

Matrix-Assisted Transplantation of Functional Beige Adipose Tissue

PubMed Central

Tharp, Kevin M.; Jha, Amit K.; Kraiczy, Judith; Yesian, Alexandra; Karateev, Grigory; Sinisi, Riccardo; Dubikovskaya, Elena A.

2015-01-01

Novel, clinically relevant, approaches to shift energy balance are urgently needed to combat metabolic disorders such as obesity and diabetes. One promising approach has been the expansion of brown adipose tissues that express uncoupling protein (UCP) 1 and thus can uncouple mitochondrial respiration from ATP synthesis. While expansion of UCP1-expressing adipose depots may be achieved in rodents via genetic and pharmacological manipulations or the transplantation of brown fat depots, these methods are difficult to use for human clinical intervention. We present a novel cell scaffold technology optimized to establish functional brown fat–like depots in vivo. We adapted the biophysical properties of hyaluronic acid–based hydrogels to support the differentiation of white adipose tissue–derived multipotent stem cells (ADMSCs) into lipid-accumulating, UCP1-expressing beige adipose tissue. Subcutaneous implantation of ADMSCs within optimized hydrogels resulted in the establishment of distinct UCP1-expressing implants that successfully attracted host vasculature and persisted for several weeks. Importantly, implant recipients demonstrated elevated core body temperature during cold challenges, enhanced respiration rates, improved glucose homeostasis, and reduced weight gain, demonstrating the therapeutic merit of this highly translatable approach. This novel approach is the first truly clinically translatable system to unlock the therapeutic potential of brown fat–like tissue expansion. PMID:26293504

Naringenin Inhibits Adipogenesis and Reduces Insulin Sensitivity and Adiponectin Expression in Adipocytes

PubMed Central

Richard, Allison J.; Ribnicky, David M.; Stephens, Jacqueline M.

2013-01-01

Adipose tissue development and function are widely studied to examine the relationship between obesity and the metabolic syndrome. It is well documented that the inability of adipose tissue to properly increase its lipid storage capacity during the obese state can lead to metabolic dysfunction. In a blind screen of 425 botanicals, we identified naringenin as an inhibitor of adipocyte differentiation. Naringenin is one of the most abundant citrus flavonoids, and recent studies have demonstrated antihyperlipidemic capabilities. These studies have largely focused on the effects of naringenin on the liver. Our biochemical studies clearly demonstrate that naringenin inhibits adipogenesis and impairs mature fat cell function. Naringenin specifically inhibited adipogenesis in a dose-dependent fashion as judged by examining lipid accumulation and induction of adipocyte marker protein expression. In mature 3T3-L1 adipocytes, naringenin reduced the ability of insulin to induce IRS-1 tyrosine phosphorylation and substantially inhibited insulin-stimulated glucose uptake in a dose-dependent manner and over a time frame of 1.5 to 24 hours. Exposure to naringenin also inhibited adiponectin protein expression in mature murine and human adipocytes. Our studies have revealed that naringenin may have a negative impact on adipocyte-related diseases by limiting differentiation of preadipocytes, by significantly inducing insulin resistance, and by decreasing adiponectin expression in mature fat cells. PMID:23983791

Sleep is not just for the brain: transcriptional responses to sleep in peripheral tissues.

PubMed

Anafi, Ron C; Pellegrino, Renata; Shockley, Keith R; Romer, Micah; Tufik, Sergio; Pack, Allan I

2013-05-30

Many have assumed that the primary function of sleep is for the brain. We evaluated the molecular consequences of sleep and sleep deprivation outside the brain, in heart and lung. Using microarrays we compared gene expression in tissue from sleeping and sleep deprived mice euthanized at the same diurnal times. In each tissue, nearly two thousand genes demonstrated statistically significant differential expression as a function of sleep/wake behavioral state. To mitigate the influence of an artificial deprivation protocol, we identified a subset of these transcripts as specifically sleep-enhanced or sleep-repressed by requiring that their expression also change over the course of unperturbed sleep. 3% and 6% of the assayed transcripts showed "sleep specific" changes in the lung and heart respectively. Sleep specific transcripts in these tissues demonstrated highly significant overlap and shared temporal dynamics. Markers of cellular stress and the unfolded protein response were reduced during sleep in both tissues. These results mirror previous findings in brain. Sleep-enhanced pathways reflected the unique metabolic functions of each tissue. Transcripts related to carbohydrate and sulfur metabolic processes were enhanced by sleep in the lung, and collectively favor buffering from oxidative stress. DNA repair and protein metabolism annotations were significantly enriched among the sleep-enhanced transcripts in the heart. Our results also suggest that sleep may provide a Zeitgeber, or synchronizing cue, in the lung as a large cluster of transcripts demonstrated systematic changes in inter-animal variability as a function of both sleep duration and circadian time. Our data support the notion that the molecular consequences of sleep/wake behavioral state extend beyond the brain to include peripheral tissues. Sleep state induces a highly overlapping response in both heart and lung. We conclude that sleep enhances organ specific molecular functions and that it has a ubiquitous role in reducing cellular metabolic stress in both brain and peripheral tissues. Finally, our data suggest a novel role for sleep in synchronizing transcription in peripheral tissues.

Novel synergic antidiabetic effects of Astragalus polysaccharides combined with Crataegus flavonoids via improvement of islet function and liver metabolism.

PubMed

Cui, Kai; Zhang, Shaobo; Jiang, Xin; Xie, Weidong

2016-06-01

The present study investigated the synergic effects and potential mechanisms of action of Astragalus polysaccharides (APS) combined with Crataegus flavonoids (CF) in the treatment of type 1 diabetes. Diabetes was induced by intraperitoneal injection of 100 mg/kg streptozotocin in mice. Normal and untreated diabetic control mice were used, and CF‑treated (200 mg/kg/day), APS‑treated (200 mg/kg/day), APS + CF (AC)‑treated (200 mg/kg/day of each) and metformin‑treated (200 mg/kg/day) diabetic mice were orally administrated the appropriate therapeutic agent for 4 weeks. The results demonstrated that AC treatment significantly reduced the fasting blood glucose, food and water intake in the diabetic mice. The AC group demonstrated increased serum insulin levels and islet cell function was restored. Furthermore, the AC‑treated mice demonstrated significant increases in the protein expression levels of pancreatic and duodenal homeobox‑1 and phosphorylated adenosine 5'‑monophosphate‑activated protein kinase in the pancreatic and liver tissue samples, respectively. In addition, AC significantly increased the mRNA expression levels of neurogenin 3, v‑maf musculoaponeurotic fibrosarcoma oncogene family, protein A and insulin, and simultaneously decreased the expressions of interleukin 6, tumor necrosis factor‑α and chemokine (C‑C motif) ligand 2 in the pancreatic islet cells of diabetic mice. The anti‑inflammatory activity of APS and the islet‑restoring effect of CF may contribute to the improvement of islet function. AC exerted greater antidiabetic effects compared with APS or CF treatments alone. These results indicated that AC treatment had a synergic antidiabetic effect, which may involve improvements in islet function and liver metabolism. These effects of AC may facilitate the treatment of type 1 or 2 diabetes, as these patients frequently experience impaired islet function and disordered extrapancreatic metabolism.

Genetic effects on gene expression across human tissues

PubMed Central

2017-01-01

Characterization of the molecular function of the human genome and its variation across individuals is essential for identifying the cellular mechanisms that underlie human genetic traits and diseases. The Genotype-Tissue Expression (GTEx) project aims to characterize variation in gene expression levels across individuals and diverse tissues of the human body, many of which are not easily accessible. Here we describe genetic effects on gene expression levels across 44 human tissues. We find that local genetic variation affects gene expression levels for the majority of genes, and we further identify inter-chromosomal genetic effects for 93 genes and 112 loci. On the basis of the identified genetic effects, we characterize patterns of tissue specificity, compare local and distal effects, and evaluate the functional properties of the genetic effects. We also demonstrate that multi-tissue, multi-individual data can be used to identify genes and pathways affected by human disease-associated variation, enabling a mechanistic interpretation of gene regulation and the genetic basis of disease. PMID:29022597

Genetic effects on gene expression across human tissues.

PubMed

Battle, Alexis; Brown, Christopher D; Engelhardt, Barbara E; Montgomery, Stephen B

2017-10-11

Characterization of the molecular function of the human genome and its variation across individuals is essential for identifying the cellular mechanisms that underlie human genetic traits and diseases. The Genotype-Tissue Expression (GTEx) project aims to characterize variation in gene expression levels across individuals and diverse tissues of the human body, many of which are not easily accessible. Here we describe genetic effects on gene expression levels across 44 human tissues. We find that local genetic variation affects gene expression levels for the majority of genes, and we further identify inter-chromosomal genetic effects for 93 genes and 112 loci. On the basis of the identified genetic effects, we characterize patterns of tissue specificity, compare local and distal effects, and evaluate the functional properties of the genetic effects. We also demonstrate that multi-tissue, multi-individual data can be used to identify genes and pathways affected by human disease-associated variation, enabling a mechanistic interpretation of gene regulation and the genetic basis of disease.

The lineage-specific gene ponzr1 is essential for zebrafish pronephric and pharyngeal arch development

PubMed Central

Bedell, Victoria M.; Person, Anthony D.; Larson, Jon D.; McLoon, Anna; Balciunas, Darius; Clark, Karl J.; Neff, Kevin I.; Nelson, Katie E.; Bill, Brent R.; Schimmenti, Lisa A.; Beiraghi, Soraya; Ekker, Stephen C.

2012-01-01

The Homeobox (Hox) and Paired box (Pax) gene families are key determinants of animal body plans and organ structure. In particular, they function within regulatory networks that control organogenesis. How these conserved genes elicit differences in organ form and function in response to evolutionary pressures is incompletely understood. We molecularly and functionally characterized one member of an evolutionarily dynamic gene family, plac8 onzin related protein 1 (ponzr1), in the zebrafish. ponzr1 mRNA is expressed early in the developing kidney and pharyngeal arches. Using ponzr1-targeting morpholinos, we show that ponzr1 is required for formation of the glomerulus. Loss of ponzr1 results in a nonfunctional glomerulus but retention of a functional pronephros, an arrangement similar to the aglomerular kidneys found in a subset of marine fish. ponzr1 is integrated into the pax2a pathway, with ponzr1 expression requiring pax2a gene function, and proper pax2a expression requiring normal ponzr1 expression. In addition to pronephric function, ponzr1 is required for pharyngeal arch formation. We functionally demonstrate that ponzr1 can act as a transcription factor or co-factor, providing the first molecular mode of action for this newly described gene family. Together, this work provides experimental evidence of an additional mechanism that incorporates evolutionarily dynamic, lineage-specific gene families into conserved regulatory gene networks to create functional organ diversity. PMID:22274699

Regulation of voltage-gated sodium channel expression in cancer: hormones, growth factors and auto-regulation

PubMed Central

Fraser, Scott P.; Ozerlat-Gunduz, Iley; Brackenbury, William J.; Fitzgerald, Elizabeth M.; Campbell, Thomas M.; Coombes, R. Charles; Djamgoz, Mustafa B. A.

2014-01-01

Although ion channels are increasingly being discovered in cancer cells in vitro and in vivo, and shown to contribute to different aspects and stages of the cancer process, much less is known about the mechanisms controlling their expression. Here, we focus on voltage-gated Na+ channels (VGSCs) which are upregulated in many types of carcinomas where their activity potentiates cell behaviours integral to the metastatic cascade. Regulation of VGSCs occurs at a hierarchy of levels from transcription to post-translation. Importantly, mainstream cancer mechanisms, especially hormones and growth factors, play a significant role in the regulation. On the whole, in major hormone-sensitive cancers, such as breast and prostate cancer, there is a negative association between genomic steroid hormone sensitivity and functional VGSC expression. Activity-dependent regulation by positive feedback has been demonstrated in strongly metastatic cells whereby the VGSC is self-sustaining, with its activity promoting further functional channel expression. Such auto-regulation is unlike normal cells in which activity-dependent regulation occurs mostly via negative feedback. Throughout, we highlight the possible clinical implications of functional VGSC expression and regulation in cancer. PMID:24493753

microRNA-497 overexpression decreases proliferation, migration and invasion of human retinoblastoma cells via targeting vascular endothelial growth factor A

PubMed Central

Li, Jianjun; Zhang, Yinghui; Wang, Xiuchao; Zhao, Ruibo

2017-01-01

The expression level and roles of microRNA-497 (miR-497) have been frequently reported in previous studies on cancer. However, its expression, function and associated molecular mechanisms in retinoblastoma remain unknown. In the present study, miR-497 expression levels in human retinoblastoma tissues, normal retinal tissues and retinoblastoma cell lines were determined using reverse transcription-quantitative polymerase chain reaction. In addition, a Cell Counting Kit-8 assay, cell migration assay, cell invasion assay, western blot analysis and Dual-Luciferase reporter assay were used to explore the expression, functions and molecular mechanisms of miR-497 in human retinoblastoma. It was demonstrated that miR-497 was significantly downregulated in retinoblastoma tissues and cell lines compared with normal retinal tissues. Ectopic expression of miR-497 decreased the proliferation, migration and invasion of retinoblastoma cells. Furthermore, VEGFA was verified as a potential direct target of miR-497 in vitro. Taken together, the results indicate that miR-497 functions as a tumor suppressor in the carcinogenesis and progression of retinoblastoma via targeting VEGFA. miR-497 should be investigated as a potential therapeutic target for the treatment of retinoblastoma. PMID:28588740

Optimum sensitivity derivatives of objective functions in nonlinear programming

NASA Technical Reports Server (NTRS)

Barthelemy, J.-F. M.; Sobieszczanski-Sobieski, J.

1983-01-01

The feasibility of eliminating second derivatives from the input of optimum sensitivity analyses of optimization problems is demonstrated. This elimination restricts the sensitivity analysis to the first-order sensitivity derivatives of the objective function. It is also shown that when a complete first-order sensitivity analysis is performed, second-order sensitivity derivatives of the objective function are available at little additional cost. An expression is derived whose application to linear programming is presented.

KSA Antigen Ep-CAM Mediates Cell–Cell Adhesion of Pancreatic Epithelial Cells: Morphoregulatory Roles in Pancreatic Islet Development

PubMed Central

Cirulli, V.; Crisa, L.; Beattie, G.M.; Mally, M.I.; Lopez, A.D.; Fannon, A.; Ptasznik, A.; Inverardi, L.; Ricordi, C.; Deerinck, T.; Ellisman, M.; Reisfeld, R.A.; Hayek, A.

1998-01-01

Cell adhesion molecules (CAMs) are important mediators of cell–cell interactions and regulate cell fate determination by influencing growth, differentiation, and organization within tissues. The human pancarcinoma antigen KSA is a glycoprotein of 40 kD originally identified as a marker of rapidly proliferating tumors of epithelial origin. Interestingly, most normal epithelia also express this antigen, although at lower levels, suggesting that a dynamic regulation of KSA may occur during cell growth and differentiation. Recently, evidence has been provided that this glycoprotein may function as an epithelial cell adhesion molecule (Ep-CAM). Here, we report that Ep-CAM exhibits the features of a morphoregulatory molecule involved in the development of human pancreatic islets. We demonstrate that Ep-CAM expression is targeted to the lateral domain of epithelial cells of the human fetal pancreas, and that it mediates calcium-independent cell–cell adhesion. Quantitative confocal immunofluorescence in fetal pancreata identified the highest levels of Ep-CAM expression in developing islet-like cell clusters budding from the ductal epithelium, a cell compartment thought to comprise endocrine progenitors. A surprisingly reversed pattern was observed in the human adult pancreas, displaying low levels of Ep-CAM in islet cells and high levels in ducts. We further demonstrate that culture conditions promoting epithelial cell growth induce upregulation of Ep-CAM, whereas endocrine differentiation of fetal pancreatic epithelial cells, transplanted in nude mice, is associated with a downregulation of Ep-CAM expression. In addition, a blockade of Ep-CAM function by KS1/4 mAb induced insulin and glucagon gene transcription and translation in fetal pancreatic cell clusters. These results indicate that developmentally regulated expression and function of Ep-CAM play a morphoregulatory role in pancreatic islet ontogeny. PMID:9508783

Direct targeting of SUZ12/ROCK2 by miR-200b/c inhibits cholangiocarcinoma tumourigenesis and metastasis

PubMed Central

Peng, F; Jiang, J; Yu, Y; Tian, R; Guo, X; Li, X; Shen, M; Xu, M; Zhu, F; Shi, C; Hu, J; Wang, M; Qin, R

2013-01-01

Background: The multidrug resistance and distant metastasis of cholangiocarcinoma result in high postoperative recurrence and low long-term survival rates. It has been demonstrated that the ectopic expression of miR-200 suppresses the multidrug resistance and metastasis of cancer. However, the expression and function of miR-200 in cholangiocarcinoma has not yet been described. Methods: In this study, we identified dysregulated microRNAs (miRNAs, miR) in cholangiocarcinoma tissue by microarray analysis, and subsequent real-time PCR and northern blot analyses validated the expression of candidate miR. We performed functional analyses and investigated the relationship between miR-200b/c expression and the properties of cholangiocarcinoma cells. A dual luciferase assay was applied to examine the effect of miRNAs on the 3′-UTR of target genes, and we demonstrated the function of the target gene by siRNA transfection identifying the downstream pathway via western blotting. Results: We found significantly downregulated expression of four miR-200 family members (miR-200a/b/c/429) and then confirmed that ectopic miR-200b/200c inhibits the migration and invasion of cholangiocarcinoma cells both in vitro and in vivo. We found that miR-200b/c influenced the tumourigenesis of cholangiocarcinoma cells including their tumour-initiating capacity, sphere formation, and drug resistance. We further found that miR-200b/c regulated migration and invasion capacities by directly targeting rho-kinase 2 and regulated tumorigenic properties by directly targeting SUZ12 (a subunit of a polycomb repressor complex). Conclusion: Our study shows that miR-200b/c has a critical role in the regulation of the tumorigenic and metastatic capacity of cholangiocarcinoma and reveals the probable underlying mechanisms. PMID:24169343

microRNA‑196b promotes cell migration and invasion by targeting FOXP2 in hepatocellular carcinoma.

PubMed

Yu, Zhaoxiang; Lin, Xiaobo; Tian, Ming; Chang, Weiping

2018-02-01

Accumulating evidence indicates that microRNAs (miRNAs) play important roles in tumorigenesis and metastasis. Recent research has shown that miR‑196b is implicated in metastasis by regulating the migration and invasion of cancer cells. However, the clinical significance of miR‑196b and its role as well as the underlying mechanisms in hepatocellular carcinoma (HCC) remain largely unknown. Here, we detected miR‑196b expression in HCC and matched non-tumor tissues with qRT‑PCR. We found that miR‑196b displayed higher expression in HCC patient tissues and cells. Clinical analysis revealed that high miR‑196 expression was correlated with venous infiltration, advanced TNM stage and poor prognosis. Functionally, we demonstrated that miR‑196b promoted the migration and invasion of HCC cells in vitro. Moreover, miR‑196b knockdown restrained pulmonary metastasis in vivo. Mechanistically, we confirmed that miR‑196b could directly bind to 3'UTR of forkhead box P2 (FOXP2) mRNA and repress its expression. miR‑196b and FOXP2 showed a negative correlation in HCC tissues. More importantly, upregulation of FOXP2 antagonized miR‑196b‑mediated migration and invasion in Hep3B cells. Furthermore, FOXP2 knockdown partially reversed the anti‑metastatic function of the miR‑196b inhibitor on HCCLM3 cells. Taken together, we demonstrated that miR‑196b may function as a prognostic biomarker and suppressed FOXP2 expression, subsequently leading to the metastasis of HCC. Our findings highlight a novel mechanism of miR‑196b in the progression of HCC and identify miR‑196b/FOXP2 axis as a promising target for HCC.

A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development

PubMed Central

Ducy, Patricia; Starbuck, Michael; Priemel, Matthias; Shen, Jianhe; Pinero, Gerald; Geoffroy, Valerie; Amling, Michael; Karsenty, Gerard

1999-01-01

The molecular mechanisms controlling bone extracellular matrix (ECM) deposition by differentiated osteoblasts in postnatal life, called hereafter bone formation, are unknown. This contrasts with the growing knowledge about the genetic control of osteoblast differentiation during embryonic development. Cbfa1, a transcriptional activator of osteoblast differentiation during embryonic development, is also expressed in differentiated osteoblasts postnatally. The perinatal lethality occurring in Cbfa1-deficient mice has prevented so far the study of its function after birth. To determine if Cbfa1 plays a role during bone formation we generated transgenic mice overexpressing Cbfa1 DNA-binding domain (ΔCbfa1) in differentiated osteoblasts only postnatally. ΔCbfa1 has a higher affinity for DNA than Cbfa1 itself, has no transcriptional activity on its own, and can act in a dominant-negative manner in DNA cotransfection assays. ΔCbfa1-expressing mice have a normal skeleton at birth but develop an osteopenic phenotype thereafter. Dynamic histomorphometric studies show that this phenotype is caused by a major decrease in the bone formation rate in the face of a normal number of osteoblasts thus indicating that once osteoblasts are differentiated Cbfa1 regulates their function. Molecular analyses reveal that the expression of the genes expressed in osteoblasts and encoding bone ECM proteins is nearly abolished in transgenic mice, and ex vivo assays demonstrated that ΔCbfa1-expressing osteoblasts were less active than wild-type osteoblasts. We also show that Cbfa1 regulates positively the activity of its own promoter, which has the highest affinity Cbfa1-binding sites characterized. This study demonstrates that beyond its differentiation function Cbfa1 is the first transcriptional activator of bone formation identified to date and illustrates that developmentally important genes control physiological processes postnatally. PMID:10215629

IFI27, a novel epidermal growth factor-stabilized protein, is functionally involved in proliferation and cell cycling of human epidermal keratinocytes.

PubMed

Hsieh, W-L; Huang, Y-H; Wang, T-M; Ming, Y-C; Tsai, C-N; Pang, J-H S

2015-04-01

IFI27 is highly expressed in psoriatic lesions but its function has not been known. The present study aimed to explore its role in proliferation of epidermal keratinocytes. IFI27 knockdown and over-expression in keratinocytes were used to compare their proliferation, by MTT assay, apoptosis (by annexin V binding) and cell cycle progression by flow cytometry. Formation of cyclin A/CDK1 complex was examined by a co-immunoprecipitaion method. Anti-proliferation effects of IFI27 were also examined in vivo by topical application of IFI27 siRNA on imiquimod-induced psoriatic lesions, in a mouse model. Epidermal growth factor was demonstrated to increase IFI27 expression by prolonging half-life of IFI27 protein. The IFI27 knockdown in keratinocytes reduced the proliferation rate, but had no effect on apoptosis nor on apoptosis-related genes. Interestingly, IFI27 knockdown resulted in S-phase arrest that was found to be associated with increased Tyr15 phosphorylation of CDK1, reduced CDC25B and reduced formation of cyclin A/CDK1 complex. In addition, IFI27 knockdown was also shown to activate p53 by Ser15 phosphorylation and increase p21 expression. Topical application of IFI27 siRNA on imiquimod-induced psoriatic lesion in a mouse model reduced epidermal thickness, formation of rete ridges and PCNA expression. Our study demonstrates for the first time, that cell function of IFI27 is involved in proliferation of skin keratinocytes both in vitro and in vivo. It suggests that IFI27 might be a suitable target for development of a novel anti-psoriasis therapy. © 2015 John Wiley & Sons Ltd.

Foxo3 activity promoted by non-coding effects of circular RNA and Foxo3 pseudogene in the inhibition of tumor growth and angiogenesis.

PubMed

Yang, W; Du, W W; Li, X; Yee, A J; Yang, B B

2016-07-28

It has recently been shown that the upregulation of a pseudogene specific to a protein-coding gene could function as a sponge to bind multiple potential targeting microRNAs (miRNAs), resulting in increased gene expression. Similarly, it was recently demonstrated that circular RNAs can function as sponges for miRNAs, and could upregulate expression of mRNAs containing an identical sequence. Furthermore, some mRNAs are now known to not only translate protein, but also function to sponge miRNA binding, facilitating gene expression. Collectively, these appear to be effective mechanisms to ensure gene expression and protein activity. Here we show that expression of a member of the forkhead family of transcription factors, Foxo3, is regulated by the Foxo3 pseudogene (Foxo3P), and Foxo3 circular RNA, both of which bind to eight miRNAs. We found that the ectopic expression of the Foxo3P, Foxo3 circular RNA and Foxo3 mRNA could all suppress tumor growth and cancer cell proliferation and survival. Our results showed that at least three mechanisms are used to ensure protein translation of Foxo3, which reflects an essential role of Foxo3 and its corresponding non-coding RNAs.

Differential downstream functions of protein kinase Ceta and -theta in EL4 mouse thymoma cells.

PubMed

Resnick, M S; Kang, B S; Luu, D; Wickham, J T; Sando, J J; Hahn, C S

1998-10-16

Sensitive EL4 mouse thymoma cells (s-EL4) respond to phorbol esters with growth inhibition, adherence to substrate, and production of cytokines including interleukin 2. Since these cells express several of the phorbol ester-sensitive protein kinase C (PKC) isozymes, the function of each isozyme remains unclear. Previous studies demonstrated that s-EL4 cells expressed substantially more PKCeta and PKCtheta than did EL4 cells resistant to phorbol esters (r-EL4). To examine potential roles for PKCeta and PKCtheta in EL4 cells, wild type and constitutively active versions of the isozymes were transiently expressed using a Sindbis virus system. Expression of constitutively active PKCeta, but not PKCtheta, in s- and r-EL4 cells altered cell morphology and cytoskeletal structure in a manner similar to that of phorbol ester treatment, suggesting a role for PKCeta in cytoskeletal organization. Prolonged treatment of s-EL4 cells with phorbol esters results in inhibition of cell cycling along with a decreased expression of most of the PKC isozymes, including PKCtheta. Introduction of virally expressed PKCtheta, but not PKCeta, overcame the inhibitory effects of the prolonged phorbol ester treatment on cell cycle progression, suggesting a possible involvement of PKCtheta in cell cycle regulation. These results support differential functions for PKCeta and PKCtheta in T cell activation.

Increased autophagy in fibroblast-like synoviocytes leads to immune enhancement potential in rheumatoid arthritis.

PubMed

Yang, Ru; Zhang, Yingzi; Wang, Lin; Hu, Ji; Wen, Jian; Xue, Leixi; Tang, Mei; Liu, Zhichun; Fu, Jinxiang

2017-02-28

The incidence of rheumatoid arthritis (RA) has been reported to be correlated with a disorder of immunregulation. Rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) play an important role in regulating the local immune microenvironment. However, the potential mechanism of RA-FLS in regulating the immnue response is not clearly understood. In this study, we demonstrated that the expression of HIF-1α was significantly up-regulated in rheumatoid arthritis tissue which indicated that the hypoxia condition in the microenvironment. We also observed that RA-FLSs demonstrated the potential to up-regulate immune activation. Meanwhile, the level of autophagy increased in RA-FLSs compared with control group. Besides that, the expression of IL-6 was up-regulated not only in RA-FLSs but also in the fibroblasts that treated with hypoxia condition. Accordingly, we found that autophagy inhibitiors could effectively inhibit the immune activation function of RA-FLSs medicated by IL-6. Taken together, the results we demonstrated above indicated that the hypoxia microenvironment could effectively induce the incidence of autophagy and then lead to the immune activation function of RA-FLSs medicated by IL-6.

Drug resistance is conferred on the model yeast Saccharomyces cerevisiae by expression of full-length melanoma-associated human ATP-binding cassette transporter ABCB5.

PubMed

Keniya, Mikhail V; Holmes, Ann R; Niimi, Masakazu; Lamping, Erwin; Gillet, Jean-Pierre; Gottesman, Michael M; Cannon, Richard D

2014-10-06

ABCB5, an ATP-binding cassette (ABC) transporter, is highly expressed in melanoma cells, and may contribute to the extreme resistance of melanomas to chemotherapy by efflux of anti-cancer drugs. Our goal was to determine whether we could functionally express human ABCB5 in the model yeast Saccharomyces cerevisiae, in order to demonstrate an efflux function for ABCB5 in the absence of background pump activity from other human transporters. Heterologous expression would also facilitate drug discovery for this important target. DNAs encoding ABCB5 sequences were cloned into the chromosomal PDR5 locus of a S. cerevisiae strain in which seven endogenous ABC transporters have been deleted. Protein expression in the yeast cells was monitored by immunodetection using both a specific anti-ABCB5 antibody and a cross-reactive anti-ABCB1 antibody. ABCB5 function in recombinant yeast cells was measured by determining whether the cells possessed increased resistance to known pump substrates, compared to the host yeast strain, in assays of yeast growth. Three ABCB5 constructs were made in yeast. One was derived from the ABCB5-β mRNA, which is highly expressed in human tissues but is a truncation of a canonical full-size ABC transporter. Two constructs contained full-length ABCB5 sequences: either a native sequence from cDNA or a synthetic sequence codon-harmonized for S. cerevisiae. Expression of all three constructs in yeast was confirmed by immunodetection. Expression of the codon-harmonized full-length ABCB5 DNA conferred increased resistance, relative to the host yeast strain, to the putative substrates rhodamine 123, daunorubicin, tetramethylrhodamine, FK506, or clorgyline. We conclude that full-length ABCB5 can be functionally expressed in S. cerevisiae and confers drug resistance.

Rapid production of functionalized recombinant proteins: marrying ligation independent cloning and in vitro protein ligation.

PubMed

Kushnir, Susanna; Marsac, Yoann; Breitling, Reinhard; Granovsky, Igor; Brok-Volchanskaya, Vera; Goody, Roger S; Becker, Christian F W; Alexandrov, Kirill

2006-01-01

Functional genomics and proteomics have been very active fields since the sequencing of several genomes was completed. To assign a physiological role to the newly discovered coding genes with unknown function, new generic methods for protein production, purification, and targeted functionalization are needed. This work presents a new vector, pCYSLIC, that allows rapid generation of Escherichia coli expression constructs via ligation-independent cloning (LIC). The vector is designed to facilitate protein purification by either Ni-NTA or GSH affinity chromatography. Subsequent proteolytic removal of affinity tags liberates an N-terminal cysteine residue that is then used for covalent modification of the target protein with different biophysical probes via protein ligation. The described system has been tested on 36 mammalian Rab GTPases, and it was demonstrated that recombinant GTPases produced with pCYSLIC could be efficiently modified with fluorescein or biotin in vitro. Finally, LIC was compared with the recently developed In-Fusion cloning method, and it was demonstrated that In-Fusion provides superior flexibility in choice of expression vector. By the application of In-Fusion cloning Cys-Rab6A GTPase with an N-terminal cysteine residue was generated employing unmodified pET30a vector and TVMV protease.

Tumor Progression Is Mediated by Thymosin-β4 through a TGFβ/MRTF Signaling Axis.

PubMed

Morita, Tsuyoshi; Hayashi, Ken'ichiro

2018-05-01

Although enhanced thymosin β4 (TMSB4X/Tβ4) expression is associated with tumor progression and metastasis, its tumor-promoting functions remain largely unknown. Here, it is demonstrated that TGFβ facilitates Tβ4 expression and leads to the activation of myocardin-related transcription factors (MRTF), which are coactivators of serum response factor (SRF) and regulate the expression of genes critical for the epithelial-mesenchymal transition (EMT) and tumor metastasis. In murine mammary gland cells (NMuMG), Tβ4 upregulation is required for full induction of a MRTF-regulated EMT gene expression program after TGFβ stimulation. Tβ4 levels are transcriptionally regulated via the novel cis -acting element AGACAAAG, which interacts with Smad and T-cell factor/lymphoid enhancer factor (TCF/LEF) to synergistically activate the Tβ4 promoter downstream of TGFβ. Murine skin melanoma cells (B16F0 and B16F1) also show the expression regulation of Tβ4 by Smad and TCF/LEF. Tβ4-knockout B16F1 (Tβ4 KO) clones show significantly diminished expression level of tumor-associated genes, which is regulated by the TGFβ/MRTFs pathway. In multiple human cancers, Tβ4 levels correlate positively with TGFβ1 and the tumor-associated gene expression levels through processes that respectively depend on TGFβ receptor 1 (TGFBR1) and MRTF expression. Kaplan-Meier survival analyses demonstrate that high Tβ4 expression associates with poor prognosis in an SRF expression-dependent manner in several cancers. In mice, Tβ4 KO clones show significantly decreased experimental metastatic potential; furthermore, ectopic expression of constitutively active MRTF-A fully restores the diminished metastatic activity. In conclusion, the TGFβ/Tβ4/MRTF/SRF pathway is critical for metastasis and tumor progression. Implications: These findings define a molecular mechanism underlying a tumor-promoting function of thymosin β4 through activation of MRTF/SRF signaling. Mol Cancer Res; 16(5); 880-93. ©2018 AACR . ©2018 American Association for Cancer Research.

Unconventional function of an Achaete-Scute homolog as a terminal selector of nociceptive neuron identity

PubMed Central

Masoudi, Neda; Tavazoie, Saeed; Glenwinkel, Lori; Ryu, Leesun; Kim, Kyuhyung

2018-01-01

Proneural genes are among the most early-acting genes in nervous system development, instructing blast cells to commit to a neuronal fate. Drosophila Atonal and Achaete-Scute complex (AS-C) genes, as well as their vertebrate orthologs, are basic helix-loop-helix (bHLH) transcription factors with such proneural activity. We show here that a C. elegans AS-C homolog, hlh-4, functions in a fundamentally different manner. In the embryonic, larval, and adult nervous systems, hlh-4 is expressed exclusively in a single nociceptive neuron class, ADL, and its expression in ADL is maintained via transcriptional autoregulation throughout the life of the animal. However, in hlh-4 null mutants, the ADL neuron is generated and still appears neuronal in overall morphology and expression of panneuronal and pansensory features. Rather than acting as a proneural gene, we find that hlh-4 is required for the ADL neuron to function properly, to adopt its correct morphology, to express its unusually large repertoire of olfactory receptor–encoding genes, and to express other known features of terminal ADL identity, including neurotransmitter phenotype, neuropeptides, ion channels, and electrical synapse proteins. hlh-4 is sufficient to induce ADL identity features upon ectopic expression in other neuron types. The expression of ADL terminal identity features is directly controlled by HLH-4 via a phylogenetically conserved E-box motif, which, through bioinformatic analysis, we find to constitute a predictive feature of ADL-expressed terminal identity markers. The lineage that produces the ADL neuron was previously shown to require the conventional, transient proneural activity of another AS-C homolog, hlh-14, demonstrating sequential activities of distinct AS-C-type bHLH genes in neuronal specification. Taken together, we have defined here an unconventional function of an AS-C-type bHLH gene as a terminal selector of neuronal identity and we speculate that such function could be reflective of an ancestral function of an “ur-” bHLH gene. PMID:29672507

Accelerated remyelination during inflammatory demyelination prevents axonal loss and improves functional recovery.

PubMed

Mei, Feng; Lehmann-Horn, Klaus; Shen, Yun-An A; Rankin, Kelsey A; Stebbins, Karin J; Lorrain, Daniel S; Pekarek, Kara; A Sagan, Sharon; Xiao, Lan; Teuscher, Cory; von Büdingen, H-Christian; Wess, Jürgen; Lawrence, J Josh; Green, Ari J; Fancy, Stephen Pj; Zamvil, Scott S; Chan, Jonah R

2016-09-27

Demyelination in MS disrupts nerve signals and contributes to axon degeneration. While remyelination promises to restore lost function, it remains unclear whether remyelination will prevent axonal loss. Inflammatory demyelination is accompanied by significant neuronal loss in the experimental autoimmune encephalomyelitis (EAE) mouse model and evidence for remyelination in this model is complicated by ongoing inflammation, degeneration and possible remyelination. Demonstrating the functional significance of remyelination necessitates selectively altering the timing of remyelination relative to inflammation and degeneration. We demonstrate accelerated remyelination after EAE induction by direct lineage analysis and hypothesize that newly formed myelin remains stable at the height of inflammation due in part to the absence of MOG expression in immature myelin. Oligodendroglial-specific genetic ablation of the M1 muscarinic receptor, a potent negative regulator of oligodendrocyte differentiation and myelination, results in accelerated remyelination, preventing axonal loss and improving functional recovery. Together our findings demonstrate that accelerated remyelination supports axonal integrity and neuronal function after inflammatory demyelination.

Taste responses in mice lacking taste receptor subunit T1R1

PubMed Central

Kusuhara, Yoko; Yoshida, Ryusuke; Ohkuri, Tadahiro; Yasumatsu, Keiko; Voigt, Anja; Hübner, Sandra; Maeda, Katsumasa; Boehm, Ulrich; Meyerhof, Wolfgang; Ninomiya, Yuzo

2013-01-01

The T1R1 receptor subunit acts as an umami taste receptor in combination with its partner, T1R3. In addition, metabotropic glutamate receptors (brain and taste variants of mGluR1 and mGluR4) are thought to function as umami taste receptors. To elucidate the function of T1R1 and the contribution of mGluRs to umami taste detection in vivo, we used newly developed knock-out (T1R1−/−) mice, which lack the entire coding region of the Tas1r1 gene and express mCherry in T1R1-expressing cells. Gustatory nerve recordings demonstrated that T1R1−/− mice exhibited a serious deficit in inosine monophosphate-elicited synergy but substantial residual responses to glutamate alone in both chorda tympani and glossopharyngeal nerves. Interestingly, chorda tympani nerve responses to sweeteners were smaller in T1R1−/− mice. Taste cell recordings demonstrated that many mCherry-expressing taste cells in T1R1+/− mice responded to sweet and umami compounds, whereas those in T1R1−/− mice responded to sweet stimuli. The proportion of sweet-responsive cells was smaller in T1R1−/− than in T1R1+/− mice. Single-cell RT-PCR demonstrated that some single mCherry-expressing cells expressed all three T1R subunits. Chorda tympani and glossopharyngeal nerve responses to glutamate were significantly inhibited by addition of mGluR antagonists in both T1R1−/− and T1R1+/− mice. Conditioned taste aversion tests demonstrated that both T1R1−/− and T1R1+/− mice were equally capable of discriminating glutamate from other basic taste stimuli. Avoidance conditioned to glutamate was significantly reduced by addition of mGluR antagonists. These results suggest that T1R1-expressing cells mainly contribute to umami taste synergism and partly to sweet sensitivity and that mGluRs are involved in the detection of umami compounds. PMID:23339178

Portraying the Expression Landscapes of B-Cell Lymphoma-Intuitive Detection of Outlier Samples and of Molecular Subtypes

PubMed Central

Hopp, Lydia; Lembcke, Kathrin; Binder, Hans; Wirth, Henry

2013-01-01

We present an analytic framework based on Self-Organizing Map (SOM) machine learning to study large scale patient data sets. The potency of the approach is demonstrated in a case study using gene expression data of more than 200 mature aggressive B-cell lymphoma patients. The method portrays each sample with individual resolution, characterizes the subtypes, disentangles the expression patterns into distinct modules, extracts their functional context using enrichment techniques and enables investigation of the similarity relations between the samples. The method also allows to detect and to correct outliers caused by contaminations. Based on our analysis, we propose a refined classification of B-cell Lymphoma into four molecular subtypes which are characterized by differential functional and clinical characteristics. PMID:24833231

Low heme oxygenase-1 levels in patients with systemic sclerosis are associated with an altered Toll-like receptor response: another role for CXCL4?

PubMed

van Bon, Lenny; Cossu, Marta; Scharstuhl, Alwin; Pennings, Bas W C; Vonk, Madelon C; Vreman, Hendrik J; Lafyatis, Robert L; van den Berg, Wim; Wagener, Frank A D T G; Radstake, Timothy R D J

2016-11-01

SSc is a disease characterized by inflammation and fibrosis. Heme Oxygenase-1 (HO-1) is a haem-degrading enzyme that mediates resolution of inflammation and is induced upon mediators abundantly present in SSc. We aimed to assess whether HO-1 expression/function is disturbed in SSc patients and could therefore be contributing to the ongoing inflammation. In total, 92 SSc patients and 48 healthy controls were included. By measuring total bilirubin in plasma in vivo, HO-activity was assessed. HO-1 expression levels were determined with western blot in monocytes before and after induction of HO-1 with cobalt protoporphyrin (CoPP) with or without CXCL4. Monocyte-derived dendritic cells (DCs) were stimulated with several Toll-like receptor (TLR) ligands with or without pre-stimulation with CoPP for 24 h. Cytokine levels were measured in the supernatants using the Luminex Bead Array. SSc patients have lower plasma levels of bilirubin, suggestive of an aberrant HO-1 function. We demonstrated low HO-1 expression in immune cells from SSc patients, whereas induction with CoPP was able to restore HO-1 levels in DCs from SSc patients, almost normalizing the increased TLR response observed in SSc. Co-exposure to CXCL4 completely abrogated CoPP-induced HO-1 expression, suggesting that the high CXCL4 levels present in SSc patients block the normal induction of HO-1 and its function. We demonstrate that HO activity in SSc patients is decreased and show its functional consequences. Since CXCL4 blocks the induction of HO-1 expression, neutralization of CXCL4 in SSc patients could have clinical benefits by diminishing overactivation of immune cells and other anti-inflammatory effects of HO-1. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Cobalt Chloride Upregulates Impaired HIF-1α Expression to Restore Sevoflurane Post-conditioning-Dependent Myocardial Protection in Diabetic Rats

PubMed Central

Wu, Jianjiang; Yang, Long; Xie, Peng; Yu, Jin; Yu, Tian; Wang, Haiying; Maimaitili, Yiliyaer; Wang, Jiang; Ma, Haiping; Yang, Yining; Zheng, Hong

2017-01-01

Previous studies from our group have demonstrated that sevoflurane post-conditioning (SPC) protects against myocardial ischemia reperfusion injury via elevating the intranuclear expression of hypoxia inducible factor-1 alpha (HIF-1α). However, diabetic SPC is associated with decreased myocardial protection and disruption of the HIF-1 signaling pathway. Previous studies have demonstrated that cobalt chloride (CoCl2) can upregulate HIF-1α expression under diabetic conditions, but whether myocardial protection by SPC can be restored afterward remains unclear. We established a rat model of type 2 diabetes and a Langendorff isolated heart model of ischemia-reperfusion injury. Prior to reperfusion, 2.4% sevoflurane was used as a post-conditioning treatment. The diabetic rats were treated with CoCl2 24 h before the experiment. At the end of reperfusion, tests were performed to assess myocardial function, infarct size, mitochondrial morphology, nitric oxide (NO), Mitochondrial reactive oxygen species (ROS), mitochondrial respiratory function and enzyme activity, HIF-1α, vascular endothelial growth factor (VEGF) and endothelial NO synthase (eNOS) protein levels. In addition, myocardial protection by SPC was monitored after the blood glucose levels were lowered by insulin. The diabetic state was associated with deficient SPC protection and decreased HIF-1α expression. After treating the diabetic rats with CoCl2, SPC significantly upregulated the expression of HIF-1α, VEGF and eNOS, which markedly improved cardiac function, NO, mitochondrial respiratory function, and enzyme activity and decreased the infarction areas and ROS. In addition, these effects were not influenced by blood glucose levels. This study proved that CoCl2activates the HIF-1α signaling pathway, which restores SPC-dependent myocardial protection under diabetic conditions, and the protective effects of SPC were independent of blood glucose levels. PMID:28659817

Variation analysis of transcriptome changes reveals cochlear genes and their associated functions in cochlear susceptibility to acoustic overstimulation.

PubMed

Yang, Shuzhi; Cai, Qunfeng; Bard, Jonathan; Jamison, Jennifer; Wang, Jianmin; Yang, Weiping; Hu, Bo Hua

2015-12-01

Individual variation in the susceptibility of the auditory system to acoustic overstimulation has been well-documented at both the functional and structural levels. However, the molecular mechanism responsible for this variation is unclear. The current investigation was designed to examine the variation patterns of cochlear gene expression using RNA-seq data and to identify the genes with expression variation that increased following acoustic trauma. This study revealed that the constitutive expressions of cochlear genes displayed diverse levels of gene-specific variation. These variation patterns were altered by acoustic trauma; approximately one-third of the examined genes displayed marked increases in their expression variation. Bioinformatics analyses revealed that the genes that exhibited increased variation were functionally related to cell death, biomolecule metabolism, and membrane function. In contrast, the stable genes were primarily related to basic cellular processes, including protein and macromolecular syntheses and transport. There was no functional overlap between the stable and variable genes. Importantly, we demonstrated that glutamate metabolism is related to the variation in the functional response of the cochlea to acoustic overstimulation. Taken together, the results indicate that our analyses of the individual variations in transcriptome changes of cochlear genes provide important information for the identification of genes that potentially contribute to the generation of individual variation in cochlear responses to acoustic overstimulation. Copyright © 2015 Elsevier B.V. All rights reserved.

The activity-dependent histone variant H2BE modulates the life span of olfactory neurons

PubMed Central

Santoro, Stephen W; Dulac, Catherine

2012-01-01

We have identified a replication-independent histone variant, Hist2h2be (referred to herein as H2be), which is expressed exclusively by olfactory chemosensory neurons. Levels of H2BE are heterogeneous among olfactory neurons, but stereotyped according to the identity of the co-expressed olfactory receptor (OR). Gain- and loss-of-function experiments demonstrate that changes in H2be expression affect olfactory function and OR representation in the adult olfactory epithelium. We show that H2BE expression is reduced by sensory activity and that it promotes neuronal cell death, such that inactive olfactory neurons display higher levels of the variant and shorter life spans. Post-translational modifications (PTMs) of H2BE differ from those of the canonical H2B, consistent with a role for H2BE in altering transcription. We propose a physiological function for H2be in modulating olfactory neuron population dynamics to adapt the OR repertoire to the environment. DOI: http://dx.doi.org/10.7554/eLife.00070.001 PMID:23240083

Optimal expression of a Fab-effector fusion protein in Escherichia coli by removing the cysteine residues responsible for an interchain disulfide bond of a Fab molecule.

PubMed

Kang, Hyeon-Ju; Kim, Hye-Jin; Jung, Mun-Sik; Han, Jae-Kyu; Cha, Sang-Hoon

2017-04-01

Development of novel bi-functional or even tri-functional Fab-effector fusion proteins would have a great potential in the biomedical sciences. However, the expression of Fab-effector fusion proteins in Escherichia coli is problematic especially when a eukaryotic effector moiety is genetically linked to a Fab due to the lack of proper chaperone proteins and an inappropriate physicochemical environment intrinsic to the microbial hosts. We previously reported that a human Fab molecule, referred to as SL335, reactive to human serum albumin has a prolonged in vivo serum half-life in rats. We, herein, tested six discrete SL335-human growth hormone (hGH) fusion constructs as a model system to define an optimal Fab-effector fusion format for E. coli expression. We found that one variant, referred to as HserG/Lser, outperformed the others in terms of a soluble expression yield and functionality in that HserG/Lser has a functional hGH bioactivity and possesses an serum albumin-binding affinity comparable to SL335. Our results clearly demonstrated that the genetic linkage of an effector domain to the C-terminus of Fd (V H +C H1 ) and the removal of cysteine (Cys) residues responsible for an interchain disulfide bond (IDB) ina Fab molecule optimize the periplasmic expression of a Fab-effector fusion protein in E. coli. We believe that our approach can contribute the development of diverse bi-functional Fab-effector fusion proteins by providing a simple strategy that enables the reliable expression of a functional fusion proteins in E. coli. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Palmitic acid suppresses apolipoprotein M gene expression via the pathway of PPAR{sub β/δ} in HepG2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Luo, Guanghua; Shi, Yuanping; Zhang, Jun

Highlights: • Palmitic acid significantly inhibited APOM gene expression in HepG2 cells. • Palmitic acid could obviously increase PPARB/D mRNA levels in HepG2 cells. • PPAR{sub β/δ} antagonist, GSK3787, had no effect on APOM expression. • GSK3787 could reverse the palmitic acid-induced down-regulation of APOM expression. • Palmitic acid induced suppression of APOM expression is mediated via the PPAR{sub β/δ} pathway. - Abstract: It has been demonstrated that apolipoprotein M (APOM) is a vasculoprotective constituent of high density lipoprotein (HDL), which could be related to the anti-atherosclerotic property of HDL. Investigation of regulation of APOM expression is of important formore » further exploring its pathophysiological function in vivo. Our previous studies indicated that expression of APOM could be regulated by platelet activating factor (PAF), transforming growth factors (TGF), insulin-like growth factor (IGF), leptin, hyperglycemia and etc., in vivo and/or in vitro. In the present study, we demonstrated that palmitic acid could significantly inhibit APOM gene expression in HepG2 cells. Further study indicated neither PI-3 kinase (PI3K) inhibitor LY294002 nor protein kinase C (PKC) inhibitor GFX could abolish palmitic acid induced down-regulation of APOM expression. In contrast, the peroxisome proliferator-activated receptor beta/delta (PPAR{sub β/δ}) antagonist GSK3787 could totally reverse the palmitic acid-induced down-regulation of APOM expression, which clearly demonstrates that down-regulation of APOM expression induced by palmitic acid is mediated via the PPAR{sub β/δ} pathway.« less

Antithrombotic Effects of Nur77 and Nor1 Are Mediated Through Upregulating Thrombomodulin Expression in Endothelial Cells.

PubMed

Yang, Ping; Wei, Xin; Zhang, Jian; Yi, Bing; Zhang, Guan-Xin; Yin, Litian; Yang, Xiao-Feng; Sun, Jianxin

2016-02-01

Thrombomodulin is highly expressed on the lumenal surface of vascular endothelial cells (ECs) and possesses potent anticoagulant, antifibrinolytic, and anti-inflammatory activities in the vessel wall. However, the regulation of thrombomodulin expression in ECs remains largely unknown. In this study, we characterized nuclear receptor 4A family as a novel regulator of thrombomodulin expression in vascular ECs. We demonstrated that both nuclear receptors 4A, Nur77 and Nor1, robustly increase thrombomodulin mRNA and protein levels in human vascular ECs and in mouse liver tissues after adenovirus-mediated transduction of Nur77 and Nor1 cDNAs. Moreover, Nur77 deficiency and knockdown of Nur77 and Nor1 expression markedly attenuated the basal and vascular endothelial growth factor165-stimulated thrombomodulin expression. Mechanistically, we found that Nur77 and Nor1 increase thrombomodulin expression by acting through 2 different mechanisms. We showed that Nur77 barely affects thrombomodulin promoter activity, but significantly increases thrombomodulin mRNA stability, whereas Nor1 enhances thrombomodulin expression mainly through induction of Kruppel-like factors 2 and 4 in vascular ECs. Furthermore, we demonstrated that both Nur77 and Nor1 significantly increase protein C activity and inhibit tumor necrosis factor α-induced prothrombotic effects in human ECs. Deficiency of Nur77 increases susceptibility to arterial thrombosis, whereas enhanced expression of Nur77 and Nor1 protects mice from arterial thrombus formation. Our results identified nuclear receptors 4A as novel regulators of thrombomodulin expression and function in vascular ECs and provided a proof-of-concept demonstration that targeted increasing expression of Nur77 and Nor1 in the vascular endothelium might represent a novel therapeutic approach for the treatment of thrombotic disorders. © 2015 American Heart Association, Inc.

Glioma IL13Rα2 Is Associated with Mesenchymal Signature Gene Expression and Poor Patient Prognosis

PubMed Central

Starr, Renate; Deng, Xutao; Badie, Behnam; Yuan, Yate-Ching; Forman, Stephen J.; Barish, Michael E.

2013-01-01

A major challenge for successful immunotherapy against glioma is the identification and characterization of validated targets. We have taken a bioinformatics approach towards understanding the biological context of IL-13 receptor α2 (IL13Rα2) expression in brain tumors, and its functional significance for patient survival. Querying multiple gene expression databases, we show that IL13Rα2 expression increases with glioma malignancy grade, and expression for high-grade tumors is bimodal, with approximately 58% of WHO grade IV gliomas over-expressing this receptor. By several measures, IL13Rα2 expression in patient samples and low-passage primary glioma lines most consistently correlates with the expression of signature genes defining mesenchymal subclass tumors and negatively correlates with proneural signature genes as defined by two studies. Positive associations were also noted with proliferative signature genes, whereas no consistent associations were found with either classical or neural signature genes. Probing the potential functional consequences of this mesenchymal association through IPA analysis suggests that IL13Rα2 expression is associated with activation of proinflammatory and immune pathways characteristic of mesenchymal subclass tumors. In addition, survival analyses indicate that IL13Rα2 over-expression is associated with poor patient prognosis, a single gene correlation ranking IL13Rα2 in the top ~1% of total gene expression probes with regard to survival association with WHO IV gliomas. This study better defines the functional consequences of IL13Rα2 expression by demonstrating association with mesenchymal signature gene expression and poor patient prognosis. It thus highlights the utility of IL13Rα2 as a therapeutic target, and helps define patient populations most likely to respond to immunotherapy in present and future clinical trials. PMID:24204956

Glioma IL13Rα2 is associated with mesenchymal signature gene expression and poor patient prognosis.

PubMed

Brown, Christine E; Warden, Charles D; Starr, Renate; Deng, Xutao; Badie, Behnam; Yuan, Yate-Ching; Forman, Stephen J; Barish, Michael E

2013-01-01

A major challenge for successful immunotherapy against glioma is the identification and characterization of validated targets. We have taken a bioinformatics approach towards understanding the biological context of IL-13 receptor α2 (IL13Rα2) expression in brain tumors, and its functional significance for patient survival. Querying multiple gene expression databases, we show that IL13Rα2 expression increases with glioma malignancy grade, and expression for high-grade tumors is bimodal, with approximately 58% of WHO grade IV gliomas over-expressing this receptor. By several measures, IL13Rα2 expression in patient samples and low-passage primary glioma lines most consistently correlates with the expression of signature genes defining mesenchymal subclass tumors and negatively correlates with proneural signature genes as defined by two studies. Positive associations were also noted with proliferative signature genes, whereas no consistent associations were found with either classical or neural signature genes. Probing the potential functional consequences of this mesenchymal association through IPA analysis suggests that IL13Rα2 expression is associated with activation of proinflammatory and immune pathways characteristic of mesenchymal subclass tumors. In addition, survival analyses indicate that IL13Rα2 over-expression is associated with poor patient prognosis, a single gene correlation ranking IL13Rα2 in the top ~1% of total gene expression probes with regard to survival association with WHO IV gliomas. This study better defines the functional consequences of IL13Rα2 expression by demonstrating association with mesenchymal signature gene expression and poor patient prognosis. It thus highlights the utility of IL13Rα2 as a therapeutic target, and helps define patient populations most likely to respond to immunotherapy in present and future clinical trials.

Matrix Metalloproteinase Stromelysin-1 Triggers a Cascade of Molecular Alterations that leads to stable epithelial-to-Mesenchymal Conversion and a Premalignant Phenotype in Mammary Epithelial Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lochter, A.; Galosy, S.; Muschler, J.

1997-08-11

Matrix metalloproteinases (MMPs) regulate ductal morphogenesis, apoptosis, and neoplastic progression in mammary epithelial cells. To elucidate the direct effects of MMPs on mammary epithelium, we generated functionally normal cells expressing an inducible autoactivating stromelysin-1 (SL-1) transgene. Induction of SL-1 expression resulted in cleavage of E-cadherin, and triggered progressive phenotypic conversion characterized by disappearance of E-cadherin and catenins from cell-cell contacts, downregulation of cytokeratins, upregulation of vimentin, induction of keratinocyte growth factor expression and activation, and upregulation of endogenous MMPs. Cells expressing SL-1 were unable to undergo lactogenic differentiation and became invasive. Once initiated, this phenotypic conversion was essentially stable, andmore » progressed even in the absence of continued SL-1 expression. These observations demonstrate that inappropriate expression of SL-1 initiates a cascade of events that may represent a coordinated program leading to loss of the differentiated epithelial phenotype and gain of some characteristics of tumor cells. Our data provide novel insights into how MMPs function in development and neoplastic conversion.« less

Role of activity-dependent BDNF expression in hippocampal–prefrontal cortical regulation of behavioral perseverance

PubMed Central

Sakata, Kazuko; Martinowich, Keri; Woo, Newton H.; Schloesser, Robert J.; Jimenez, Dennisse V.; Ji, Yuanyuan; Shen, Liya; Lu, Bai

2013-01-01

Activity-dependent gene transcription, including that of the brain-derived neurotrophic factor (Bdnf) gene, has been implicated in various cognitive functions. We previously demonstrated that mutant mice with selective disruption of activity-dependent BDNF expression (BDNF-KIV mice) exhibit deficits in GABA-mediated inhibition in the prefrontal cortex (PFC). Here, we show that disruption of activity-dependent BDNF expression impairs BDNF-dependent late-phase long-term potentiation (L-LTP) in CA1, a site of hippocampal output to the PFC. Interestingly, early-phase LTP and conventional L-LTP induced by strong tetanic stimulation were completely normal in BDNF-KIV mice. In parallel, attenuation of activity-dependent BDNF expression significantly impairs spatial memory reversal and contextual memory extinction, two executive functions that require intact hippocampal–PFC circuitry. In contrast, spatial and contextual memory per se were not affected. Thus, activity-dependent BDNF expression in the hippocampus and PFC may contribute to cognitive and behavioral flexibility. These results suggest distinct roles for different forms of L-LTP and provide a link between activity-dependent BDNF expression and behavioral perseverance, a hallmark of several psychiatric disorders. PMID:23980178

Overexpression of Interleukin-4 in the Thyroid of Transgenic Mice Upregulates the Expression of Duox1 and the Anion Transporter Pendrin

PubMed Central

Achouri, Younes; Hahn, Stephan; Many, Marie-Christine; Craps, Julie; Refetoff, Samuel; Liao, Xiao-Hui; Dumont, Jacques E.; Van Sande, Jacqueline; Corvilain, Bernard; Miot, Françoise; De Deken, Xavier

2016-01-01

Background: The dual oxidases (Duox) are involved in hydrogen peroxide generation, which is essential for thyroid hormone synthesis, and therefore they are markers of thyroid function. During inflammation, cytokines upregulate DUOX gene expression in the airway and the intestine, suggesting a role for these proteins in innate immunity. It was previously demonstrated that interleukin-4 (IL-4) upregulates DUOX gene expression in thyrocytes. Although the role of IL-4 in autoimmune thyroid diseases has been studied extensively, the effects of IL-4 on thyroid physiology remain largely unknown. Therefore, a new animal model was generated to study the impact of IL-4 on thyroid function. Methods: Transgenic (Thyr-IL-4) mice with thyroid-targeted expression of murine IL-4 were generated. Transgene expression was verified at the mRNA and protein level in thyroid tissues and primary cultures. The phenotype of the Thyr-IL-4 animals was characterized by measuring serum thyroxine (T4) and thyrotropin levels and performing thyroid morphometric analysis, immunohistochemistry, whole transcriptome sequencing, quantitative reverse transcription polymerase chain reaction, and ex vivo thyroid function assays. Results: Thyrocytes from two Thyr-IL-4 mouse lines (#30 and #52) expressed IL-4, which was secreted into the extracellular space. Although 10-month-old transgenic animals had T4 and thyrotropin serum levels in the normal range, they had altered thyroid follicular structure with enlarged follicles composed of elongated thyrocytes containing numerous endocytic vesicles. These follicles were positive for T4 staining the colloid, indicating their capacity to produce thyroid hormones. RNA profiling of Thyr-IL-4 thyroid samples revealed modulation of multiple genes involved in inflammation, while no major leukocyte infiltration could be detected. Upregulated expression of Duox1, Duoxa1, and the pendrin anion exchanger gene (Slc26a4) was detected. In contrast, the iodide symporter gene Slc5a5 was markedly downregulated resulting in impaired iodide uptake and reduced thyroid hormone levels in transgenic thyroid tissue. Hydrogen peroxide production was increased in Thyr-IL-4 thyroid tissue compared with wild-type animals, but no significant oxidative stress could be detected. Conclusions: This is the first study to show that ectopic expression of IL-4 in thyroid tissue upregulates Duox1/Duoxa1 and Slc26a4 expression in the thyroid. The present data demonstrate that IL-4 could affect thyroid morphology and function, mainly by downregulating Slc5a5 expression, while maintaining a normal euthyroid phenotype. PMID:27599561

The orphan nuclear receptor DAX-1 acts as a novel transcriptional corepressor of PPAR{gamma}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Gwang Sik; Lee, Gha Young; Nedumaran, Balachandar

2008-05-30

DAX-1 is an atypical nuclear receptor (NR) which functions primarily as a transcriptional corepressor of other NRs via heterodimerization. Peroxisome proliferator-activated receptor (PPAR) {gamma} is a ligand-dependent NR which performs a key function in adipogenesis. In this study, we evaluated a novel cross-talk mechanism between DAX-1 and PPAR{gamma}. Transient transfection assays demonstrated that DAX-1 inhibits the transactivity of PPAR{gamma} in a dose-dependent manner. DAX-1 directly competed with the PPAR{gamma} coactivator (PGC)-1{alpha} for binding to PPAR{gamma}. Endogenous levels of DAX-1 were significantly lower in differentiated 3T3-L1 adipocytes as compared to preadipocytes. Using a retroviral expression system, we demonstrated that DAX-1 overexpressionmore » downregulates the expression of PPAR{gamma} target genes, resulting in an attenuation of adipogenesis in 3T3-L1 cells. Our results suggest that DAX-1 acts as a corepressor of PPAR{gamma} and performs a potential function in the regulation of PPAR{gamma}-mediated cellular differentiation.« less

Tributyltin chloride induces renal dysfunction by inflammation and oxidative stress in female rats.

PubMed

Coutinho, João V S; Freitas-Lima, Leandro C; Freitas, Frederico F C T; Freitas, Flávia P S; Podratz, Priscila L; Magnago, Rafaella P L; Porto, Marcella L; Meyrelles, Silvana S; Vasquez, Elisardo C; Brandão, Poliane A A; Carneiro, Maria T W D; Paiva-Melo, Francisca D; Miranda-Alves, Leandro; Silva, Ian V; Gava, Agata L; Graceli, Jones B

2016-10-17

Tributyltin chloride (TBT) is an organometallic pollutant that is used as a biocide in antifouling paints. TBT induces several toxic and endocrine-disrupting effects. However, studies evaluating the effects of TBT on renal function are rare. This study demonstrates that TBT exposure is responsible for improper renal function as well as the development of abnormal morphophysiology in mammalian kidneys. Female rats were treated with TBT, and their renal morphophysiology was assessed. Morphophysiological abnormalities such as decreased glomerular filtration rate and increased proteinuria levels were observed in TBT rats. In addition, increases in inflammation, collagen deposition and α-smooth muscle actin (α-SMA) protein expression were observed in TBT kidneys. A disrupted cellular redox balance and apoptosis in kidney tissue were also observed in TBT rats. TBT rats demonstrated reduced serum estrogen levels and estrogen receptor-α (ERα) protein expression in renal cortex. Together, these data provide in vivo evidence that TBT is toxic to normal renal function and that these effects may be associated with renal histopathology complications, such as inflammation and fibrosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

The Neuroendocrine Peptide Catestatin Is a Cutaneous Antimicrobial and Induced in the Skin after Injury

PubMed Central

Radek, Katherine A.; Lopez-Garcia, Belen; Hupe, Melanie; Niesman, Ingrid R.; Elias, Peter M.; Taupenot, Laurent; Mahata, Sushil K.; O’Connor, Daniel T.; Gallo, Richard L.

2009-01-01

Epithelia establish a microbial barrier against infection through the production of antimicrobial peptides (AMPs). In this study, we investigated whether catestatin (Cst), a peptide derived from the neuroendocrine protein chromogranin A (CHGA), is a functional AMP and is present in the epidermis. We show that Cst is antimicrobial against relevant skin microbes, including Gram-positive and Gram-negative bacteria, yeast, and fungi. The antimicrobial mechanism of Cst was found to be similar to other AMPs, as it was dependent on bacterial charge and growth conditions, and induced membrane disruption. The potential relevance of Cst against skin pathogens was supported by the observation that CHGA was expressed in keratinocytes. In human skin, CHGA was found to be proteolytically processed into the antimicrobial fragment Cst, thus enabling its AMP function. Furthermore, Cst expression in murine skin increased in response to injury and infection, providing potential for increased protection against infection. These data demonstrate that a neuroendocrine peptide has antimicrobial function against a wide assortment of skin pathogens and is upregulated upon injury, thus demonstrating a direct link between the neuroendocrine and cutaneous immune systems. PMID:18185531

Modeling inelastic phonon scattering in atomic- and molecular-wire junctions

NASA Astrophysics Data System (ADS)

Paulsson, Magnus; Frederiksen, Thomas; Brandbyge, Mads

2005-11-01

Computationally inexpensive approximations describing electron-phonon scattering in molecular-scale conductors are derived from the nonequilibrium Green’s function method. The accuracy is demonstrated with a first-principles calculation on an atomic gold wire. Quantitative agreement between the full nonequilibrium Green’s function calculation and the newly derived expressions is obtained while simplifying the computational burden by several orders of magnitude. In addition, analytical models provide intuitive understanding of the conductance including nonequilibrium heating and provide a convenient way of parameterizing the physics. This is exemplified by fitting the expressions to the experimentally observed conductances through both an atomic gold wire and a hydrogen molecule.

Transplantation of autologous bone marrow-derived mesenchymal stem cells for traumatic brain injury☆

PubMed Central

Jiang, Jindou; Bu, Xingyao; Liu, Meng; Cheng, Peixun

2012-01-01

Results from the present study demonstrated that transplantation of autologous bone marrow-derived mesenchymal stem cells into the lesion site in rat brain significantly ameliorated brain tissue pathological changes and brain edema, attenuated glial cell proliferation, and increased brain-derived neurotrophic factor expression. In addition, the number of cells double-labeled for 5-bromodeoxyuridine/glial fibrillary acidic protein and cells expressing nestin increased. Finally, blood vessels were newly generated, and the rats exhibited improved motor and cognitive functions. These results suggested that transplantation of autologous bone marrow-derived mesenchymal stem cells promoted brain remodeling and improved neurological functions following traumatic brain injury. PMID:25806058

Angiotensin converting enzyme over expression in myelocytes enhances the immune response

PubMed Central

Bernstein, Kenneth E.; Gonzalez-Villalobos, Romer A.; Giani, Jorge F.; Shah, Kandarp; Bernstein, Ellen; Janjulia, Tea; Koronyo, Yosef; Shi, Peng D.; Koronyo-Hamaoui, Maya; Fuchs, Sebastien; Shen, Xiao Z.

2015-01-01

Angiotensin converting enzyme (ACE) plays an important role in blood pressure control. ACE also has effects on renal function, reproduction, hematopoiesis and several aspects of the immune response. ACE 10/10 mice over express ACE in monocytic cells; macrophages from ACE 10/10 mice demonstrate increased polarization towards a proinflammatory phenotype. As a result, ACE 10/10 mice have a highly effective immune response following challenge with either melanoma, bacterial infection or Alzheimer’s disease. The ACE 10/10 mice suggest that enhanced monocytic function greatly contributes to the ability of the immune response to defend against a wide variety of antigenic and non-antigenic challenges. PMID:24633750

MicroRNA-424/E2F6 feedback loop modulates cell invasion, migration and EMT in endometrial carcinoma

PubMed Central

Lu, Zheng; Nian, Zhou; Jingjing, Zhang; Tao, Luo; Quan, Li

2017-01-01

Our previous study explored the roles of microRNA-424 (miR-424) in the development of endometrial carcinoma (EC) and analyzed the miR-424/E2F7 axis in EC cell growth. In this study, we investigated the status of miR-424 in human endometrial cancer tissues, which were collected from a cohort of Zunyi patients. We found that the expression level of miR-424 was associated with clinical tumor stage, cell differentiation, lymph node metastasis and cell migration ability. Cell function experiments demonstrated that miR-424 overexpression suppressed the invasion and migration abilities of endometrial carcinoma cells in vitro. Bioinformatic predictions and dual-luciferase reporter assays suggested E2F6 as a possible target of miR-424. RT-PCR and western blot assays demonstrated that miR-424 transfection reduced the expression level of E2F6, while inhibiting miR-424 with ASO-miR-424 (antisense oligonucleotides of miR-424) increased the expression level of E2F6. Cell function experiments indicated that E2F6 transfection rescued the EC cell phenotype induced by miR-424. In addition, we also found that E2F6 negatively regulated miR-424 expression in EC cells. In summary, our results demonstrated that the miR-424/E2F6 feedback loop modulates cell invasion, migration and EMT in EC and that the miR-424/E2Fs regulation network may serve as a new and potentially important therapeutic target in EC. PMID:29371986

Extracellular Purines Promote the Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells to the Osteogenic and Adipogenic Lineages

PubMed Central

Zini, Roberta; Rossi, Lara; Salvestrini, Valentina; Ferrari, Davide; Manfredini, Rossella; Lemoli, Roberto M.

2013-01-01

Extracellular nucleotides are potent signaling molecules mediating cell-specific biological functions, mostly within the processes of tissue damage and repair and flogosis. We previously demonstrated that adenosine 5′-triphosphate (ATP) inhibits the proliferation of human bone marrow-derived mesenchymal stem cells (BM-hMSCs), while stimulating, in vitro and in vivo, their migration. Here, we investigated the effects of ATP on BM-hMSC differentiation capacity. Molecular analysis showed that ATP treatment modulated the expression of several genes governing adipogenic and osteoblastic (ie, WNT-pathway-related genes) differentiation of MSCs. Functional studies demonstrated that ATP, under specific culture conditions, stimulated adipogenesis by significantly increasing the lipid accumulation and the expression levels of the adipogenic master gene PPARγ (peroxisome proliferator-activated receptor-gamma). In addition, ATP stimulated osteogenic differentiation by promoting mineralization and expression of the osteoblast-related gene RUNX2 (runt-related transcription factor 2). Furthermore, we demonstrated that ATP stimulated adipogenesis via its triphosphate form, while osteogenic differentiation was induced by the nucleoside adenosine, resulting from ATP degradation induced by CD39 and CD73 ectonucleotidases expressed on the MSC membrane. The pharmacological profile of P2 purinergic receptors (P2Rs) suggests that adipogenic differentiation is mainly mediated by the engagement of P2Y1 and P2Y4 receptors, while stimulation of the P1R adenosine-specific subtype A2B is involved in adenosine-induced osteogenic differentiation. Thus, we provide new insights into molecular regulation of MSC differentiation. PMID:23259837

Divergent Pseudomonas exotoxin A sensitivity in normal and transformed liver cells is correlated with low-density lipoprotein receptor-related protein expression.

PubMed

Laithwaite, J E; Benn, S J; Marshall, W S; FitzGerald, D J; LaMarre, J

2001-09-01

Pseudomonas exotoxin A (PEA) is an extracellular virulence factor produced by the opportunistic human pathogen Pseudomonas aerguinosa. PEA intoxification begins when PEA binds to the low-density lipoprotein receptor-related protein (LRP). The liver is the primary target of systemic PEA, due largely to the high levels of functional LRP expressed by liver cells. Using a 3H-leucine incorporation assay to measure inhibition of protein synthesis we have demonstrated that normal (BNL CL.2) and transformed (BNL 1ME A7R.1) liver cells exhibit divergent PEA sensitivity; with BNL 1ME A7R.1 cells demonstrating greater PEA sensitivity than their non-transformed counterparts. The receptor-associated protein, a LRP antagonist, decreased PEA toxicity in BNL 1ME A7R.1 cells, confirming the importance of the LRP in PEA intoxification in this cell type. Increased PEA sensitivity in BNL 1ME A7R.1 cells was associated with increased functional cell surface LRP expression, as measured by alpha2-macroglobulin binding and internalization studies, and increased LRP mRNA levels, as determined by Northern blot analysis. Interestingly, BNL CL.2 cells were more sensitive than BNL 1ME A7R.1 cells to conjugate and mutant PEA toxins that do not utilize the LRP for cellular entry. These data demonstrate that increased LRP expression is an important mechanism by which PEA sensitivity is increased in BNL 1ME A7R.1 transformed liver cells.

Calcium-Alginate Hydrogel-Encapsulated Fibroblasts Provide Sustained Release of Vascular Endothelial Growth Factor

PubMed Central

Hunt, Nicola C.; Shelton, Richard M.; Henderson, Deborah J.

2013-01-01

Vascularization of engineered or damaged tissues is essential to maintain cell viability and proper tissue function. Revascularization of the left ventricle (LV) of the heart after myocardial infarction is particularly important, since hypoxia can give rise to chronic heart failure due to inappropriate remodeling of the LV after death of cardiomyocytes (CMs). Fibroblasts can express vascular endothelial growth factor (VEGF), which plays a major role in angiogenesis and also acts as a chemoattractant and survival factor for CMs and cardiac progenitors. In this in vitro model study, mouse NIH 3T3 fibroblasts encapsulated in 2% w/v Ca-alginate were shown to remain viable for 150 days. Semiquantitative reverse transcription–polymerase chain reaction and immunohistochemistry demonstrated that over 21 days of encapsulation, fibroblasts continued to express VEGF, while enzyme-linked immunosorbent assay showed that there was sustained release of VEGF from the Ca-alginate during this period. The scaffold degraded gradually over the 21 days, without reduction in volume. Cells released from the Ca-alginate at 7 and 21 days as a result of scaffold degradation were shown to retain viability, to adhere to fibronectin in a normal manner, and continue to express VEGF, demonstrating their potential to further contribute to maintenance of cardiac function after scaffold degradation. This model in vitro study therefore demonstrates that fibroblasts encapsulated in Ca-alginate provide sustained release of VEGF. PMID:23082964

Expression of Wise in chick embryos.

PubMed

Shigetani, Y; Itasaki, N

2007-08-01

We have performed in situ hybridization to study the expression of Wise in early chick embryos. Wise expression is first detectable in the ectoderm at posterior levels of late neurula. As development proceeds, Wise expression is seen in specific patterns in the ectoderm of the trunk region, pharyngeal arches, limb buds, and feather buds. In addition to these areas, particular cartilages such as the ones in the maxillary process and limbs start to express Wise at the late pharyngula stage, and the expression in these cartilages becomes stronger than that in epidermal components at later stages. Importantly, Wise is expressed in regions where other signaling molecules such as Wnt, Bmp, and Shh are known to function in morphogenesis and differentiation. Direct comparisons of the expression of Wise and these genes are also demonstrated. (c) 2007 Wiley-Liss, Inc.

Mammalian enabled (Mena) is a critical regulator of cardiac function

PubMed Central

Aguilar, Frédérick; Belmonte, Stephen L.; Ram, Rashmi; Noujaim, Sami F.; Dunaevsky, Olga; Protack, Tricia L.; Jalife, Jose; Todd Massey, H.; Gertler, Frank B.

2011-01-01

Mammalian enabled (Mena) of the Drosophila enabled/vasodilator-stimulated phosphoprotein gene family is a cytoskeletal protein implicated in actin regulation and cell motility. Cardiac Mena expression is enriched in intercalated discs (ICD), the critical intercellular communication nexus between adjacent muscle cells. We previously identified Mena gene expression to be a key predictor of human and murine heart failure (HF). To determine the in vivo function of Mena in the heart, we assessed Mena protein expression in multiple HF models and characterized the effects of genetic Mena deletion on cardiac structure and function. Immunoblot analysis revealed significant upregulation of Mena protein expression in left ventricle tissue from patients with end-stage HF, calsequestrin-overexpressing mice, and isoproterenol-infused mice. Characterization of the baseline cardiac function of adult Mena knockout mice (Mena−/−) via echocardiography demonstrated persistent cardiac dysfunction, including a significant reduction in percent fractional shortening compared with wild-type littermates. Electrocardiogram PR and QRS intervals were significantly prolonged in Mena−/− mice, manifested by slowed conduction on optical mapping studies. Ultrastructural analysis of Mena−/− hearts revealed disrupted organization and widening of ICD structures, mislocalization of the gap junction protein connexin 43 (Cx43) to the lateral borders of cardiomyoycytes, and increased Cx43 expression. Furthermore, the expression of vinculin (an adherens junction protein) was significantly reduced in Mena−/− mice. We report for the first time that genetic ablation of Mena results in cardiac dysfunction, highlighted by diminished contractile performance, disrupted ICD structure, and slowed electrical conduction. PMID:21335464

Mammalian enabled (Mena) is a critical regulator of cardiac function.

PubMed

Aguilar, Frédérick; Belmonte, Stephen L; Ram, Rashmi; Noujaim, Sami F; Dunaevsky, Olga; Protack, Tricia L; Jalife, Jose; Todd Massey, H; Gertler, Frank B; Blaxall, Burns C

2011-05-01

Mammalian enabled (Mena) of the Drosophila enabled/vasodilator-stimulated phosphoprotein gene family is a cytoskeletal protein implicated in actin regulation and cell motility. Cardiac Mena expression is enriched in intercalated discs (ICD), the critical intercellular communication nexus between adjacent muscle cells. We previously identified Mena gene expression to be a key predictor of human and murine heart failure (HF). To determine the in vivo function of Mena in the heart, we assessed Mena protein expression in multiple HF models and characterized the effects of genetic Mena deletion on cardiac structure and function. Immunoblot analysis revealed significant upregulation of Mena protein expression in left ventricle tissue from patients with end-stage HF, calsequestrin-overexpressing mice, and isoproterenol-infused mice. Characterization of the baseline cardiac function of adult Mena knockout mice (Mena(-/-)) via echocardiography demonstrated persistent cardiac dysfunction, including a significant reduction in percent fractional shortening compared with wild-type littermates. Electrocardiogram PR and QRS intervals were significantly prolonged in Mena(-/-) mice, manifested by slowed conduction on optical mapping studies. Ultrastructural analysis of Mena(-/-) hearts revealed disrupted organization and widening of ICD structures, mislocalization of the gap junction protein connexin 43 (Cx43) to the lateral borders of cardiomyoycytes, and increased Cx43 expression. Furthermore, the expression of vinculin (an adherens junction protein) was significantly reduced in Mena(-/-) mice. We report for the first time that genetic ablation of Mena results in cardiac dysfunction, highlighted by diminished contractile performance, disrupted ICD structure, and slowed electrical conduction.

Heat shock protein 27 regulates human prostate cancer cell motility and metastatic progression

PubMed Central

Voll, Eric A; Ogden, Irene M; Pavese, Janet M; Huang, XiaoKe; Xu, Li; Jovanovic, Borko D; Bergan, Raymond C

2014-01-01

Prostate cancer (PCa) is the most common form of cancer in American men. Mortality from PCa is caused by the movement of cancer cells from the primary organ to form metastatic tumors at distant sites. Heat shock protein 27 (HSP27) is known to increase human PCa cell invasion and its overexpression is associated with metastatic disease. The role of HSP27 in driving PCa cell movement from the prostate to distant metastatic sites is unknown. Increased HSP27 expression increased metastasis as well as primary tumor mass. In vitro studies further examined the mechanism of HSP27-induced metastatic behavior. HSP27 did not affect cell detachment, adhesion, or migration, but did increase cell invasion. Cell invasion was dependent upon matrix metalloproteinase 2 (MMP-2), whose expression was increased by HSP27. In vivo, HSP27 induced commensurate changes in MMP-2 expression in tumors. These findings demonstrate that HSP27 drives metastatic spread of cancer cells from the prostate to distant sites, does so across a continuum of expression levels, and identifies HSP27-driven increases in MMP-2 expression as functionally relevant. These findings add to prior studies demonstrating that HSP27 increases PCa cell motility, growth and survival. Together, they demonstrate that HSP27 plays an important role in PCa progression. PMID:24798191

Integrative functional transcriptomic analyses implicate specific molecular pathways in pulmonary toxicity from exposure to aluminum oxide nanoparticles.

PubMed

Li, Xiaobo; Zhang, Chengcheng; Bian, Qian; Gao, Na; Zhang, Xin; Meng, Qingtao; Wu, Shenshen; Wang, Shizhi; Xia, Yankai; Chen, Rui

2016-09-01

Gene expression profiling has developed rapidly in recent years and it can predict and define mechanisms underlying chemical toxicity. Here, RNA microarray and computational technology were used to show that aluminum oxide nanoparticles (Al2O3 NPs) were capable of triggering up-regulation of genes related to the cell cycle and cell death in a human A549 lung adenocarcinoma cell line. Gene expression levels were validated in Al2O3 NPs exposed A549 cells and mice lung tissues, most of which showed consistent trends in regulation. Gene-transcription factor network analysis coupled with cell- and animal-based assays demonstrated that the genes encoding PTPN6, RTN4, BAX and IER play a role in the biological responses induced by the nanoparticle exposure, which caused cell death and cell cycle arrest in the G2/S phase. Further, down-regulated PTPN6 expression demonstrated a core role in the network, thus expression level of PTPN6 was rescued by plasmid transfection, which showed ameliorative effects of A549 cells against cell death and cell cycle arrest. These results demonstrate the feasibility of using gene expression profiling to predict cellular responses induced by nanomaterials, which could be used to develop a comprehensive knowledge of nanotoxicity.

Is fear in your head? A comparison of instructed and real-life expressions of emotion in the face and body.

PubMed

Abramson, Lior; Marom, Inbal; Petranker, Rotem; Aviezer, Hillel

2017-04-01

The majority of emotion perception studies utilize instructed and stereotypical expressions of faces or bodies. While such stimuli are highly standardized and well-recognized, their resemblance to real-life expressions of emotion remains unknown. Here we examined facial and body expressions of fear and anger during real-life situations and compared their recognition to that of instructed expressions of the same emotions. In order to examine the source of the affective signal, expressions of emotion were presented as faces alone, bodies alone, and naturally, as faces with bodies. The results demonstrated striking deviations between recognition of instructed and real-life stimuli, which differed as a function of the emotion expressed. In real-life fearful expressions of emotion, bodies were far better recognized than faces, a pattern not found with instructed expressions of emotion. Anger reactions were better recognized from the body than from the face in both real-life and instructed stimuli. However, the real-life stimuli were overall better recognized than their instructed counterparts. These results indicate that differences between instructed and real-life expressions of emotion are prevalent and raise caution against an overreliance of researchers on instructed affective stimuli. The findings also demonstrate that in real life, facial expression perception may rely heavily on information from the contextualizing body. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

Critical role of γ4 chain in the expression of functional Vγ4Vδ1 T cell receptor of gastric tumour-infiltrating γδT lymphocytes.

PubMed

Jiang, Y; Tang, F; Li, Z; Cui, L; He, W

2012-01-01

Vγ4Vδ1 T cell receptor (TCRγ4δ1)-expressing γδT cells were the most dominant subset in gastric tumour-infiltrating γδT cells (γδTIL) we recently analyzed. To study the essential roles of γ and δ chains in assembly and function of TCRγ4δ1, we sequenced and constructed them into lentiviral vectors for the reconstitution of TCRγ4δ1 using different modalities of transduction. We were able to efficiently reconstitute TCRγ4δ1 with functional activities when both γ4 and δ1 chains are coexpressed in TCR-negative J.RT3-T3.5 cells. However, the expression of δ1 chain is greatly diminished when γ4 expression is absent, suggesting that the coexpressing γ4 is critical in maintaining the folding and stability of δ1 product. To functionally study the reconstituted TCRγ4δ1, we examined the cytolytic activity of TCRγ4δ1-reconstituted J.RT3-T3.5 cells and cytokine secretion and found the receptors are fully functional, but their functionality also requires the presence of γ4. Our results demonstrated that γ4 is critical for the stability of δ1 and the function of TCRγ4δ1. © 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.

Ectopic lymphoid structures support ongoing production of class-switched autoantibodies in rheumatoid synovium.

PubMed

Humby, Frances; Bombardieri, Michele; Manzo, Antonio; Kelly, Stephen; Blades, Mark C; Kirkham, Bruce; Spencer, Jo; Pitzalis, Costantino

2009-01-13

Follicular structures resembling germinal centres (GCs) that are characterized by follicular dendritic cell (FDC) networks have long been recognized in chronically inflamed tissues in autoimmune diseases, including the synovium of rheumatoid arthritis (RA). However, it is debated whether these ectopic structures promote autoimmunity and chronic inflammation driving the production of pathogenic autoantibodies. Anti-citrullinated protein/peptide antibodies (ACPA) are highly specific markers of RA, predict a poor prognosis, and have been suggested to be pathogenic. Therefore, the main study objectives were to determine whether ectopic lymphoid structures in RA synovium: (i) express activation-induced cytidine deaminase (AID), the enzyme required for somatic hypermutation and class-switch recombination (CSR) of Ig genes; (ii) support ongoing CSR and ACPA production; and (iii) remain functional in a RA/severe combined immunodeficiency (SCID) chimera model devoid of new immune cell influx into the synovium. Using immunohistochemistry (IHC) and quantitative Taqman real-time PCR (QT-PCR) in synovial tissue from 55 patients with RA, we demonstrated that FDC+ structures invariably expressed AID with a distribution resembling secondary lymphoid organs. Further, AID+/CD21+ follicular structures were surrounded by ACPA+/CD138+ plasma cells, as demonstrated by immune reactivity to citrullinated fibrinogen. Moreover, we identified a novel subset of synovial AID+/CD20+ B cells outside GCs resembling interfollicular large B cells. In order to gain direct functional evidence that AID+ structures support CSR and in situ manufacturing of class-switched ACPA, 34 SCID mice were transplanted with RA synovium and humanely killed at 4 wk for harvesting of transplants and sera. Persistent expression of AID and Igamma-Cmu circular transcripts (identifying ongoing IgM-IgG class-switching) was observed in synovial grafts expressing FDCs/CD21L. Furthermore, synovial mRNA levels of AID were closely associated with circulating human IgG ACPA in mouse sera. Finally, the survival and proliferation of functional B cell niches was associated with persistent overexpression of genes regulating ectopic lymphoneogenesis. Our demonstration that FDC+ follicular units invariably express AID and are surrounded by ACPA-producing plasma cells provides strong evidence that ectopic lymphoid structures in the RA synovium are functional and support autoantibody production. This concept is further confirmed by evidence of sustained AID expression, B cell proliferation, ongoing CSR, and production of human IgG ACPA from GC+ synovial tissue transplanted into SCID mice, independently of new B cell influx from the systemic circulation. These data identify AID as a potential therapeutic target in RA and suggest that survival of functional synovial B cell niches may profoundly influence chronic inflammation, autoimmunity, and response to B cell-depleting therapies.

The extracellular matrix controls gap junction protein expression and function in postnatal hippocampal neural progenitor cells

PubMed Central

Imbeault, Sophie; Gauvin, Lianne G; Toeg, Hadi D; Pettit, Alexandra; Sorbara, Catherine D; Migahed, Lamiaa; DesRoches, Rebecca; Menzies, A Sheila; Nishii, Kiyomasa; Paul, David L; Simon, Alexander M; Bennett, Steffany AL

2009-01-01

Background Gap junction protein and extracellular matrix signalling systems act in concert to influence developmental specification of neural stem and progenitor cells. It is not known how these two signalling systems interact. Here, we examined the role of ECM components in regulating connexin expression and function in postnatal hippocampal progenitor cells. Results We found that Cx26, Cx29, Cx30, Cx37, Cx40, Cx43, Cx45, and Cx47 mRNA and protein but only Cx32 and Cx36 mRNA are detected in distinct neural progenitor cell populations cultured in the absence of exogenous ECM. Multipotential Type 1 cells express Cx26, Cx30, and Cx43 protein. Their Type 2a progeny but not Type 2b and 3 neuronally committed progenitor cells additionally express Cx37, Cx40, and Cx45. Cx29 and Cx47 protein is detected in early oligodendrocyte progenitors and mature oligodendrocytes respectively. Engagement with a laminin substrate markedly increases Cx26 protein expression, decreases Cx40, Cx43, Cx45, and Cx47 protein expression, and alters subcellular localization of Cx30. These changes are associated with decreased neurogenesis. Further, laminin elicits the appearance of Cx32 protein in early oligodendrocyte progenitors and Cx36 protein in immature neurons. These changes impact upon functional connexin-mediated hemichannel activity but not gap junctional intercellular communication. Conclusion Together, these findings demonstrate a new role for extracellular matrix-cell interaction, specifically laminin, in the regulation of intrinsic connexin expression and function in postnatal neural progenitor cells. PMID:19236721

Robust, synergistic regulation of human gene expression using TALE activators.

PubMed

Maeder, Morgan L; Linder, Samantha J; Reyon, Deepak; Angstman, James F; Fu, Yanfang; Sander, Jeffry D; Joung, J Keith

2013-03-01

Artificial activators designed using transcription activator-like effector (TALE) technology have broad utility, but previous studies suggest that these monomeric proteins often exhibit low activities. Here we demonstrate that TALE activators can robustly function individually or in synergistic combinations to increase expression of endogenous human genes over wide dynamic ranges. These findings will encourage applications of TALE activators for research and therapy, and guide design of monomeric TALE-based fusion proteins.

Functional Genome Mining for Metabolites Encoded by Large Gene Clusters through Heterologous Expression of a Whole-Genome Bacterial Artificial Chromosome Library in Streptomyces spp.

PubMed Central

Xu, Min; Wang, Yemin; Zhao, Zhilong; Gao, Guixi; Huang, Sheng-Xiong; Kang, Qianjin; He, Xinyi; Lin, Shuangjun; Pang, Xiuhua; Deng, Zixin

2016-01-01

ABSTRACT Genome sequencing projects in the last decade revealed numerous cryptic biosynthetic pathways for unknown secondary metabolites in microbes, revitalizing drug discovery from microbial metabolites by approaches called genome mining. In this work, we developed a heterologous expression and functional screening approach for genome mining from genomic bacterial artificial chromosome (BAC) libraries in Streptomyces spp. We demonstrate mining from a strain of Streptomyces rochei, which is known to produce streptothricins and borrelidin, by expressing its BAC library in the surrogate host Streptomyces lividans SBT5, and screening for antimicrobial activity. In addition to the successful capture of the streptothricin and borrelidin biosynthetic gene clusters, we discovered two novel linear lipopeptides and their corresponding biosynthetic gene cluster, as well as a novel cryptic gene cluster for an unknown antibiotic from S. rochei. This high-throughput functional genome mining approach can be easily applied to other streptomycetes, and it is very suitable for the large-scale screening of genomic BAC libraries for bioactive natural products and the corresponding biosynthetic pathways. IMPORTANCE Microbial genomes encode numerous cryptic biosynthetic gene clusters for unknown small metabolites with potential biological activities. Several genome mining approaches have been developed to activate and bring these cryptic metabolites to biological tests for future drug discovery. Previous sequence-guided procedures relied on bioinformatic analysis to predict potentially interesting biosynthetic gene clusters. In this study, we describe an efficient approach based on heterologous expression and functional screening of a whole-genome library for the mining of bioactive metabolites from Streptomyces. The usefulness of this function-driven approach was demonstrated by the capture of four large biosynthetic gene clusters for metabolites of various chemical types, including streptothricins, borrelidin, two novel lipopeptides, and one unknown antibiotic from Streptomyces rochei Sal35. The transfer, expression, and screening of the library were all performed in a high-throughput way, so that this approach is scalable and adaptable to industrial automation for next-generation antibiotic discovery. PMID:27451447

The role of TGFβ receptor 1-smad3 signaling in regulating the osteoclastic mode affected by fluoride.

PubMed

Yu, Haolan; Jiang, Ningning; Yu, XiuHua; Zhao, Zhitao; Zhang, Xiuyun; Xu, Hui

2018-01-15

Studies that have focused on the role TGFβ signaling plays in osteoclast activity are gradually increasing; however, literature is rare in terms of fluorosis. The aim of this study is to observe the role the TβR1/Smad3 pathway plays in fluoride regulating cellsosteoclast-like cells that are under the treatment of TGFβ receptor 1 kinase. The RANKL-mediated osteoclast-like cells from RAW264.7 cells were used as osteoclast precursor model. The profile of miRNA expression in fluoride-treated osteoclast-like cells exhibited 303 upregulated miRNAs, 61 downregulated miRNAs, and further drew 37 signaling pathway maps by KEGG and Biocarta pathway enrichment analysis. TGFβ and its downstream effectors were included among them. Osteoclast viability, formation and function were detected via MTT method, bone resorption pit and tartrate-resistant acid phosphatase (TRACP) staining, respectively. Results demonstrated that different doses of fluoride exhibited a biphasic effect on osteoclast cell viability, differentiation, formation and function. It indicated that a low dose of fluoride treatment stimulated them, but high dose inhibited them. SB431542 acted as TβR1 kinase inhibitor and blocked viability, formation and function of osteoclast-like cells regulated by fluoride. The expression of the osteoclast marker, RANK, and TβR1/Smad3 at gene and protein level was analyzed under fluoride with and without SB431542 treatment. Fluoride treatment indicated little effect on the RANK protein expression; however it significantly influenced TRACP expression in osteoclast-like cells. The stimulation of fluoride on the expression of Smad3 gene and phosphorylated Smad3 protein exhibited dose-dependent manner. SB431542 significantly impeded phosphorylation of Smad3 protein and TRACP expression in osteoclast-like cells that were exposed to fluoride. Our work demonstrated that TGFβ signaling played a key role in fluoride regulating osteoclast differentiation, formation and function. It elucidated that TβR1/Smad3 pathway participated in the mechanism of biphasic modulation of osteoclast mode regulated by fluoride. Copyright © 2017 Elsevier B.V. All rights reserved.

Protoplast isolation, transient transformation of leaf mesophyll protoplasts and improved Agrobacterium-mediated leaf disc infiltration of Phaseolus vulgaris: tools for rapid gene expression analysis.

PubMed

Nanjareddy, Kalpana; Arthikala, Manoj-Kumar; Blanco, Lourdes; Arellano, Elizabeth S; Lara, Miguel

2016-06-24

Phaseolus vulgaris is one of the most extensively studied model legumes in the world. The P. vulgaris genome sequence is available; therefore, the need for an efficient and rapid transformation system is more imperative than ever. The functional characterization of P. vulgaris genes is impeded chiefly due to the non-amenable nature of Phaseolus sp. to stable genetic transformation. Transient transformation systems are convenient and versatile alternatives for rapid gene functional characterization studies. Hence, the present work focuses on standardizing methodologies for protoplast isolation from multiple tissues and transient transformation protocols for rapid gene expression analysis in the recalcitrant grain legume P. vulgaris. Herein, we provide methodologies for the high-throughput isolation of leaf mesophyll-, flower petal-, hypocotyl-, root- and nodule-derived protoplasts from P. vulgaris. The highly efficient polyethylene glycol-mannitol magnesium (PEG-MMG)-mediated transformation of leaf mesophyll protoplasts was optimized using a GUS reporter gene. We used the P. vulgaris SNF1-related protein kinase 1 (PvSnRK1) gene as proof of concept to demonstrate rapid gene functional analysis. An RT-qPCR analysis of protoplasts that had been transformed with PvSnRK1-RNAi and PvSnRK1-OE vectors showed the significant downregulation and ectopic constitutive expression (overexpression), respectively, of the PvSnRK1 transcript. We also demonstrated an improved transient transformation approach, sonication-assisted Agrobacterium-mediated transformation (SAAT), for the leaf disc infiltration of P. vulgaris. Interestingly, this method resulted in a 90 % transformation efficiency and transformed 60-85 % of the cells in a given area of the leaf surface. The constitutive expression of YFP further confirmed the amenability of the system to gene functional characterization studies. We present simple and efficient methodologies for protoplast isolation from multiple P. vulgaris tissues. We also provide a high-efficiency and amenable method for leaf mesophyll transformation for rapid gene functional characterization studies. Furthermore, a modified SAAT leaf disc infiltration approach aids in validating genes and their functions. Together, these methods help to rapidly unravel novel gene functions and are promising tools for P. vulgaris research.

Dissecting the genetics of the human transcriptome identifies novel trait-related trans-eQTLs and corroborates the regulatory relevance of non-protein coding loci†.

PubMed

Kirsten, Holger; Al-Hasani, Hoor; Holdt, Lesca; Gross, Arnd; Beutner, Frank; Krohn, Knut; Horn, Katrin; Ahnert, Peter; Burkhardt, Ralph; Reiche, Kristin; Hackermüller, Jörg; Löffler, Markus; Teupser, Daniel; Thiery, Joachim; Scholz, Markus

2015-08-15

Genetics of gene expression (eQTLs or expression QTLs) has proved an indispensable tool for understanding biological pathways and pathomechanisms of trait-associated SNPs. However, power of most genome-wide eQTL studies is still limited. We performed a large eQTL study in peripheral blood mononuclear cells of 2112 individuals increasing the power to detect trans-effects genome-wide. Going beyond univariate SNP-transcript associations, we analyse relations of eQTLs to biological pathways, polygenetic effects of expression regulation, trans-clusters and enrichment of co-localized functional elements. We found eQTLs for about 85% of analysed genes, and 18% of genes were trans-regulated. Local eSNPs were enriched up to a distance of 5 Mb to the transcript challenging typically implemented ranges of cis-regulations. Pathway enrichment within regulated genes of GWAS-related eSNPs supported functional relevance of identified eQTLs. We demonstrate that nearest genes of GWAS-SNPs might frequently be misleading functional candidates. We identified novel trans-clusters of potential functional relevance for GWAS-SNPs of several phenotypes including obesity-related traits, HDL-cholesterol levels and haematological phenotypes. We used chromatin immunoprecipitation data for demonstrating biological effects. Yet, we show for strongly heritable transcripts that still little trans-chromosomal heritability is explained by all identified trans-eSNPs; however, our data suggest that most cis-heritability of these transcripts seems explained. Dissection of co-localized functional elements indicated a prominent role of SNPs in loci of pseudogenes and non-coding RNAs for the regulation of coding genes. In summary, our study substantially increases the catalogue of human eQTLs and improves our understanding of the complex genetic regulation of gene expression, pathways and disease-related processes. © The Author 2015. Published by Oxford University Press.

Glucagon-like peptide 1 receptor expression in primary porcine proximal tubular cells.

PubMed

Schlatter, P; Beglinger, C; Drewe, J; Gutmann, H

2007-06-07

GLP-1 is secreted into the circulation after food intake. The main biological effects of GLP-1 include stimulation of glucose dependent insulin secretion and induction of satiety feelings. Recently, it was demonstrated in rats and humans that GLP-1 can stimulate renal excretion of sodium. Based on these data, the existence of a renal GLP-1 receptor (GLP-1R) was postulated. However, the exact localization of the GLP-1R and the mechanism of this GLP-1 action have not yet been investigated. Primary porcine proximal tubular cells were isolated from porcine kidneys. Expression of GLP-1R was measured at the mRNA level by quantitative RT-PCR. Protein expression of GLP-1R was verified with immunocytochemistry, immunohistochemistry and Western blot analysis. Functional studies included transport assessments of sodium and glucose using three different GLP-1 concentrations (200 pM, 2 nM and 20 nM), 200 pM exendin-4 (GLP-1 analogue) and an inhibitor of the dipeptidylpeptidase IV (DPPIV) enzyme (P32/98 at 10 microM). Finally, the expression of NHE3, the predominant Na(+)/H(+) exchanger in proximal tubular cells, was also investigated. GLP-1R, NHE3 and DPPIV were expressed at the mRNA level in porcine proximal tubular kidney cells. GLP-1R expression was confirmed at the protein level. Staining of human and pig kidney cortex revealed that GLP-1R was predominantly expressed in proximal tubular cells. Functional assays demonstrated an inhibition of sodium re-absorption with GLP-1 after 3 h of incubation. Exendin-4 and GLP-1 in combination with P32/98 co-administration had no clear influence on glucose and sodium uptake and transport. GLP-1R is functionally expressed in porcine proximal tubular kidney cells. Addition of GLP-1 to these cells resulted in a reduced sodium re-absorption. GLP-1 had no effect on glucose re-absorption. We conclude that GLP-1 modulates sodium homeostasis in the kidney most likely through a direct action via its GLP-1R in proximal tubular cells.

Functional characterization of EZH2β reveals the increased complexity of EZH2 isoforms involved in the regulation of mammalian gene expression

PubMed Central

2013-01-01

Background Histone methyltransferase enhancer of zeste homologue 2 (EZH2) forms an obligate repressive complex with suppressor of zeste 12 and embryonic ectoderm development, which is thought, along with EZH1, to be primarily responsible for mediating Polycomb-dependent gene silencing. Polycomb-mediated repression influences gene expression across the entire gamut of biological processes, including development, differentiation and cellular proliferation. Deregulation of EZH2 expression is implicated in numerous complex human diseases. To date, most EZH2-mediated function has been primarily ascribed to a single protein product of the EZH2 locus. Results We report that the EZH2 locus undergoes alternative splicing to yield at least two structurally and functionally distinct EZH2 methyltransferases. The longest protein encoded by this locus is the conventional enzyme, which we refer to as EZH2α, whereas EZH2β, characterized here, represents a novel isoform. We find that EZH2β localizes to the cell nucleus, complexes with embryonic ectoderm development and suppressor of zeste 12, trimethylates histone 3 at lysine 27, and mediates silencing of target promoters. At the cell biological level, we find that increased EZH2β induces cell proliferation, demonstrating that this protein is functional in the regulation of processes previously attributed to EZH2α. Biochemically, through the use of genome-wide expression profiling, we demonstrate that EZH2β governs a pattern of gene repression that is often ontologically redundant from that of EZH2α, but also divergent for a wide variety of specific target genes. Conclusions Combined, these results demonstrate that an expanded repertoire of EZH2 writers can modulate histone code instruction during histone 3 lysine 27-mediated gene silencing. These data support the notion that the regulation of EZH2-mediated gene silencing is more complex than previously anticipated and should guide the design and interpretation of future studies aimed at understanding the biochemical and biological roles of this important family of epigenomic regulators. PMID:23448518

Androgen receptor (AR) promotes clear cell renal cell carcinoma (ccRCC) migration and invasion via altering the circHIAT1/miR-195-5p/29a-3p/29c-3p/CDC42 signals.

PubMed

Wang, Kefeng; Sun, Yin; Tao, Wei; Fei, Xiang; Chang, Chawnshang

2017-05-28

Increasing evidence has demonstrated that the androgen receptor (AR) plays important roles to promote the metastasis of clear cell renal cell carcinoma (ccRCC). The detailed mechanisms, especially how AR functions via altering the circular RNAs (circRNAs) remain unclear. Here we identified a new circRNA (named as circHIAT1) whose expression was lower in ccRCCs than adjacent normal tissues. Targeting AR could suppress ccRCC cell progression via increasing circHIAT1 expression. ChIP assay and luciferase assay demonstrated that AR suppressed circHIAT1 expression via regulating its host gene, Hippocampus Abundant Transcript 1 (HIAT1) expression at the transcriptional level. The consequences of AR-suppressed circHIAT1 resulted in deregulating miR-195-5p/29a-3p/29c-3p expressions, which increased CDC42 expression to enhance ccRCC cell migration and invasion. Increasing this newly identified signal via circHIAT1 suppressed AR-enhanced ccRCC cell migration and invasion. Together, these results suggested that circHIAT1 functioned as a metastatic inhibitor to suppress AR-enhanced ccRCC cell migration and invasion. Targeting this newly identified AR-circHIAT1-mediated miR-195-5p/29a-3p/29c-3p/CDC42 signals may help us develop potential new therapies to better suppress ccRCC metastasis. Copyright © 2017 Elsevier B.V. All rights reserved.

Msx-2 expression and glucocorticoid-induced overexpression in embryonic mouse submandibular glands.

PubMed

Jaskoll, T; Luo, W; Snead, M L

1998-01-01

It is well known that the process of branching morphogenesis requires epithelial-mesenchymal interactions. One outstanding model for the study of tissue interactions during branching morphogenesis is the embryonic mouse submandibular gland (SMG). Although it has been clearly demonstrated that the branching pattern is dependent on interactions between the epithelium and the surrounding mesenchyme, little is known about the molecular mechanism underlying the branching process. One group of transcription factors that likely participates in the control of epithelial-mesenchymal inductive interactions are the Msx-class of homeodomain-containing proteins. In this paper, we focus on Msx-2 because its developmental expression is correlated with inductive interactions, suggesting that Msx-2 may play a functional role during cell-cell interactions. We demonstrate the expression of Msx-2 mRNA and protein to be primarily in the branching epithelia with progressive embryonic (E13 to E15) SMG development and, to a lesser extent, in the mesenchyme. We also show that Msx-2 is expressed by embryonic SMG primordia cultured under defined conditions. In addition, to begin to delineate a functional role for Msx-2, we employed an experimental strategy by using exogenous glucocorticoid (CORT) treatment of embryonic SMGs in vitro and in vivo to significantly enhance branching morphogenesis and evaluate the effect of CORT treatment on embryonic SMG Msx-2 expression. A marked increase in Msx-2 transcripts and protein is detected with in vitro and in vivo CORT treatment. Our studies indicate that one mechanism of CORT regulation of salivary gland morphogenesis is likely through the modulation of Msx-2 gene expression.

Increased Calcium Levels and Prolonged Shelf Life in Tomatoes Expressing Arabidopsis H+/Ca2+ Transporters1

PubMed Central

Park, Sunghun; Cheng, Ning Hui; Pittman, Jon K.; Yoo, Kil Sun; Park, Jungeun; Smith, Roberta H.; Hirschi, Kendal D.

2005-01-01

Here we demonstrate that fruit from tomato (Lycopersicon esculentum) plants expressing Arabidopsis (Arabidopsis thaliana) H+/cation exchangers (CAX) have more calcium (Ca2+) and prolonged shelf life when compared to controls. Previously, using the prototypical CAX1, it has been demonstrated that, in yeast (Saccharomyces cerevisiae) cells, CAX transporters are activated when the N-terminal autoinhibitory region is deleted, to give an N-terminally truncated CAX (sCAX), or altered through specific manipulations. To continue to understand the diversity of CAX function, we used yeast assays to characterize the putative transport properties of CAX4 and N-terminal variants of CAX4. CAX4 variants can suppress the Ca2+ hypersensitive yeast phenotypes and also appear to be more specific Ca2+ transporters than sCAX1. We then compared the phenotypes of sCAX1- and CAX4-expressing tomato lines. The sCAX1-expressing tomato lines demonstrate increased vacuolar H+/Ca2+ transport, when measured in root tissue, elevated fruit Ca2+ level, and prolonged shelf life but have severe alterations in plant development and morphology, including increased incidence of blossom-end rot. The CAX4-expressing plants demonstrate more modest increases in Ca2+ levels and shelf life but no deleterious effects on plant growth. These findings suggest that CAX expression may fortify plants with Ca2+ and may serve as an alternative to the application of CaCl2 used to extend the shelf life of numerous agriculturally important commodities. However, judicious regulation of CAX transport is required to assure optimal plant growth. PMID:16244156

TGF-beta1 inhibits Cx43 expression and formation of functional syncytia in cultured smooth muscle cells from human detrusor.

PubMed

Neuhaus, Jochen; Heinrich, Marco; Schwalenberg, Thilo; Stolzenburg, Jens-Uwe

2009-02-01

Human detrusor smooth muscle cells (hBSMCs) are coupled by connexin 43 (Cx43)-positive gap junctions to form functional syncytia. Gap junctional communication likely is necessary for synchronised detrusor contractions and is supposed to be altered in voiding disturbances. Other authors have shown that the pleiotropic cytokine TGF-beta1 upregulates Cx43 expression in human aortic smooth muscle cells. In this study, we examined the TGF-beta1 effects on Cx43 expression in cultured hBSMCs. hBSMC cultures, established from patients undergoing cystectomy, were treated with recombinant human TGF-beta1. Cx43 expression was then examined by Western blotting, real-time PCR, and immunocytochemistry. Dye-injection experiments were used to study the size of functional syncytia. Dye-coupling experiments revealed stable formation of functional syncytia in passaged cell cultures (P1-P4). Stimulation with TGF-beta1 led to significant reduction of Cx43 immunoreactivity and coupling. Cx43 protein expression was significantly downregulated and Cx43 mRNA was only 30% of the control level. Interestingly, low phosphorylation species of Cx43 were particularly affected. Our experiments demonstrated a significant down regulation of connexin 43 by TGF-beta1 in cultured hBSMCs. These findings support the view that TGF-beta1 is involved in the pathophysiology of urinary bladder dysfunction.

Ionophore and Biometal Modulation of P-glycoprotein Expression and Function in Human Brain Microvascular Endothelial Cells.

PubMed

McInerney, Mitchell P; Volitakis, Irene; Bush, Ashley I; Banks, William A; Short, Jennifer L; Nicolazzo, Joseph A

2018-03-05

Biometals such as zinc and copper have been shown to affect tight junction expression and subsequently blood-brain barrier (BBB) integrity. Whether these biometals also influence the expression and function of BBB transporters such as P-glycoprotein (P-gp) however is currently unknown. Using the immortalised human cerebral microvascular endothelial (hCMEC/D3) cell line, an in-cell western assay (alongside western blotting) assessed relative P-gp expression after treatment with the metal ionophore clioquinol and biometals zinc and copper. The fluorescent P-gp substrate rhodamine-123 was employed to observe functional modulation, and inductively coupled plasma mass spectrometry (ICP-MS) provided information on biometal trafficking. A 24-h treatment with clioquinol, zinc and copper (0.5, 0.5 and 0.1 μM) induced a significant upregulation of P-gp (1.7-fold) assessed by in-cell western and this was confirmed with western blotting (1.8-fold increase). This same treatment resulted in a 23% decrease in rhodamine-123 accumulation over a 1 h incubation. ICP-MS demonstrated that while t8his combination treatment had no effect on intracellular zinc concentrations, the treatment significantly enhanced bioavailable copper (4.6-fold). Enhanced delivery of copper to human brain microvascular endothelial cells is associated with enhanced expression and function of the important efflux pump P-gp, which may provide therapeutic opportunities for P-gp modulation.

P2Y12 expression and function in alternatively activated human microglia

PubMed Central

Ase, Ariel R.; Kinsara, Angham; Rao, Vijayaraghava T.S.; Michell-Robinson, Mackenzie; Leong, Soo Yuen; Butovsky, Oleg; Ludwin, Samuel K.; Séguéla, Philippe; Bar-Or, Amit; Antel, Jack P.

2015-01-01

Objective: To investigate and measure the functional significance of altered P2Y12 expression in the context of human microglia activation. Methods: We performed in vitro and in situ experiments to measure how P2Y12 expression can influence disease-relevant functional properties of classically activated (M1) and alternatively activated (M2) human microglia in the inflamed brain. Results: We demonstrated that compared to resting and classically activated (M1) human microglia, P2Y12 expression is increased under alternatively activated (M2) conditions. In response to ADP, the endogenous ligand of P2Y12, M2 microglia have increased ligand-mediated calcium responses, which are blocked by selective P2Y12 antagonism. P2Y12 antagonism was also shown to decrease migratory and inflammatory responses in human microglia upon exposure to nucleotides that are released during CNS injury; no effects were observed in human monocytes or macrophages. In situ experiments confirm that P2Y12 is selectively expressed on human microglia and elevated under neuropathologic conditions that promote Th2 responses, such as parasitic CNS infection. Conclusion: These findings provide insight into the roles of M2 microglia in the context of neuroinflammation and suggest a mechanism to selectively target a functionally unique population of myeloid cells in the CNS. PMID:25821842

Silencing hsp25/hsp27 gene expression augments proteasome activity and increases CD8+ T cell-mediated tumor killing and memory responses

PubMed Central

Nagaraja, Ganachari M.; Kaur, Punit; Neumann, William; Asea, Edwina E.; Bausero, María A.; Multhoff, Gabriele; Asea, Alexzander

2011-01-01

Relatively high expression of Hsp27 in breast and prostate cancer is a predictor of poor clinical outcome. This study elucidates a hitherto unknown mechanism by which Hsp27 regulates proteasome function and modulates tumor-specific T cell responses. Here we demonstrated that short term silencing of Hsp25 or Hsp27 using siRNA or permanent silencing of Hsp25 using lentivirus-RNAi technology enhanced PA28α mRNA expression, PA28α protein expression, proteasome activity, abrogated metastatic potential, induced the regression of established breast tumors by tumor-specific CD8+ T cells and stimulated long-lasting memory responses. The adoptive transfer of reactive CD8+ T cells from mice bearing Hsp25-silenced tumors efficiently induced the regression of established tumors in non-treated mice which normally succumb to tumor burden. The overexpression of Hsp25 and Hsp27 resulted in the repression of normal proteasome function, induced poor antigen presentation and resulted in increased tumor burden. Taken together, this study establishes a paradigm shift in our understanding of the role of Hsp27 in the regulation of proteasome function and tumor-specific T cell responses and paves the way for the development of molecular targets to enhance proteasome function and concomitantly inhibit Hsp27 expression in tumors for therapeutic gain. PMID:22185976

Hes repressors are essential regulators of hematopoietic stem cell development downstream of Notch signaling

PubMed Central

Guiu, Jordi; Shimizu, Ritsuko; D’Altri, Teresa; Fraser, Stuart T.; Hatakeyama, Jun; Bresnick, Emery H.; Kageyama, Ryoichiro; Dzierzak, Elaine; Yamamoto, Masayuki; Espinosa, Lluis

2013-01-01

Previous studies have identified Notch as a key regulator of hematopoietic stem cell (HSC) development, but the underlying downstream mechanisms remain unknown. The Notch target Hes1 is widely expressed in the aortic endothelium and hematopoietic clusters, though Hes1-deficient mice show no overt hematopoietic abnormalities. We now demonstrate that Hes is required for the development of HSC in the mouse embryo, a function previously undetected as the result of functional compensation by de novo expression of Hes5 in the aorta/gonad/mesonephros (AGM) region of Hes1 mutants. Analysis of embryos deficient for Hes1 and Hes5 reveals an intact arterial program with overproduction of nonfunctional hematopoietic precursors and total absence of HSC activity. These alterations were associated with increased expression of the hematopoietic regulators Runx1, c-myb, and the previously identified Notch target Gata2. By analyzing the Gata2 locus, we have identified functional RBPJ-binding sites, which mutation results in loss of Gata2 reporter expression in transgenic embryos, and functional Hes-binding sites, which mutation leads to specific Gata2 up-regulation in the hematopoietic precursors. Together, our findings show that Notch activation in the AGM triggers Gata2 and Hes1 transcription, and next HES-1 protein represses Gata2, creating an incoherent feed-forward loop required to restrict Gata2 expression in the emerging HSCs. PMID:23267012

An integrative approach to inferring biologically meaningful gene modules.

PubMed

Cho, Ji-Hoon; Wang, Kai; Galas, David J

2011-07-26

The ability to construct biologically meaningful gene networks and modules is critical for contemporary systems biology. Though recent studies have demonstrated the power of using gene modules to shed light on the functioning of complex biological systems, most modules in these networks have shown little association with meaningful biological function. We have devised a method which directly incorporates gene ontology (GO) annotation in construction of gene modules in order to gain better functional association. We have devised a method, Semantic Similarity-Integrated approach for Modularization (SSIM) that integrates various gene-gene pairwise similarity values, including information obtained from gene expression, protein-protein interactions and GO annotations, in the construction of modules using affinity propagation clustering. We demonstrated the performance of the proposed method using data from two complex biological responses: 1. the osmotic shock response in Saccharomyces cerevisiae, and 2. the prion-induced pathogenic mouse model. In comparison with two previously reported algorithms, modules identified by SSIM showed significantly stronger association with biological functions. The incorporation of semantic similarity based on GO annotation with gene expression and protein-protein interaction data can greatly enhance the functional relevance of inferred gene modules. In addition, the SSIM approach can also reveal the hierarchical structure of gene modules to gain a broader functional view of the biological system. Hence, the proposed method can facilitate comprehensive and in-depth analysis of high throughput experimental data at the gene network level.

Tongxinluo ameliorates renal structure and function by regulating miR-21-induced epithelial-to-mesenchymal transition in diabetic nephropathy.

PubMed

Wang, Jin-yang; Gao, Yan-bin; Zhang, Na; Zou, Da-wei; Xu, Li-ping; Zhu, Zhi-yao; Li, Jiao-yang; Zhou, Sheng-nan; Cui, Fang-qiang; Zeng, Xiang-jun; Geng, Jian-guo; Yang, Jin-kui

2014-03-01

Diabetic nephropathy (DN) is one of the most important diabetic microangiopathies. The epithelial-to-mesenchymal transition (EMT) plays an important role in DN. The physiological role of microRNA-21 (miR-21) was closely linked to EMT. However, it remained elusive whether tongxinluo (TXL) ameliorated renal structure and function by regulating miR-21-induced EMT in DN. This study aimed to determine the effect of TXL on miR-21-induced renal tubular EMT and to explore the relationship between miR-21 and TGF-β1/smads signals. Real-time RT-PCR, cell transfection, in situ hybridization (ISH), and laser confocal microscopy were used, respectively. Here, we revealed that TXL dose dependently lowered miR-21 expression in tissue, serum, and cells. Overexpression of miR-21 can enhance α-smooth muscle actin (SMA) expression and decrease E-cadherin expression by upregulating smad3/p-smad3 expression and downregulating smad7 expression. Interestingly, TXL also increased E-cadherin expression and decreased α-SMA expression by regulating miR-21 expression. More importantly, TXL decreased collagen IV, fibronectin, glomerular basement membrane, glomerular area, and the albumin/creatinine ratio, whereas it increased the creatinine clearance ratio. The results demonstrated that TXL ameliorated renal structure and function by regulating miR-21-induced EMT, which was one of the mechanisms to protect against DN, and that miR-21 may be one of the therapeutic targets for TXL in DN.

A versatile expression vector for the growth and amplification of unmodified phage display polypeptides.

PubMed

Winton, Alexander J; Baptiste, Janae L; Allen, Mark A

2018-09-01

Proteins and polypeptides represent nature's most complex and versatile polymer. They provide complicated shapes, diverse chemical functionalities, and tightly regulated and controlled sizes. Several disease states are related to the misfolding or overproduction of polypeptides and yet polypeptides are present in several therapeutic molecules. In addition to biological roles; short chain polypeptides have been shown to interact with and drive the bio-inspired synthesis or modification of inorganic materials. This paper outlines the development of a versatile cloning vector which allows for the expression of a short polypeptide by controlling the incorporation of a desired DNA coding insert. As a demonstration of the efficacy of the expression system, a solid binding polypeptide identified from M13 phage display was expressed and purified. The solid binding polypeptide was expressed as a soluble 6xHis-SUMO tagged construct. Expression was performed in E. coli using auto-induction followed by Ni-NTA affinity chromatography and ULP1 protease cleavage. Methodology demonstrates the production of greater than 8 mg of purified polypeptide per liter of E. coli culture. Isotopic labeling of the peptide is also demonstrated. The versatility of the designed cloning vector, use of the 6xHis-SUMO solubility partner, bacterial expression in auto-inducing media and the purification methodology make this expressionun vector a readily scalable and user-friendly system for the creation of desired peptide domains. Copyright © 2018. Published by Elsevier Inc.

Intron retention generates ANKRD1 splice variants that are co-regulated with the main transcript in normal and failing myocardium.

PubMed

Torrado, Mario; Iglesias, Raquel; Nespereira, Beatriz; Centeno, Alberto; López, Eduardo; Mikhailov, Alexander T

2009-07-01

The cardiac ankyrin repeat domain 1 protein (ANKRD1, also known as CARP) has been extensively characterized with regard to its proposed functions as a cardio-enriched transcriptional co-factor and stress-inducible myofibrillar protein. The present results show the occurrence of alternative splicing by intron retention events in the pig and human ankrd1 gene. In pig heart, ankrd1 is expressed as four alternatively spliced transcripts, three of which have non-excised introns: ankrd1-contained introns 6, 7 and 8 (i.e., ankrd1-i6,7,8), ankrd1-contained introns 7 and 8 (i.e., ankrd1-i7,8), and ankrd1 retained only intron 8 (i.e., ankrd1-i8). In the human heart, two orthologues of porcine intron-retaining ankrd1 variants (i.e., ankrd1-i8 and ankrd1-i7,8) are detected. We demonstrate that these newly-identified intron-retaining ankrd1 transcripts are functionally intact, efficiently translated into protein in vitro and exported to the cytoplasm in cardiomyocytes in vivo. In the piglet heart, both the intronless and intron-retaining ankrd1 mRNAs are co-expressed in a chamber-dependent manner being more abundant in the left as compared to the right myocardium. Our data further indicate co-upregulation of the ankrd1 spliced variants in myocardium in the porcine model of diastolic heart failure. Most significantly, we demonstrate that in vivo forced expression of recombinant intronless ankrd1 markedly increases the levels of intron-retaining ankrd1 variants (but not of the endogenous main transcript) in piglet myocardium, suggesting that ANKRD1 may positively regulate the expression of its own intron-containing RNAs in response to cardiac stress. Overall, our findings demonstrate that in cardiomyocytes ANKRD1 can exist in multiple isoforms which may contribute to the functional diversity of this factor in heart development and disease.

Ubiquitination by the Membrane-associated RING-CH-8 (MARCH-8) Ligase Controls Steady-state Cell Surface Expression of Tumor Necrosis Factor-related Apoptosis Inducing Ligand (TRAIL) Receptor 1*

PubMed Central

van de Kooij, Bert; Verbrugge, Inge; de Vries, Evert; Gijsen, Merel; Montserrat, Veronica; Maas, Chiel; Neefjes, Jacques; Borst, Jannie

2013-01-01

The eleven members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family are relatively unexplored. Upon exogenous (over)expression, a number of these ligases can affect the trafficking of membrane molecules. However, only for MARCH-1 endogenous functions have been demonstrated. For the other endogenous MARCH proteins, no functions or substrates are known. We report here that TRAIL-R1 is a physiological substrate of the endogenous MARCH-8 ligase. Human TRAIL-R1 and R2 play a role in immunosurveillance and are targets for cancer therapy, because they selectively induce apoptosis in tumor cells. We demonstrate that TRAIL-R1 is down-regulated from the cell surface, with great preference over TRAIL-R2, by exogenous expression of MARCH ligases that are implicated in endosomal trafficking, such as MARCH-1 and -8. MARCH-8 attenuated TRAIL-R1 cell surface expression and apoptosis signaling by virtue of its ligase activity. This suggested that ubiquitination of TRAIL-R1 was instrumental in its down-regulation by MARCH-8. Indeed, in cells with endogenous MARCH expression, TRAIL-R1 was ubiquitinated at steady-state, with the conserved membrane-proximal lysine 273 as one of the potential acceptor sites. This residue was also essential for the interaction of TRAIL-R1 with MARCH-1 and MARCH-8 and its down-regulation by these ligases. Gene silencing identified MARCH-8 as the endogenous ligase that ubiquitinates TRAIL-R1 and attenuates its cell surface expression. These findings reveal that endogenous MARCH-8 regulates the steady-state cell surface expression of TRAIL-R1. PMID:23300075

Efficient -2 frameshifting by mammalian ribosomes to synthesize an additional arterivirus protein.

PubMed

Fang, Ying; Treffers, Emmely E; Li, Yanhua; Tas, Ali; Sun, Zhi; van der Meer, Yvonne; de Ru, Arnoud H; van Veelen, Peter A; Atkins, John F; Snijder, Eric J; Firth, Andrew E

2012-10-23

Programmed -1 ribosomal frameshifting (-1 PRF) is a gene-expression mechanism used to express many viral and some cellular genes. In contrast, efficient natural utilization of -2 PRF has not been demonstrated previously in eukaryotic systems. Like all nidoviruses, members of the Arteriviridae (a family of positive-stranded RNA viruses) express their replicase polyproteins pp1a and pp1ab from two long ORFs (1a and 1b), where synthesis of pp1ab depends on -1 PRF. These polyproteins are posttranslationally cleaved into at least 13 functional nonstructural proteins. Here we report that porcine reproductive and respiratory syndrome virus (PRRSV), and apparently most other arteriviruses, use an additional PRF mechanism to access a conserved alternative ORF that overlaps the nsp2-encoding region of ORF1a in the +1 frame. We show here that this ORF is translated via -2 PRF at a conserved G_GUU_UUU sequence (underscores separate ORF1a codons) at an estimated efficiency of around 20%, yielding a transframe fusion (nsp2TF) with the N-terminal two thirds of nsp2. Expression of nsp2TF in PRRSV-infected cells was verified using specific Abs, and the site and direction of frameshifting were determined via mass spectrometric analysis of nsp2TF. Further, mutagenesis showed that the frameshift site and an unusual frameshift-stimulatory element (a conserved CCCANCUCC motif 11 nucleotides downstream) are required to direct efficient -2 PRF. Mutations preventing nsp2TF expression impair PRRSV replication and produce a small-plaque phenotype. Our findings demonstrate that -2 PRF is a functional gene-expression mechanism in eukaryotes and add another layer to the complexity of arterivirus genome expression.

Vitamin K2 improves proliferation and migration of bovine skeletal muscle cells in vitro.

PubMed

Rønning, Sissel Beate; Pedersen, Mona Elisabeth; Berg, Ragnhild Stenberg; Kirkhus, Bente; Rødbotten, Rune

2018-01-01

Skeletal muscle function is highly dependent on the ability to regenerate, however, during ageing or disease, the proliferative capacity is reduced, leading to loss of muscle function. We have previously demonstrated the presence of vitamin K2 in bovine skeletal muscles, but whether vitamin K has a role in muscle regulation and function is unknown. In this study, we used primary bovine skeletal muscle cells, cultured in monolayers in vitro, to assess a potential effect of vitamin K2 (MK-4) during myogenesis of muscle cells. Cell viability experiments demonstrate that the amount of ATP produced by the cells was unchanged when MK-4 was added, indicating viable cells. Cytotoxicity analysis show that MK-4 reduced the lactate dehydrogenase (LDH) released into the media, suggesting that MK-4 was beneficial to the muscle cells. Cell migration, proliferation and differentiation was characterised after MK-4 incubation using wound scratch analysis, immunocytochemistry and real-time PCR analysis. Adding MK-4 to the cells led to an increased muscle proliferation, increased gene expression of the myogenic transcription factor myod as well as increased cell migration. In addition, we observed a reduction in the fusion index and relative gene expression of muscle differentiation markers, with fewer complex myotubes formed in MK-4 stimulated cells compared to control cells, indicating that the MK-4 plays a significant role during the early phases of muscle proliferation. Likewise, we see the same pattern for the relative gene expression of collagen 1A, showing increased gene expression in proliferating cells, and reduced expression in differentiating cells. Our results also suggest that MK-4 incubation affect low density lipoprotein receptor-related protein 1 (LRP1) and the low-density lipoprotein receptor (LDLR) with a peak in gene expression after 45 min of MK-4 incubation. Altogether, our experiments show that MK-4 has a positive effect on muscle cell migration and proliferation, which are two important steps during early myogenesis.

Expression of the Hsp23 chaperone during Drosophila embryogenesis: association to distinct neural and glial lineages

PubMed Central

Michaud, Sébastien; Tanguay, Robert M

2003-01-01

Background In addition to their strong induction following stress, small heat shock proteins (Hsp) are also expressed during development in a wide variety of organisms. However, the precise identity of cell(s) expressing these proteins and the functional contribution of small heat shock proteins in such developmental context remain to be determined. The present study provides a detailed description of the Drosophila small heat shock protein Hsp23 expression pattern during embryogenesis and evaluates its functional contribution to central nervous system development. Results Throughout embryogenesis, Hsp23 is expressed in a stage-specific manner by a restricted number of neuronal and glial lineages of the central nervous system. Hsp23 is also detected in the amnioserosa and within a single lateral chordotonal organ. Its expression within the MP2 lineage does not require the presence of a functional midline nor the activity of the Notch signaling pathway. Transactivation assays demonstrate that transcription factors implicated in the differentiation of the midline also regulate hsp23 promoter activity. Phenotypic analysis of a transgenic line exhibiting loss of Hsp23 expression in the central nervous system suggests that Hsp23 is not required for development and function of this tissue. Likewise, its overexpression does not cause deleterious effects, as development remains unaffected. Conclusions Based on the presented data, we suggest that the tightly regulated developmental expression of Hsp23 is not actively involved in cell differentiation and central nervous system development per se but rather reflects a putative role in preventive "pre-stress" neuroprotection or in non-vital process(es) common to the identified cell lineages. PMID:14617383

The cotton transcription factor TCP14 functions in auxin-mediated epidermal cell differentiation and elongation.

PubMed

Wang, Miao-Ying; Zhao, Pi-Ming; Cheng, Huan-Qing; Han, Li-Bo; Wu, Xiao-Min; Gao, Peng; Wang, Hai-Yun; Yang, Chun-Lin; Zhong, Nai-Qin; Zuo, Jian-Ru; Xia, Gui-Xian

2013-07-01

Plant-specific TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors play crucial roles in development, but their functional mechanisms remain largely unknown. Here, we characterized the cellular functions of the class I TCP transcription factor GhTCP14 from upland cotton (Gossypium hirsutum). GhTCP14 is expressed predominantly in fiber cells, especially at the initiation and elongation stages of development, and its expression increased in response to exogenous auxin. Induced heterologous overexpression of GhTCP14 in Arabidopsis (Arabidopsis thaliana) enhanced initiation and elongation of trichomes and root hairs. In addition, root gravitropism was severely affected, similar to mutant of the auxin efflux carrier PIN-FORMED2 (PIN2) gene. Examination of auxin distribution in GhTCP14-expressing Arabidopsis by observation of auxin-responsive reporters revealed substantial alterations in auxin distribution in sepal trichomes and root cortical regions. Consistent with these changes, expression of the auxin uptake carrier AUXIN1 (AUX1) was up-regulated and PIN2 expression was down-regulated in the GhTCP14-expressing plants. The association of GhTCP14 with auxin responses was also evidenced by the enhanced expression of auxin response gene IAA3, a gene in the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) family. Electrophoretic mobility shift assays showed that GhTCP14 bound the promoters of PIN2, IAA3, and AUX1, and transactivation assays indicated that GhTCP14 had transcription activation activity. Taken together, these results demonstrate that GhTCP14 is a dual-function transcription factor able to positively or negatively regulate expression of auxin response and transporter genes, thus potentially acting as a crucial regulator in auxin-mediated differentiation and elongation of cotton fiber cells.

The Cotton Transcription Factor TCP14 Functions in Auxin-Mediated Epidermal Cell Differentiation and Elongation1[C][W

PubMed Central

Wang, Miao-Ying; Zhao, Pi-Ming; Cheng, Huan-Qing; Han, Li-Bo; Wu, Xiao-Min; Gao, Peng; Wang, Hai-Yun; Yang, Chun-Lin; Zhong, Nai-Qin; Zuo, Jian-Ru; Xia, Gui-Xian

2013-01-01

Plant-specific TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors play crucial roles in development, but their functional mechanisms remain largely unknown. Here, we characterized the cellular functions of the class I TCP transcription factor GhTCP14 from upland cotton (Gossypium hirsutum). GhTCP14 is expressed predominantly in fiber cells, especially at the initiation and elongation stages of development, and its expression increased in response to exogenous auxin. Induced heterologous overexpression of GhTCP14 in Arabidopsis (Arabidopsis thaliana) enhanced initiation and elongation of trichomes and root hairs. In addition, root gravitropism was severely affected, similar to mutant of the auxin efflux carrier PIN-FORMED2 (PIN2) gene. Examination of auxin distribution in GhTCP14-expressing Arabidopsis by observation of auxin-responsive reporters revealed substantial alterations in auxin distribution in sepal trichomes and root cortical regions. Consistent with these changes, expression of the auxin uptake carrier AUXIN1 (AUX1) was up-regulated and PIN2 expression was down-regulated in the GhTCP14-expressing plants. The association of GhTCP14 with auxin responses was also evidenced by the enhanced expression of auxin response gene IAA3, a gene in the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) family. Electrophoretic mobility shift assays showed that GhTCP14 bound the promoters of PIN2, IAA3, and AUX1, and transactivation assays indicated that GhTCP14 had transcription activation activity. Taken together, these results demonstrate that GhTCP14 is a dual-function transcription factor able to positively or negatively regulate expression of auxin response and transporter genes, thus potentially acting as a crucial regulator in auxin-mediated differentiation and elongation of cotton fiber cells. PMID:23715527

Human Urine Decreases Function and Expression of Type 1 Pili in Uropathogenic Escherichia coli

PubMed Central

Greene, Sarah E.; Hibbing, Michael E.; Janetka, James; Chen, Swaine L.

2015-01-01

ABSTRACT Uropathogenic Escherichia coli (UPEC) is the primary cause of community-acquired urinary tract infections (UTIs). UPEC bind the bladder using type 1 pili, encoded by the fim operon in nearly all E. coli. Assembled type 1 pili terminate in the FimH adhesin, which specifically binds to mannosylated glycoproteins on the bladder epithelium. Expression of type 1 pili is regulated in part by phase-variable inversion of the genomic element containing the fimS promoter, resulting in phase ON (expressing) and OFF (nonexpressing) orientations. Type 1 pili are essential for virulence in murine models of UTI; however, studies of urine samples from human UTI patients demonstrate variable expression of type 1 pili. We provide insight into this paradox by showing that human urine specifically inhibits both expression and function of type 1 pili. Growth in urine induces the fimS phase OFF orientation, preventing fim expression. Urine also contains inhibitors of FimH function, and this inhibition leads to a further bias in fimS orientation toward the phase OFF state. The dual effect of urine on fimS regulation and FimH binding presents a potential barrier to type 1 pilus-mediated colonization and invasion of the bladder epithelium. However, FimH-mediated attachment to human bladder cells during growth in urine reverses these effects such that fim expression remains ON and/or turns ON. Interestingly, FimH inhibitors called mannosides also induce the fimS phase OFF orientation. Thus, the transduction of FimH protein attachment or inhibition into epigenetic regulation of type 1 pilus expression has important implications for the development of therapeutics targeting FimH function. PMID:26126855

HIF-1α activates hypoxia-induced BCL-9 expression in human colorectal cancer cells

PubMed Central

Chen, Tian-Rui; Wei, Hai-feng; Song, Dian-Wen; Liu, Tie-Long; Yang, Xing-Hai; Fu, Chuan-Gang; Hu, Zhi-qian; Zhou, Wang; Yan, Wang-Jun; Xiao, Jian-Ru

2017-01-01

B-cell CLL/lymphoma 9 protein (BCL-9), a multi-functional co-factor in Wnt signaling, induced carcinogenesis as well as promoting tumor progression, metastasis and chemo-resistance in colorectal cancer (CRC). However, the mechanisms for increased BCL-9 expression in CRC were not well understood. Here, we report that hypoxia, a hallmark of solid tumors, induced BCL-9 mRNA expression in human CRC cells. Analysis of BCL-9 promoter revealed two functional hypoxia-responsive elements (HRE-B and HRE-C) that can be specifically bound with and be transactivated by hypoxia inducible factors (HIF) -1α but not HIF-2α. Consistently, ectopic expression of HIF-1α but not HIF-2α transcriptionally induced BCL-9 expression levels in cells. Knockdown of endogenous HIF-1α but not HIF-2α by siRNA largely abolished the induction of HIF by hypoxia. Furthermore, there was a strong association of HIF-1α expression with BCL-9 expression in human CRC specimens. In summary, results from this study demonstrated that hypoxia induced BCL-9 expression in human CRC cells mainly through HIF-1α, which could be an important underlying mechanism for increased BCL-9 expression in CRC. PMID:27121066

G protein-coupled receptor kinase-2 (GRK-2) regulates serotonin metabolism through the monoamine oxidase AMX-2 in Caenorhabditis elegans.

PubMed

Wang, Jianjun; Luo, Jiansong; Aryal, Dipendra K; Wetsel, William C; Nass, Richard; Benovic, Jeffrey L

2017-04-07

G protein-coupled receptors (GPCRs) regulate many animal behaviors. GPCR signaling is mediated by agonist-promoted interactions of GPCRs with heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. To further elucidate the role of GRKs in regulating GPCR-mediated behaviors, we utilized the genetic model system Caenorhabditis elegans Our studies demonstrate that grk-2 loss-of-function strains are egg laying-defective and contain low levels of serotonin (5-HT) and high levels of the 5-HT metabolite 5-hydroxyindole acetic acid (5-HIAA). The egg laying defect could be rescued by the expression of wild type but not by catalytically inactive grk-2 or by the selective expression of grk-2 in hermaphrodite-specific neurons. The addition of 5-HT or inhibition of 5-HT metabolism also rescued the egg laying defect. Furthermore, we demonstrate that AMX-2 is the primary monoamine oxidase that metabolizes 5-HT in C. elegans , and we also found that grk-2 loss-of-function strains have abnormally high levels of AMX-2 compared with wild-type nematodes. Interestingly, GRK-2 was also found to interact with and promote the phosphorylation of AMX-2. Additional studies reveal that 5-HIAA functions to inhibit egg laying in a manner dependent on the 5-HT receptor SER-1 and the G protein GOA-1. These results demonstrate that GRK-2 modulates 5-HT metabolism by regulating AMX-2 function and that 5-HIAA may function in the SER-1 signaling pathway. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Sumoylation of FOXP2 regulates motor function and vocal communication through Purkinje cell development

PubMed Central

Usui, Noriyoshi; Co, Marissa; Harper, Matthew; Rieger, Michael A.; Dougherty, Joseph D.; Konopka, Genevieve

2016-01-01

Background Mutations in the gene encoding the transcription factor forkhead box P2, FOXP2, result in brain developmental abnormalities including reduced gray matter in both human patients and rodent models, and speech and language deficits. However, neither the region-specific function of FOXP2 in the brain, in particular the cerebellum, nor the effects of any post-translational modifications of FOXP2 in the brain and disorders have been explored. Methods We characterized sumoylation of FOXP2 biochemically, and analyzed the region-specific function and sumoylation of FOXP2 in the developing mouse cerebellum. Using in utero electroporation to manipulate the sumoylation-state of Foxp2 as well as Foxp2 expression levels in Purkinje cells (PCs) of the cerebellum in vivo, we reduced Foxp2 expression approximately 40% in the mouse cerebellum. Such a reduction approximates the haploinsufficiency observed in human patients who demonstrate speech and language impairments. Results We identified sumoylation of FOXP2 at K674 (K673 in mouse) in the cerebellum of neonates. In vitro co-immunoprecipitation and in vivo colocalization experiments suggest that PIAS3 acts as the SUMO E3 ligase for FOXP2 sumoylation. This sumoylation modifies transcriptional regulation by FOXP2. We demonstrate that Foxp2 sumoylation is required for regulation of cerebellar motor function and vocal communication, likely through dendritic outgrowth and arborization of PCs in the mouse cerebellum. Conclusions Sumoylation of Foxp2 in neonatal mouse cerebellum regulates PC development as well as motor functions and vocal communication, demonstrating evidence for sumoylation in regulating mammalian behaviors. PMID:27009683

Selection based on CD133 and high aldehyde dehydrogenase activity isolates long-term reconstituting human hematopoietic stem cells

PubMed Central

Hess, David A.; Wirthlin, Louisa; Craft, Timothy P.; Herrbrich, Phillip E.; Hohm, Sarah A.; Lahey, Ryan; Eades, William C.; Creer, Michael H.; Nolta, Jan A.

2006-01-01

The development of novel cell-based therapies requires understanding of distinct human hematopoietic stem and progenitor cell populations. We recently isolated reconstituting hematopoietic stem cells (HSCs) by lineage depletion and purification based on high aldehyde dehydrogenase activity (ALDHhiLin- cells). Here, we further dissected the ALDHhi-Lin- population by selection for CD133, a surface molecule expressed on progenitors from hematopoietic, endothelial, and neural lineages. ALDHhiCD133+Lin- cells were primarily CD34+, but also included CD34-CD38-CD133+ cells, a phenotype previously associated with repopulating function. Both ALDHhiCD133-Lin- and ALDHhiCD133+Lin- cells demonstrated distinct clonogenic progenitor function in vitro, whereas only the ALDHhiCD133+Lin- population seeded the murine bone marrow 48 hours after transplantation. Significant human cell repopulation was observed only in NOD/SCID and NOD/SCID β2M-null mice that received transplants of ALDHhiCD133+Lin- cells. Limiting dilution analysis demonstrated a 10-fold increase in the frequency of NOD/SCID repopulating cells compared with CD133+Lin- cells, suggesting that high ALDH activity further purified cells with repopulating function. Transplanted ALDHhiCD133+Lin- cells also maintained primitive hematopoietic phenotypes (CD34+CD38-) and demonstrated enhanced repopulating function in recipients of serial, secondary transplants. Cell selection based on ALDH activity and CD133 expression provides a novel purification of HSCs with long-term repopulating function and may be considered an alternative to CD34 cell selection for stem cell therapies. PMID:16269619

G protein-coupled receptor kinase-2 (GRK-2) regulates serotonin metabolism through the monoamine oxidase AMX-2 in Caenorhabditis elegans

PubMed Central

Wang, Jianjun; Luo, Jiansong; Aryal, Dipendra K.; Wetsel, William C.; Nass, Richard; Benovic, Jeffrey L.

2017-01-01

G protein-coupled receptors (GPCRs) regulate many animal behaviors. GPCR signaling is mediated by agonist-promoted interactions of GPCRs with heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. To further elucidate the role of GRKs in regulating GPCR-mediated behaviors, we utilized the genetic model system Caenorhabditis elegans. Our studies demonstrate that grk-2 loss-of-function strains are egg laying-defective and contain low levels of serotonin (5-HT) and high levels of the 5-HT metabolite 5-hydroxyindole acetic acid (5-HIAA). The egg laying defect could be rescued by the expression of wild type but not by catalytically inactive grk-2 or by the selective expression of grk-2 in hermaphrodite-specific neurons. The addition of 5-HT or inhibition of 5-HT metabolism also rescued the egg laying defect. Furthermore, we demonstrate that AMX-2 is the primary monoamine oxidase that metabolizes 5-HT in C. elegans, and we also found that grk-2 loss-of-function strains have abnormally high levels of AMX-2 compared with wild-type nematodes. Interestingly, GRK-2 was also found to interact with and promote the phosphorylation of AMX-2. Additional studies reveal that 5-HIAA functions to inhibit egg laying in a manner dependent on the 5-HT receptor SER-1 and the G protein GOA-1. These results demonstrate that GRK-2 modulates 5-HT metabolism by regulating AMX-2 function and that 5-HIAA may function in the SER-1 signaling pathway. PMID:28213524

Adult Restoration of Shank3 Expression Rescues Selective Autistic-Like Phenotypes

PubMed Central

Mei, Yuan; Monteiro, Patricia; Zhou, Yang; Kim, Jin-Ah; Gao, Xian; Fu, Zhanyan; Feng, Guoping

2016-01-01

Because ASD is a neurodevelopmental disorder and patients typically display symptoms before the age of three1, one of the key questions in autism research is whether the pathology is reversible in adults. Here we investigated the developmental requirement of Shank3, one of the most prominent monogenic ASD genes that is estimated to contribute to ~1% of all ASD cases2–6. SHANK3 is a postsynaptic scaffold protein that regulates synaptic development, function and plasticity by orchestrating the assembly of postsynaptic density (PSD) macromolecular signaling complex7–9. Disruptions of the Shank3 gene in mouse models have resulted in synaptic defects and autistic-like behaviors including anxiety, social interaction deficits, and repetitive behavior10–13. We generated a novel Shank3 conditional knock-in mouse model and used it to demonstrate that re-expression of the Shank3 gene in adult led to improvements in synaptic protein composition, spine density and neural function in the striatum. We also provided behavioral evidence that certain behavioral abnormalities including social interaction deficit and repetitive grooming behavior could be rescued, while anxiety and motor coordination deficit could not be recovered in adulthood. Together, these results elucidate the profound impact of post-developmental activation of Shank3 expression on neural function and demonstrate certain degree of continued plasticity in the adult diseased brain. PMID:26886798

Virus-induced dysfunction of CD4+CD25+ T cells in patients with HTLV-I-associated neuroimmunological disease.

PubMed

Yamano, Yoshihisa; Takenouchi, Norihiro; Li, Hong-Chuan; Tomaru, Utano; Yao, Karen; Grant, Christian W; Maric, Dragan A; Jacobson, Steven

2005-05-01

CD4(+)CD25(+) Tregs are important in the maintenance of immunological self tolerance and in the prevention of autoimmune diseases. As the CD4(+)CD25(+) T cell population in patients with human T cell lymphotropic virus type I-associated (HTLV-I-associated) myelopathy/tropical spastic paraparesis (HAM/TSP) has been shown to be a major reservoir for this virus, it was of interest to determine whether the frequency and function of CD4(+)CD25(+) Tregs in HAM/TSP patients might be affected. In these cells, both mRNA and protein expression of the forkhead transcription factor Foxp3, a specific marker of Tregs, were lower than those in CD4(+)CD25(+) T cells from healthy individuals. The virus-encoded transactivating HTLV-I tax gene was demonstrated to have a direct inhibitory effect on Foxp3 expression and function of CD4(+)CD25(+) T cells. This is the first report to our knowledge demonstrating the role of a specific viral gene product (HTLV-I Tax) on the expression of genes associated with Tregs (in particular, foxp3) resulting in inhibition of Treg function. These results suggest that direct human retroviral infection of CD4(+)CD25(+) T cells may be associated with the pathogenesis of HTLV-I-associated neurologic disease.

Hyaluronan 35kDa treatment protects mice from Citrobacter rodentium infection and induces epithelial tight junction protein ZO-1 in vivo.

PubMed

Kim, Yeojung; Kessler, Sean P; Obery, Dana R; Homer, Craig R; McDonald, Christine; de la Motte, Carol A

2017-10-01

Maintaining a healthy intestinal barrier, the primary physical barrier between intestinal microbiota and the underlying lamina propria, is critical for optimal health. Epithelial integrity is essential for the prevention of the entrance of luminal contents, such as bacteria and their products, through the large intestinal barrier. In this study, we investigated the protective functions of biosynthetic, specific sized, hyaluronan around 35kDa (HA35) on intestinal epithelium in healthy mice, as well as mice infected Citrobacter rodentium, an established model that mimics infection with a serious human pathogen, enteropathogenic E. coli (EPEC). Our results reveal that treatment with HA35 protects mice from Citrobacter infection and enhances the epithelial barrier function. In particular, we have found that HA35 induces the expression of tight junction protein zonula occludens (ZO)-1 in both healthy and Citrobacter infected mice, as demonstrated by immunoflurorescence and Western blot analyses. Furthermore, we determined that HA35 treatment enhances ZO-1 expression and reduces intestinal permeability at the early stages of dextran sulfate sodium (DSS)-induced colitis in mice. Together, our data demonstrate that the expression and functionality of tight junctions, are increased by HA35 treatment, suggesting a novel mechanism for the protection from Citrobacter infection. Copyright © 2016 Elsevier B.V. All rights reserved.

Sox5 Functions as a Fate Switch in Medaka Pigment Cell Development

PubMed Central

Nagao, Yusuke; Suzuki, Takao; Shimizu, Atsushi; Kimura, Tetsuaki; Seki, Ryoko; Adachi, Tomoko; Inoue, Chikako; Omae, Yoshihiro; Kamei, Yasuhiro; Hara, Ikuyo; Taniguchi, Yoshihito; Naruse, Kiyoshi; Wakamatsu, Yuko; Kelsh, Robert N.; Hibi, Masahiko; Hashimoto, Hisashi

2014-01-01

Mechanisms generating diverse cell types from multipotent progenitors are crucial for normal development. Neural crest cells (NCCs) are multipotent stem cells that give rise to numerous cell-types, including pigment cells. Medaka has four types of NCC-derived pigment cells (xanthophores, leucophores, melanophores and iridophores), making medaka pigment cell development an excellent model for studying the mechanisms controlling specification of distinct cell types from a multipotent progenitor. Medaka many leucophores-3 (ml-3) mutant embryos exhibit a unique phenotype characterized by excessive formation of leucophores and absence of xanthophores. We show that ml-3 encodes sox5, which is expressed in premigratory NCCs and differentiating xanthophores. Cell transplantation studies reveal a cell-autonomous role of sox5 in the xanthophore lineage. pax7a is expressed in NCCs and required for both xanthophore and leucophore lineages; we demonstrate that Sox5 functions downstream of Pax7a. We propose a model in which multipotent NCCs first give rise to pax7a-positive partially fate-restricted intermediate progenitors for xanthophores and leucophores; some of these progenitors then express sox5, and as a result of Sox5 action develop into xanthophores. Our results provide the first demonstration that Sox5 can function as a molecular switch driving specification of a specific cell-fate (xanthophore) from a partially-restricted, but still multipotent, progenitor (the shared xanthophore-leucophore progenitor). PMID:24699463

Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system.

PubMed

Lee, Young Je; Hoynes-O'Connor, Allison; Leong, Matthew C; Moon, Tae Seok

2016-03-18

A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA-asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

The expression of asparaginyl endopeptidase promotes growth potential in epithelial ovarian cancer.

PubMed

Zhu, Qinyi; Tang, Meiling; Wang, Xipeng

2017-04-03

Epithelial ovarian cancer (EOC) is the most common and lethal cancer-related death among females in the world. Asparaginyl endopeptidase (AEP) is a member of C13 family peptidases and expressed in the extracellular matrix and tumor cells. The aim of this article is to explore the function of asparaginyl endopeptidase in epithelial ovarian cancer. The expression of AEP was examined in 20 EOC samples, 3 EOC metastasis samples, 6 fallopian tube metastasis samples, 4 peritoneum metastasis samples and 20 benign ovarian tumor samples by immunohistochemistry. The expression of AEP was also evaluated in serum and ascites of EOC patients by elisa. And we used a lentiviral vector to overexpress AEP in human epithelial ovarian cancer cell lines SKOV3ip and detected the function of AEP-SKOV3ip cells both in vitro and in vivo. The growth of AEP-SKOV3ip cells was observed by MTT, migration and tube formation assays in vitro. Additionally, the subcutaneous mice model was used to identify the tumor growth and metastasis in vivo. Mice tumors were stained for CD31 to determine the microvessel density (MVD). We demonstrated that AEP was highly expressed in the EOC patient tissues and ascites. The AEP transfected SKOV3ip cells could both promote tumor growth in vitro and in vivo. The MVD in AEP-SKOV3ip group was higher than that in NC-SKOV3ip group. Therefore, our results demonstrated that AEP could induce EOC growth and progressionboth in vitro and in vivo.

Gene Expression Profile Change and Associated Physiological and Pathological Effects in Mouse Liver Induced by Fasting and Refeeding

PubMed Central

Zhang, Fang; Xu, Xiang; Zhou, Ben; He, Zhishui; Zhai, Qiwei

2011-01-01

Food availability regulates basal metabolism and progression of many diseases, and liver plays an important role in these processes. The effects of food availability on digital gene expression profile, physiological and pathological functions in liver are yet to be further elucidated. In this study, we applied high-throughput sequencing technology to detect digital gene expression profile of mouse liver in fed, fasted and refed states. Totally 12162 genes were detected, and 2305 genes were significantly regulated by food availability. Biological process and pathway analysis showed that fasting mainly affected lipid and carboxylic acid metabolic processes in liver. Moreover, the genes regulated by fasting and refeeding in liver were mainly enriched in lipid metabolic process or fatty acid metabolism. Network analysis demonstrated that fasting mainly regulated Drug Metabolism, Small Molecule Biochemistry and Endocrine System Development and Function, and the networks including Lipid Metabolism, Small Molecule Biochemistry and Gene Expression were affected by refeeding. In addition, FunDo analysis showed that liver cancer and diabetes mellitus were most likely to be affected by food availability. This study provides the digital gene expression profile of mouse liver regulated by food availability, and demonstrates the main biological processes, pathways, gene networks and potential hepatic diseases regulated by fasting and refeeding. These results show that food availability mainly regulates hepatic lipid metabolism and is highly correlated with liver-related diseases including liver cancer and diabetes. PMID:22096593

Effects of Freeze-Thawing and Intravenous Infusion on Mesenchymal Stromal Cell Gene Expression.

PubMed

Hoogduijn, Martin J; de Witte, Samantha F H; Luk, Franka; van den Hout-van Vroonhoven, Mirjam C G N; Ignatowicz, Lech; Catar, Rusan; Strini, Tanja; Korevaar, Sander S; van IJcken, Wilfred F J; Betjes, Michiel G H; Franquesa, Marcella; Moll, Guido; Baan, Carla C

2016-04-15

Mesenchymal stromal cells (MSC) are increasingly used as an investigative therapeutic product for immune disorders and degenerative disease. Typically, MSC are isolated from human tissue, expanded in culture, and cryopreserved until usage. The safety and efficacy of MSC therapy will depend on the phenotypical and functional characteristics of MSC. The freeze-thawing procedure may change these characteristics. Furthermore, the cells encounter a microenvironment after administration that may impact their properties. It has been demonstrated that the majority of MSC localize to the lungs after intravenous infusion, making this the site to study the effects of the in vivo milieu on administered MSC. In this study, we investigated the effect of freeze-thawing and the mouse lung microenvironment on human adipose tissue-derived MSC. There were effects of freeze-thawing on the whole genome expression profile of MSC, although the effects did not exceed interdonor differences. There were no major changes in the expression of hemostatic regulators on transcriptional level, but significantly increased expression of procoagulant tissue factor on the surface of thawed adipose MSC, correlating with increased procoagulant activity of thawed cells. Exposure for 2 h to the lung microenvironment had a major effect on MSC gene expression and affected several immunological pathways. This indicates that MSC undergo functional changes shortly after infusion and this may influence the efficacy of MSC to modulate inflammatory responses. The results of this study demonstrate that MSC rapidly alter in response to the local milieu and disease-specific conditions may shape MSC after administration.

Lectin-Like Oxidized LDL Receptor-1 Is an Enhancer of Tumor Angiogenesis in Human Prostate Cancer Cells

PubMed Central

González-Chavarría, Iván; Cerro, Rita P.; Parra, Natalie P.; Sandoval, Felipe A.; Zuñiga, Felipe A.; Omazábal, Valeska A.; Lamperti, Liliana I.; Jiménez, Silvana P.; Fernandez, Edelmira A.; Gutiérrez, Nicolas A.; Rodriguez, Federico S.; Onate, Sergio A.; Sánchez, Oliberto; Vera, Juan C.; Toledo, Jorge R.

2014-01-01

Altered expression and function of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) has been associated with several diseases such as endothelial dysfunction, atherosclerosis and obesity. In these pathologies, oxLDL/LOX-1 activates signaling pathways that promote cell proliferation, cell motility and angiogenesis. Recent studies have indicated that olr1 mRNA is over-expressed in stage III and IV of human prostatic adenocarcinomas. However, the function of LOX-1 in prostate cancer angiogenesis remains to be determined. Our aim was to analyze the contribution of oxLDL and LOX-1 to tumor angiogenesis using C4-2 prostate cancer cells. We analyzed the expression of pro-angiogenic molecules and angiogenesis on prostate cancer tumor xenografts, using prostate cancer cell models with overexpression or knockdown of LOX-1 receptor. Our results demonstrate that the activation of LOX-1 using oxLDL increases cell proliferation, and the expression of the pro-angiogenic molecules VEGF, MMP-2, and MMP-9 in a dose-dependent manner. Noticeably, these effects were prevented in the C4-2 prostate cancer model when LOX-1 expression was knocked down. The angiogenic effect of LOX-1 activated with oxLDL was further demonstrated using the aortic ring assay and the xenograft model of tumor growth on chorioallantoic membrane of chicken embryos. Consequently, we propose that LOX-1 activation by oxLDL is an important event that enhances tumor angiogenesis in human prostate cancer cells. PMID:25170920

Gene expression profile change and associated physiological and pathological effects in mouse liver induced by fasting and refeeding.

PubMed

Zhang, Fang; Xu, Xiang; Zhou, Ben; He, Zhishui; Zhai, Qiwei

2011-01-01

Food availability regulates basal metabolism and progression of many diseases, and liver plays an important role in these processes. The effects of food availability on digital gene expression profile, physiological and pathological functions in liver are yet to be further elucidated. In this study, we applied high-throughput sequencing technology to detect digital gene expression profile of mouse liver in fed, fasted and refed states. Totally 12162 genes were detected, and 2305 genes were significantly regulated by food availability. Biological process and pathway analysis showed that fasting mainly affected lipid and carboxylic acid metabolic processes in liver. Moreover, the genes regulated by fasting and refeeding in liver were mainly enriched in lipid metabolic process or fatty acid metabolism. Network analysis demonstrated that fasting mainly regulated Drug Metabolism, Small Molecule Biochemistry and Endocrine System Development and Function, and the networks including Lipid Metabolism, Small Molecule Biochemistry and Gene Expression were affected by refeeding. In addition, FunDo analysis showed that liver cancer and diabetes mellitus were most likely to be affected by food availability. This study provides the digital gene expression profile of mouse liver regulated by food availability, and demonstrates the main biological processes, pathways, gene networks and potential hepatic diseases regulated by fasting and refeeding. These results show that food availability mainly regulates hepatic lipid metabolism and is highly correlated with liver-related diseases including liver cancer and diabetes.

Evaluating the consistency of gene sets used in the analysis of bacterial gene expression data.

PubMed

Tintle, Nathan L; Sitarik, Alexandra; Boerema, Benjamin; Young, Kylie; Best, Aaron A; Dejongh, Matthew

2012-08-08

Statistical analyses of whole genome expression data require functional information about genes in order to yield meaningful biological conclusions. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) are common sources of functionally grouped gene sets. For bacteria, the SEED and MicrobesOnline provide alternative, complementary sources of gene sets. To date, no comprehensive evaluation of the data obtained from these resources has been performed. We define a series of gene set consistency metrics directly related to the most common classes of statistical analyses for gene expression data, and then perform a comprehensive analysis of 3581 Affymetrix® gene expression arrays across 17 diverse bacteria. We find that gene sets obtained from GO and KEGG demonstrate lower consistency than those obtained from the SEED and MicrobesOnline, regardless of gene set size. Despite the widespread use of GO and KEGG gene sets in bacterial gene expression data analysis, the SEED and MicrobesOnline provide more consistent sets for a wide variety of statistical analyses. Increased use of the SEED and MicrobesOnline gene sets in the analysis of bacterial gene expression data may improve statistical power and utility of expression data.

Prognostic significance of MYCN gene amplification and protein expression in primary brain tumors: Astrocytoma and meningioma.

PubMed

Estiar, Mehrdad Asghari; Javan, Firouzeh; Zekri, Ali; Mehrazin, Masoud; Mehdipour, Parvin

2017-07-04

Astrocytoma and meningioma are the most common primary brain tumors. MYCN as a member of MYC proto-oncogenes has recently appeared as an attractive therapeutic target. Functions of MYCN are critical for growth of nervous system and neural differentiation. We examined MYCN amplification and protein expression in astrocytoma and meningioma cases. In this study, we used real-time PCR, FISH assay and flowcytometry to analyze DNA amplification and protein expression of MYCN. Among 30 samples of brain tumor, 14 cases (46.6%) revealed MYCN amplification. High-protein expression of MYCN was also observed in 43.3% of patients. There was a significant correlation between MYCN gene amplification and protein expression (r= 0.523; p= 0.003), interestingly five case showed discrepancy between the gene amplification and protein expression. Although MYCN amplification fails to show correlation with poor prognosis (p= 0.305), protein high-expression of MYCN significantly reduce disease-free survival (p= 0.019). Our results challenge the concept of the neural specificity of MYCN by demonstrating contribution of MYCN in meningioma. Moreover, this study highlights the importance of research at both level of DNA and protein, to determine the biological functions and medical impacts of MYCN.

Epidermal Expression and Regulation of Interleukin-33 during Homeostasis and Inflammation: Strong Species Differences.

PubMed

Sundnes, Olav; Pietka, Wojciech; Loos, Tamara; Sponheim, Jon; Rankin, Andrew L; Pflanz, Stefan; Bertelsen, Vibeke; Sitek, Jan C; Hol, Johanna; Haraldsen, Guttorm; Khnykin, Denis

2015-07-01

IL-33 is a novel IL-1 family member with a putative role in inflammatory skin disorders and a complex biology. Therefore, recent conflicting data regarding its function in experimental models justify a close assessment of its tissue expression and regulation. Indeed, we report here that there are strong species differences in the expression and regulation of epidermal IL-33. In murine epidermis, IL-33 behaved similar to an alarmin, being constitutively expressed in keratinocyte nuclei and rapidly lost during acute inflammation. By contrast, human and porcine IL-33 were weakly expressed or absent in keratinocytes of noninflamed skin but induced during acute inflammation. To this end, we observed that expression of IL-33 in human keratinocytes but not murine keratinocytes was strongly induced by IFN-γ, and this upregulation completely depended on the presence of EGFR ligands. Accordingly, IFN-γ increased the expression of IL-33 in the basal layers of the epidermis in human ex vivo skin cultures only, despite good evidence of IFN-γ activity in cultures from both species. Together these findings demonstrate that a full understanding of IL-33 function in clinical settings must take species-specific differences into account.

Expression pattern and function of tyrosine receptor kinase B isoforms in rat mesenteric arterial smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Otani, Kosuke; Okada, Muneyoshi; Yamawaki, Hideyuki, E-mail: yamawaki@vmas.kitasato-u.ac.jp

Tyrosine receptor kinaseB (TrkB) is a high affinity receptor for brain-derived neurotrophic factor (BDNF). TrkB isoforms involve full length TrkB (TrkB FL) and truncated TrkB type1 (TrkB T1) and type 2 (TrkB T2) in rats. The aim of present study was to explore their expression pattern and function in mesenteric arterial smooth muscle cells (MASMCs). The expression of TrkB isoform protein and mRNA was examined by Western blotting, immunofluorescence and quantitative RT-PCR analyses. Cell proliferation was measured by a bromodeoxyuridine (BrdU) incorporation assay. Cell migration was measured by a Boyden chamber assay. Cell morphology was observed with a phase-contrast microscope.more » Protein and mRNA expression of BDNF and TrkB isoforms was confirmed in MASMCs. Expression level of TrkB FL was less, while that of TrkB T1 was the highest in MASMCs. Although BDNF increased phosphorylation of ERK, it had no influence on migration and proliferation of MASMCs. TrkB T1 gene knockdown by a RNA interference induced morphological changes and reduced expression level of α-smooth muscle actin (α-SMA) in MASMCs. Similar morphological changes and reduced α-SMA expression were induced in MASMCs by a Rho kinase inhibitor, Y-27632. In conclusion, we for the first time demonstrate that TrkB T1 expressed highly in MASMCs contributes to maintain normal cell morphology possibly via regulation of Rho activity. This study firstly defined expression level of TrkB isoforms and partly revealed their functions in peripheral vascular cells. - Highlights: • BDNF-TrkB axis mediates neurogenesis, growth, differentiation and survival. • Expression pattern and function of TrkB in vascular smooth muscle remain unclear. • Expression of TrkB FL is low, while that of TrkB T1 is the highest. • TrkB T1 contributes to maintain normal morphology possibly via activating Rho.« less

Functional role of Runx3 in the regulation of aggrecan expression during cartilage development.

PubMed

Wigner, Nathan A; Soung, Do Y; Einhorn, Thomas A; Drissi, Hicham; Gerstenfeld, Louis C

2013-11-01

Runx2 and Runx3 are known to be expressed in the growth plate during endochondral bone formation. Here we addressed the functional role of Runx3 as distinct from Runx2 by using two models of postnatal bone repair: fracture healing that proceeds by an endochondral process and marrow ablation that proceeds by only an intramembranous process. Both Runx2 and Runx3 mRNAs were differentially up regulated during fracture healing. In contrast, only Runx2 showed increased expression after marrow ablation. During fracture healing, Runx3 was expressed earlier than Runx2, was concurrent with the period of chondrogenesis, and coincident with maximal aggrecan expression a protein associated with proliferating and permanent cartilage. Immunohistological analysis showed Runx3 protein was also expressed by chondrocytes in vivo. In contrast, Runx2 was expressed later during chondrocyte hypertrophy, and primary bone formation. The functional activities of Runx3 during chondrocyte differentiation were assessed by examining its regulatory actions on aggrecan gene expression. Aggrecan mRNA levels and aggrecan promoter activity were enhanced in response to the over-expression of either Runx2 and Runx3 in ATDC5 chondrogenic cell line, while sh-RNA knocked down of each Runx protein showed that only Runx3 knock down specifically suppressed aggrecan mRNA expression and promoter activity. ChIP assay demonstrated that Runx3 interactions were selective to sites within the aggrecan promoter and were only observed during early periods of chondrogenesis before hypertrophy. Our studies suggest that Runx3 positively regulates aggrecan expression and suggest that its function is more limited to cartilage development than to bone. In aggregate these data further suggest that the various members of the Runx transcription factors are involved in the coordination of chondrocyte development, maturation, and hypertrophy during endochondral bone formation. Copyright © 2013 Wiley Periodicals, Inc.

(Implicitly) judging a book by its cover: the power of pride and shame expressions in shaping judgments of social status.

PubMed

Shariff, Azim F; Tracy, Jessica L; Markusoff, Jeffrey L

2012-09-01

How do we decide who merits social status? According to functionalist theories of emotion, the nonverbal expressions of pride and shame play a key role, functioning as automatically perceived status signals. In this view, observers automatically make status inferences about expressers on the basis of these expressions, even when contradictory contextual information about the expressers' status is available. In four studies, the authors tested whether implicit and explicit status perceptions are influenced by pride and shame expressions even when these expressions' status-related messages are contradicted by contextual information. Results indicate that emotion expressions powerfully influence implicit and explicit status inferences, at times neutralizing or even overriding situational knowledge. These findings demonstrate the irrepressible communicative power of emotion displays and indicate that status judgments can be informed as much (and often more) by automatic responses to nonverbal expressions of emotion as by rational, contextually bound knowledge.

HOX genes in human lung: altered expression in primary pulmonary hypertension and emphysema.

PubMed

Golpon, H A; Geraci, M W; Moore, M D; Miller, H L; Miller, G J; Tuder, R M; Voelkel, N F

2001-03-01

HOX genes belong to the large family of homeodomain genes that function as transcription factors. Animal studies indicate that they play an essential role in lung development. We investigated the expression pattern of HOX genes in human lung tissue by using microarray and degenerate reverse transcriptase-polymerase chain reaction survey techniques. HOX genes predominantly from the 3' end of clusters A and B were expressed in normal human adult lung and among them HOXA5 was the most abundant, followed by HOXB2 and HOXB6. In fetal (12 weeks old) and diseased lung specimens (emphysema, primary pulmonary hypertension) additional HOX genes from clusters C and D were expressed. Using in situ hybridization, transcripts for HOXA5 were predominantly found in alveolar septal and epithelial cells, both in normal and diseased lungs. A 2.5-fold increase in HOXA5 mRNA expression was demonstrated by quantitative reverse transcriptase-polymerase chain reaction in primary pulmonary hypertension lung specimens when compared to normal lung tissue. In conclusion, we demonstrate that HOX genes are selectively expressed in the human lung. Differences in the pattern of HOX gene expression exist among fetal, adult, and diseased lung specimens. The altered pattern of HOX gene expression may contribute to the development of pulmonary diseases.

Regulation of the macrophage oxytocin receptor in response to inflammation

PubMed Central

Szeto, Angela; Sun-Suslow, Ni; Mendez, Armando J.; Hernandez, Rosa I.; Wagner, Klaus V.

2017-01-01

It has been demonstrated that the neuropeptide oxytocin (OT) attenuates oxidative stress and inflammation in macrophages. In the current study, we examined the role of inflammation on the expression of the oxytocin receptor (OXTR). We hypothesized that OXTR expression is increased during the inflammation through a nuclear factor-κB (NF-κB)-mediated pathway, thus responding as an acute-phase protein. Inflammation was induced by treating macrophages (human primary, THP-1, and murine) with lipopolysaccharide (LPS) and monitored by expression of IL-6. Expression of OXTR and vasopressin receptors was assessed by qPCR, and OXTR expression was confirmed by immunoblotting. Inflammation upregulated OXTR transcription 10- to 250-fold relative to control in THP-1 and human primary macrophages and increased OXTR protein expression. In contrast, vasopressin receptor-2 mRNA expression was reduced following LPS treatment. Blocking NF-κB activation prevented the increase in OXTR transcription. OT treatment of control cells and LPS-treated cells increased ERK1/2 phosphorylation, demonstrating activation of the OXTR/Gαq/11 signaling pathway. OT activation of OXTR reduced secretion of IL-6 in LPS-activated macrophages. Collectively, these findings suggest that OXTR is an acute-phase protein and that its increased expression is regulated by NF-κB and functions to attenuate cellular inflammatory responses in macrophages. PMID:28049625

Achieving large dynamic range control of gene expression with a compact RNA transcription–translation regulator

PubMed Central

2017-01-01

Abstract RNA transcriptional regulators are emerging as versatile components for genetic network construction. However, these regulators suffer from incomplete repression in their OFF state, making their dynamic range less than that of their protein counterparts. This incomplete repression causes expression leak, which impedes the construction of larger synthetic regulatory networks as leak propagation can interfere with desired network function. To address this, we demonstrate how naturally derived antisense RNA-mediated transcriptional regulators can be configured to regulate both transcription and translation in a single compact RNA mechanism that functions in Escherichia coli. Using in vivo gene expression assays, we show that a combination of transcriptional termination and ribosome binding site sequestration increases repression from 85% to 98%, or activation from 10-fold to over 900-fold, in response to cognate antisense RNAs. We also show that orthogonal repressive versions of this mechanism can be created through engineering minimal antisense RNAs. Finally, to demonstrate the utility of this mechanism, we use it to reduce network leak in an RNA-only cascade. We anticipate these regulators will find broad use as synthetic biology moves beyond parts engineering to the design and construction of more sophisticated regulatory networks. PMID:28387839

Astrocyte-neuron crosstalk regulates the expression and subcellular localization of carbohydrate metabolism enzymes.

PubMed

Mamczur, Piotr; Borsuk, Borys; Paszko, Jadwiga; Sas, Zuzanna; Mozrzymas, Jerzy; Wiśniewski, Jacek R; Gizak, Agnieszka; Rakus, Dariusz

2015-02-01

Astrocytes releasing glucose- and/or glycogen-derived lactate and glutamine play a crucial role in shaping neuronal function and plasticity. Little is known, however, how metabolic functions of astrocytes, e.g., their ability to degrade glucosyl units, are affected by the presence of neurons. To address this issue we carried out experiments which demonstrated that co-culturing of rat hippocampal astrocytes with neurons significantly elevates the level of mRNA and protein for crucial enzymes of glycolysis (phosphofructokinase, aldolase, and pyruvate kinase), glycogen metabolism (glycogen synthase and glycogen phosphorylase), and glutamine synthetase in astrocytes. Simultaneously, the decrease of the capability of neurons to metabolize glucose and glutamine is observed. We provide evidence that neurons alter the expression of astrocytic enzymes by secretion of as yet unknown molecule(s) into the extracellular fluid. Moreover, our data demonstrate that almost all studied enzymes may localize in astrocytic nuclei and this localization is affected by the co-culturing with neurons which also reduces proliferative activity of astrocytes. Our results provide the first experimental evidence that the astrocyte-neuron crosstalk substantially affects the expression of basal metabolic enzymes in the both types of cells and influences their subcellular localization in astrocytes. © 2014 Wiley Periodicals, Inc.

Functional Division of Hippocampal Area CA1 Via Modulatory Gating of Entorhinal Cortical Inputs

PubMed Central

Ito, Hiroshi T.; Schuman, Erin M.

2013-01-01

The hippocampus receives two streams of information, spatial and nonspatial, via major afferent inputs from the medial (MEC) and lateral entorhinal cortexes (LEC). The MEC and LEC projections in the temporoammonic pathway are topographically organized along the transverse-axis of area CA1. The potential for functional segregation of area CA1, however, remains relatively unexplored. Here, we demonstrated differential novelty-induced c-Fos expression along the transverse-axis of area CA1 corresponding to topographic projections of MEC and LEC inputs. We found that, while novel place exposure induced a uniform c-Fos expression along the transverse-axis of area CA1, novel object exposure primarily activated the distal half of CA1 neurons. In hippocampal slices, we observed distinct presynaptic properties between LEC and MEC terminals, and application of either DA or NE produced a largely selective influence on one set of inputs (LEC). Finally, we demonstrated that differential c-Fos expression along the transverse axis of area CA1 was largely abolished by an antagonist of neuromodulatory receptors, clozapine. Our results suggest that neuromodulators can control topographic TA projections allowing the hippocampus to differentially encode new information along the transverse axis of area CA1. PMID:21240920

The fibronectin leucine-rich repeat transmembrane protein Flrt2 is required in the epicardium to promote heart morphogenesis

PubMed Central

Müller, Pari-Sima; Schulz, Ramona; Maretto, Silvia; Costello, Ita; Srinivas, Shankar; Bikoff, Elizabeth; Robertson, Elizabeth

2011-01-01

The epicardium, the outermost tissue layer that envelops the developing heart and provides essential trophic signals for the myocardium, derives from the pro-epicardial organ (PEO). Two of the three members of the Flrt family of transmembrane glycoproteins, Flrt2 and Flrt3, are strongly co-expressed in the PEO. However, beginning at around day 10 of mouse development, following attachment and outgrowth, Flrt3 is selectively downregulated, and only Flrt2 is exclusively expressed in the fully delaminated epicardium. The present gene-targeting experiments demonstrate that mouse embryos lacking Flrt2 expression arrest at mid-gestation owing to cardiac insufficiency. The defects in integrity of the epicardial sheet and disturbed organization of the underlying basement membrane closely resemble those described in Flrt3-deficient embryos that fail to maintain cell-cell contacts in the anterior visceral endoderm (AVE) signalling centre that normally establishes the A-P axis. Using in vitro and in vivo reconstitution assays, we demonstrate that Flrt2 and Flrt3 are functionally interchangeable. When acting alone, either of these proteins is sufficient to rescue functional activities in the AVE and the developing epicardium. PMID:21350012

The Dual Functions of WLIM1a in Cell Elongation and Secondary Wall Formation in Developing Cotton Fibers[C][W

PubMed Central

Han, Li-Bo; Li, Yuan-Bao; Wang, Hai-Yun; Wu, Xiao-Min; Li, Chun-Li; Luo, Ming; Wu, Shen-Jie; Kong, Zhao-Sheng; Pei, Yan; Jiao, Gai-Li; Xia, Gui-Xian

2013-01-01

LIN-11, Isl1 and MEC-3 (LIM)-domain proteins play pivotal roles in a variety of cellular processes in animals, but plant LIM functions remain largely unexplored. Here, we demonstrate dual roles of the WLIM1a gene in fiber development in upland cotton (Gossypium hirsutum). WLIM1a is preferentially expressed during the elongation and secondary wall synthesis stages in developing fibers. Overexpression of WLIM1a in cotton led to significant changes in fiber length and secondary wall structure. Compared with the wild type, fibers of WLIM1a-overexpressing plants grew longer and formed a thinner and more compact secondary cell wall, which contributed to improved fiber strength and fineness. Functional studies demonstrated that (1) WLIM1a acts as an actin bundler to facilitate elongation of fiber cells and (2) WLIM1a also functions as a transcription factor to activate expression of Phe ammonia lyase–box genes involved in phenylpropanoid biosynthesis to build up the secondary cell wall. WLIM1a localizes in the cytosol and nucleus and moves into the nucleus in response to hydrogen peroxide. Taken together, these results demonstrate that WLIM1a has dual roles in cotton fiber development, elongation, and secondary wall formation. Moreover, our study shows that lignin/lignin-like phenolics may substantially affect cotton fiber quality; this finding may guide cotton breeding for improved fiber traits. PMID:24220634

EVI2B is a C/EBPα target gene required for granulocytic differentiation and functionality of hematopoietic progenitors.

PubMed

Zjablovskaja, Polina; Kardosova, Miroslava; Danek, Petr; Angelisova, Pavla; Benoukraf, Touati; Wurm, Alexander A; Kalina, Tomas; Sian, Stephanie; Balastik, Martin; Delwel, Ruud; Brdicka, Tomas; Tenen, Daniel G; Behre, Gerhard; Fiore, Fréderic; Malissen, Bernard; Horejsi, Vaclav; Alberich-Jorda, Meritxell

2017-04-01

Development of hematopoietic populations through the process of differentiation is critical for proper hematopoiesis. The transcription factor CCAAT/enhancer binding protein alpha (C/EBPα) is a master regulator of myeloid differentiation, and the identification of C/EBPα target genes is key to understand this process. Here we identified the Ecotropic Viral Integration Site 2B (EVI2B) gene as a direct target of C/EBPα. We showed that the product of the gene, the transmembrane glycoprotein EVI2B (CD361), is abundantly expressed on the surface of primary hematopoietic cells, the highest levels of expression being reached in mature granulocytes. Using shRNA-mediated downregulation of EVI2B in human and murine cell lines and in primary hematopoietic stem and progenitor cells, we demonstrated impaired myeloid lineage development and altered progenitor functions in EVI2B-silenced cells. We showed that the compromised progenitor functionality in Evi2b-depleted cells can be in part explained by deregulation of cell proliferation and apoptosis. In addition, we generated an Evi2b knockout murine model and demonstrated altered properties of hematopoietic progenitors, as well as impaired G-CSF dependent myeloid colony formation in the knockout cells. Remarkably, we found that EVI2B is significantly downregulated in human acute myeloid leukemia samples characterized by defects in CEBPA. Altogether, our data demonstrate that EVI2B is a downstream target of C/EBPα, which regulates myeloid differentiation and functionality of hematopoietic progenitors.

AKT regulates BRCA1 stability in response to hormone signaling.

PubMed

Nelson, Andrew C; Lyons, Traci R; Young, Christian D; Hansen, Kirk C; Anderson, Steven M; Holt, Jeffrey T

2010-05-05

The observation that inherited mutations within BRCA1 result in breast and ovarian cancers suggests a functional relationship may exist between hormone signaling and BRCA1 function. We demonstrate that AKT activation promotes the expression of BRCA1 in response to estrogen and IGF-1 receptor signaling, and the rapid increase in BRCA1 protein levels appears to occur independently of new protein synthesis. Further, we identify a novel AKT phosphorylation site in BRCA1 at S694 which is responsive to activation of these signaling pathways. These data suggest AKT phosphorylation of BRCA1 increases total protein expression by preventing proteasomal degradation. AKT activation also appears to support nuclear localization of BRCA1, and co-expression of activated AKT with BRCA1 decreases radiation sensitivity, suggesting this interaction has functional consequences for BRCA1's role in DNA repair. Targets within this pathway could provide strategies for modulation of BRCA1 protein, which may prove therapeutically beneficial for breast and ovarian cancer treatment. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Engineering biosynthetic excitable tissues from unexcitable cells for electrophysiological and cell therapy studies

PubMed Central

Kirkton, Robert D.; Bursac, Nenad

2012-01-01

Patch-clamp recordings in single-cell expression systems have been traditionally used to study the function of ion channels. However, this experimental setting does not enable assessment of tissue-level function such as action potential (AP) conduction. Here we introduce a biosynthetic system that permits studies of both channel activity in single cells and electrical conduction in multicellular networks. We convert unexcitable somatic cells into an autonomous source of electrically excitable and conducting cells by stably expressing only three membrane channels. The specific roles that these expressed channels have on AP shape and conduction are revealed by different pharmacological and pacing protocols. Furthermore, we demonstrate that biosynthetic excitable cells and tissues can repair large conduction defects within primary 2- and 3-dimensional cardiac cell cultures. This approach enables novel studies of ion channel function in a reproducible tissue-level setting and may stimulate the development of new cell-based therapies for excitable tissue repair. PMID:21556054

The N-Methyl-D-Aspartate Receptor in Heart Development: A Gene Knockdown Model Using siRNA

PubMed Central

Lie, Octavian V.; Bennett, Gregory D.; Rosenquist, Thomas H

2009-01-01

Antagonists of the N-methyl-D-aspartate receptor (NMDAR) may disrupt the development of the cardiac neural crest (CNC) and contribute to conotruncal heart defects. To test this interaction, a loss-of-function model was generated using small interfering RNAs (siRNA) directed against the critical NR1-subunit of this receptor in avian embryos. The coding sequence of the chicken NR1-gene and predicted protein sequences were characterized and found to be homologous with other vertebrate species. Analysis of its spatiotemporal expression demonstrated its expression within the neural tube at pre-migratory CNC sites. siRNA targeted to the NR1-mRNA in pre-migratory CNC lead to a significant decrease in NR1 protein expression. However, embryo survival and heart development were not adversely affected. These results indicate that the CNC may function normally in the absence of functional NMDAR, and that NMDAR antagonists may have a complex impact upon the CNC that transcends impairment of a single receptor type. PMID:19737608

Multiple functions of the leucine-rich repeat protein LrrA of Treponema denticola.

PubMed

Ikegami, Akihiko; Honma, Kiyonobu; Sharma, Ashu; Kuramitsu, Howard K

2004-08-01

The gene lrrA, encoding a leucine-rich repeat protein, LrrA, that contains eight consensus tandem repeats of 23 amino acid residues, has been identified in Treponema denticola ATCC 35405. A leucine-rich repeat is a generally useful protein-binding motif, and proteins containing this repeat are typically involved in protein-protein interactions. Southern blot analysis demonstrated that T. denticola ATCC 35405 expresses the lrrA gene, but the gene was not identified in T. denticola ATCC 33520. In order to analyze the functions of LrrA in T. denticola, an lrrA-inactivated mutant of strain ATCC 35405 and an lrrA gene expression transformant of strain ATCC 33520 were constructed. Characterization of the mutant and transformant demonstrated that LrrA is associated with the extracytoplasmic fraction of T. denticola and expresses multifunctional properties. It was demonstrated that the attachment of strain ATCC 35405 to HEp-2 cell cultures and coaggregation with Tannerella forsythensis were attenuated by the lrrA mutation. In addition, an in vitro binding assay demonstrated specific binding of LrrA to a portion of the Tannerella forsythensis leucine-rich repeat protein, BspA, which is mediated by the N-terminal region of LrrA. It was also observed that the lrrA mutation caused a reduction of swarming in T. denticola ATCC 35405 and consequently attenuated tissue penetration. These results suggest that the leucine-rich repeat protein LrrA plays a role in the attachment and penetration of human epithelial cells and coaggregation with Tannerella forsythensis. These properties may play important roles in the virulence of T. denticola.

Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32)

PubMed Central

2011-01-01

Background Elevated numbers of regulatory T cells (Tregs) have been implicated in certain cancers. Depletion of Tregs has been shown to increase anti-tumor immunity. Tregs also play a critical role in the suppression of autoimmune responses. The study of Tregs has been hampered by a lack of adequate surface markers. Leucine Rich Repeat Containing 32 (LRRC32), also known as Glycoprotein A Repetitions Predominant (GARP), has been postulated as a novel surface marker of activated Tregs. However, there is limited information regarding the processing of LRRC32 or the regulatory phenotype and functional activity of Tregs expressing LRRC32. Results Using naturally-occurring freshly isolated Tregs, we demonstrate that low levels of LRRC32 are present intracellularly prior to activation and that freshly isolated LRRC32+ Tregs are distinct from LRRC32- Tregs with respect to the expression of surface CD62L. Using LRRC32 transfectants of HEK cells, we demonstrate that the N-terminus of LRRC32 is cleaved prior to expression of the protein at the cell surface. Furthermore, we demonstrate using a construct containing a deleted putative signal peptide region that the presence of a signal peptide region is critical to cell surface expression of LRRC32. Finally, mixed lymphocyte assays demonstrate that LRRC32+ Tregs are more potent suppressors than LRRC32- Tregs. Conclusions A cleaved signal peptide site in LRRC32 is necessary for surface localization of native LRRC32 following activation of naturally-occurring freshly-isolated regulatory T cells. LRRC32 expression appears to alter the surface expression of activation markers of T cells such as CD62L. LRRC32 surface expression may be useful as a marker that selects for more potent Treg populations. In summary, understanding the processing and expression of LRRC32 may provide insight into the mechanism of action of Tregs and the refinement of immunotherapeutic strategies aimed at targeting these cells. PMID:21615933

Loss of MyoD and Myf5 in Skeletal Muscle Stem Cells Results in Altered Myogenic Programming and Failed Regeneration.

PubMed

Yamamoto, Masakazu; Legendre, Nicholas P; Biswas, Arpita A; Lawton, Alexander; Yamamoto, Shoko; Tajbakhsh, Shahragim; Kardon, Gabrielle; Goldhamer, David J

2018-03-13

MyoD and Myf5 are fundamental regulators of skeletal muscle lineage determination in the embryo, and their expression is induced in satellite cells following muscle injury. MyoD and Myf5 are also expressed by satellite cell precursors developmentally, although the relative contribution of historical and injury-induced expression to satellite cell function is unknown. We show that satellite cells lacking both MyoD and Myf5 (double knockout [dKO]) are maintained with aging in uninjured muscle. However, injured muscle fails to regenerate and dKO satellite cell progeny accumulate in damaged muscle but do not undergo muscle differentiation. dKO satellite cell progeny continue to express markers of myoblast identity, although their myogenic programming is labile, as demonstrated by dramatic morphological changes and increased propensity for non-myogenic differentiation. These data demonstrate an absolute requirement for either MyoD or Myf5 in muscle regeneration and indicate that their expression after injury stabilizes myogenic identity and confers the capacity for muscle differentiation. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Stress-response transcription factors Msn2 and Msn4 couple TORC2-Ypk1 signaling and mitochondrial respiration to ATG8 gene expression and autophagy.

PubMed

Vlahakis, Ariadne; Lopez Muniozguren, Nerea; Powers, Ted

2017-01-01

Macroautophagy/autophagy is a starvation and stress-induced catabolic process critical for cellular homeostasis and adaptation. Several Atg proteins are involved in the formation of the autophagosome and subsequent degradation of cytoplasmic components, a process termed autophagy flux. Additionally, the expression of several Atg proteins, in particular Atg8, is modulated transcriptionally, yet the regulatory mechanisms involved remain poorly understood. Here we demonstrate that the AGC kinase Ypk1, target of the rapamycin-insensitive TORC2 signaling pathway, controls ATG8 expression by repressing the heterodimeric Zinc-finger transcription factors Msn2 and Msn4. We find that Msn2 and Msn4 promote ATG8 expression downstream of the histone deacetylase complex (HDAC) subunit Ume6, a previously identified negative regulator of ATG8 expression. Moreover, we demonstrate that TORC2-Ypk1 signaling is functionally linked to distinct mitochondrial respiratory complexes. Surprisingly, we find that autophagy flux during amino acid starvation is also dependent upon Msn2-Msn4 activity, revealing a broad role for these transcription factors in the autophagy response.

Endoribonuclease-Based Two-Component Repressor Systems for Tight Gene Expression Control in Plants

DOE PAGES

Liang, Yan; Richardson, Sarah; Yan, Jingwei; ...

2017-01-17

Tight control and multifactorial regulation of gene expression are important challenges in genetic engineering and are critical for the development of regulatory circuits. In meeting these challenges we will facilitate transgene expression regulation and support the fine-tuning of metabolic pathways to avoid the accumulation of undesired intermediates. By employing the endoribonuclease Csy4 and its recognition sequence from Pseudomonas aeruginosa and manipulating 5'UTR of mRNA, we developed a two-component expression–repression system to tightly control synthesis of transgene products. We demonstrated that this regulatory device was functional in monocotyledonous and dicotyledonous plant species, and showed that it can be used to repressmore » transgene expression by >400-fold and to synchronize transgene repression. In addition to tissue-specific transgene repression, this system offers stimuli-dependent expression control. Here, we identified 54 orthologous systems from various bacteria, using a bioinformatics approach and then validated in planta the activity for a few of those systems, demonstrating the potential diversity of such a two-component repressor system.« less

Endoribonuclease-Based Two-Component Repressor Systems for Tight Gene Expression Control in Plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang, Yan; Richardson, Sarah; Yan, Jingwei

Tight control and multifactorial regulation of gene expression are important challenges in genetic engineering and are critical for the development of regulatory circuits. In meeting these challenges we will facilitate transgene expression regulation and support the fine-tuning of metabolic pathways to avoid the accumulation of undesired intermediates. By employing the endoribonuclease Csy4 and its recognition sequence from Pseudomonas aeruginosa and manipulating 5'UTR of mRNA, we developed a two-component expression–repression system to tightly control synthesis of transgene products. We demonstrated that this regulatory device was functional in monocotyledonous and dicotyledonous plant species, and showed that it can be used to repressmore » transgene expression by >400-fold and to synchronize transgene repression. In addition to tissue-specific transgene repression, this system offers stimuli-dependent expression control. Here, we identified 54 orthologous systems from various bacteria, using a bioinformatics approach and then validated in planta the activity for a few of those systems, demonstrating the potential diversity of such a two-component repressor system.« less

DEK oncogene is overexpressed during melanoma progression.

PubMed

Riveiro-Falkenbach, Erica; Ruano, Yolanda; García-Martín, Rosa M; Lora, David; Cifdaloz, Metehan; Acquadro, Francesco; Ballestín, Claudio; Ortiz-Romero, Pablo L; Soengas, María S; Rodríguez-Peralto, José L

2017-03-01

DEK is an oncoprotein involved in a variety of cellular functions, such as DNA repair, replication, and transcriptional control. DEK is preferentially expressed in actively proliferating and malignant cells, including melanoma cell lines in which DEK was previously demonstrated to play a critical role in proliferation and chemoresistance. Still, the impact of this protein in melanoma progression remains unclear. Thus, we performed a comprehensive analysis of DEK expression in different melanocytic tumors. The immunostaining results of 303 tumors demonstrated negligible DEK expression in benign lesions. Conversely, malignant lesions, particularly in metastatic cases, were largely positive for DEK expression, which was partially associated with genomic amplification. Importantly, DEK overexpression was correlated with histological features of aggressiveness in primary tumors and poor prognosis in melanoma patients. In conclusion, our study provides new insight into the involvement of DEK in melanoma progression, as well as proof of concept for its potential application as a marker and therapeutic target of melanoma. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The SKI proto-oncogene enhances the in vivo repopulation of hematopoietic stem cells and causes myeloproliferative disease.

PubMed

Singbrant, Sofie; Wall, Meaghan; Moody, Jennifer; Karlsson, Göran; Chalk, Alistair M; Liddicoat, Brian; Russell, Megan R; Walkley, Carl R; Karlsson, Stefan

2014-04-01

The proto-oncogene SKI is highly expressed in human myeloid leukemia and also in murine hematopoietic stem cells. However, its operative relevance in these cells remains elusive. We have over-expressed SKI to define its intrinsic role in hematopoiesis and myeloid neoplasms, which resulted in a robust competitive advantage upon transplantation, a complete dominance of the stem and progenitor compartments, and a marked enhancement of myeloid differentiation at the expense of other lineages. Accordingly, enforced expression of SKI induced a gene signature associated with hematopoietic stem cells and myeloid differentiation, as well as hepatocyte growth factor signaling. Here we demonstrate that, in contrast to what has generally been assumed, the significant impact of SKI on hematopoiesis is independent of its ability to inhibit TGF-beta signaling. Instead, myeloid progenitors expressing SKI are partially dependent on functional hepatocyte growth factor signaling. Collectively our results demonstrate that SKI is an important regulator of hematopoietic stem cell activity and its overexpression leads to myeloproliferative disease.

Transcriptional and functional studies of Human Endogenous Retrovirus envelope EnvP(b) and EnvV genes in human trophoblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vargas, Amandine, E-mail: amandine.vargas@voila.fr; Thiery, Maxime, E-mail: thiery.maxime@courrier.uqam.ca; Lafond, Julie, E-mail: lafond.julie@uqam.ca

2012-03-30

HERV (Human Endogenous Retrovirus)-encoded envelope proteins are implicated in the development of the placenta. Indeed, Syncytin-1 and -2 play a crucial role in the fusion of human trophoblasts, a key step in placentation. Other studies have identified two other HERV env proteins, namely EnvP(b) and EnvV, both expressed in the placenta. In this study, we have fully characterized both env transcripts and their expression pattern and have assessed their implication in trophoblast fusion. Through RACE analyses, standard spliced transcripts were detected, while EnvV transcripts demonstrated alternative splicing at its 3 Prime end. Promoter activity and expression of both genes weremore » induced in forskolin-stimulated BeWo cells and in primary trophoblasts. Although we have confirmed the fusogenic activity of EnvP(b), overexpression or silencing experiments revealed no impact of this protein on trophoblast fusion. Our results demonstrate that both env genes are expressed in human trophoblasts but are not required for syncytialization.« less

The SKI proto-oncogene enhances the in vivo repopulation of hematopoietic stem cells and causes myeloproliferative disease

PubMed Central

Singbrant, Sofie; Wall, Meaghan; Moody, Jennifer; Karlsson, Göran; Chalk, Alistair M.; Liddicoat, Brian; Russell, Megan R.; Walkley, Carl R.; Karlsson, Stefan

2014-01-01

The proto-oncogene SKI is highly expressed in human myeloid leukemia and also in murine hematopoietic stem cells. However, its operative relevance in these cells remains elusive. We have over-expressed SKI to define its intrinsic role in hematopoiesis and myeloid neoplasms, which resulted in a robust competitive advantage upon transplantation, a complete dominance of the stem and progenitor compartments, and a marked enhancement of myeloid differentiation at the expense of other lineages. Accordingly, enforced expression of SKI induced a gene signature associated with hematopoietic stem cells and myeloid differentiation, as well as hepatocyte growth factor signaling. Here we demonstrate that, in contrast to what has generally been assumed, the significant impact of SKI on hematopoiesis is independent of its ability to inhibit TGF-beta signaling. Instead, myeloid progenitors expressing SKI are partially dependent on functional hepatocyte growth factor signaling. Collectively our results demonstrate that SKI is an important regulator of hematopoietic stem cell activity and its overexpression leads to myeloproliferative disease. PMID:24415629

Altered LARK Expression Perturbs Development and Physiology of the Drosophila PDF Clock Neurons

PubMed Central

Huang, Yanmei; Howlett, Eric; Stern, Michael; Jackson, F. Rob

2009-01-01

The LARK RNA-binding protein (RBP) has well documented roles in the circadian systems of Drosophila and mammals. Recent studies have demonstrated that the Drosophila LARK RBP is associated with many mRNA targets, in vivo, including those that regulate either neurophysiology or development of the nervous system. In the present study, we have employed conditional expression techniques to distinguish developmental and physiological functions of LARK for a defined class of neurons: the Pigment Dispersing Factor (PDF)-containing LNv clock neurons. We found that increased LARK expression during development dramatically alters the small LNv class of neurons with no obvious effects on the large LNv cells. Conversely, conditional expression of LARK at the adult stage results in altered clock protein rhythms and circadian locomotor activity, even though neural morphology is normal in such animals. Electrophysiological analyses at the larval neuromuscular junction indicate a role for LARK in regulating neuronal excitability. Altogether, our results demonstrate that LARK activity is critical for neuronal development and physiology. PMID:19303442

Reactant conversion in homogeneous turbulence: Mathematical modeling, computational validations and practical applications

NASA Technical Reports Server (NTRS)

Madnia, C. K.; Frankel, S. H.; Givi, P.

1992-01-01

Closed form analytical expressions are obtained for predicting the limited rate of reactant conversion in a binary reaction of the type F + rO yields (1 + r) Product in unpremixed homogeneous turbulence. These relations are obtained by means of a single point Probability Density Function (PDF) method based on the Amplitude Mapping Closure. It is demonstrated that with this model, the maximum rate of the reactants' decay can be conveniently expressed in terms of definite integrals of the Parabolic Cylinder Functions. For the cases with complete initial segregation, it is shown that the results agree very closely with those predicted by employing a Beta density of the first kind for an appropriately defined Shvab-Zeldovich scalar variable. With this assumption, the final results can also be expressed in terms of closed form analytical expressions which are based on the Incomplete Beta Functions. With both models, the dependence of the results on the stoichiometric coefficient and the equivalence ratio can be expressed in an explicit manner. For a stoichiometric mixture, the analytical results simplify significantly. In the mapping closure, these results are expressed in terms of simple trigonometric functions. For the Beta density model, they are in the form of Gamma Functions. In all the cases considered, the results are shown to agree well with data generated by Direct Numerical Simulations (DNS). Due to the simplicity of these expressions and because of nice mathematical features of the Parabolic Cylinder and the Incomplete Beta Functions, these models are recommended for estimating the limiting rate of reactant conversion in homogeneous reacting flows. These results also provide useful insights in assessing the extent of validity of turbulence closures in the modeling of unpremixed reacting flows. Some discussions are provided on the extension of the model for treating more complicated reacting systems including realistic kinetics schemes and multi-scalar mixing with finite rate chemical reactions in more complex configurations.

Effects of human rhinovirus on epithelial barrier integrity and function in children with asthma.

PubMed

Looi, K; Buckley, A G; Rigby, P J; Garratt, L W; Iosifidis, T; Zosky, G R; Larcombe, A N; Lannigan, F J; Ling, K-M; Martinovich, K M; Kicic-Starcevich, E; Shaw, N C; Sutanto, E N; Knight, D A; Kicic, A; Stick, S M

2018-05-01

Bronchial epithelial tight junctions (TJ) have been extensively assessed in healthy airway epithelium. However, no studies have yet assessed the effect of human rhinovirus (HRV) infection on the expression and resultant barrier function in epithelial tight junctions (TJ) in childhood asthma. To investigate the impact of HRV infection on airway epithelial TJ expression and barrier function in airway epithelial cells (AECs) of children with and without asthma. Furthermore, to test the hypothesis that barrier integrity and function is compromised to a greater extent by HRV in AECs from asthmatic children. Primary AECs were obtained from children with and without asthma, differentiated into air-liquid interface (ALI) cultures and infected with rhinovirus. Expression of claudin-1, occludin and zonula occluden-1 (ZO-1) was assessed via qPCR, immunocytochemistry (ICC), in-cell western (ICW) and confocal microscopy. Barrier function was assessed by transepithelial electrical resistance (TER; R T ) and permeability to fluorescent dextran. Basal TJ gene expression of claudin-1 and occludin was significantly upregulated in asthmatic children compared to non-asthmatics; however, no difference was seen with ZO-1. Interestingly, claudin-1, occludin and ZO-1 protein expression was significantly reduced in AEC of asthmatic children compared to non-asthmatic controls suggesting possible post-transcriptional inherent differences. HRV infection resulted in a transient dissociation of TJ and airway barrier integrity in non-asthmatic children. Although similar dissociation of TJ was observed in asthmatic children, a significant and sustained reduction in TJ expression concurrent with both a significant decrease in TER and an increase in permeability in asthmatic children was observed. This study demonstrates novel intrinsic differences in TJ gene and protein expression between AEC of children with and without asthma. Furthermore, it correlates directly the relationship between HRV infection and the resultant dissociation of epithelial TJ that causes a continued altered barrier function in children with asthma. © 2018 John Wiley & Sons Ltd.

Correlations of occupation numbers in the canonical ensemble and application to a Bose-Einstein condensate in a one-dimensional harmonic trap

NASA Astrophysics Data System (ADS)

Giraud, Olivier; Grabsch, Aurélien; Texier, Christophe

2018-05-01

We study statistical properties of N noninteracting identical bosons or fermions in the canonical ensemble. We derive several general representations for the p -point correlation function of occupation numbers n1⋯np ¯. We demonstrate that it can be expressed as a ratio of two p ×p determinants involving the (canonical) mean occupations n1¯, ..., np¯, which can themselves be conveniently expressed in terms of the k -body partition functions (with k ≤N ). We draw some connection with the theory of symmetric functions and obtain an expression of the correlation function in terms of Schur functions. Our findings are illustrated by revisiting the problem of Bose-Einstein condensation in a one-dimensional harmonic trap, for which we get analytical results. We get the moments of the occupation numbers and the correlation between ground-state and excited-state occupancies. In the temperature regime dominated by quantum correlations, the distribution of the ground-state occupancy is shown to be a truncated Gumbel law. The Gumbel law, describing extreme-value statistics, is obtained when the temperature is much smaller than the Bose-Einstein temperature.

Laminar shear stress regulates endothelial kinin B1 receptor expression and function: potential implication in atherogenesis

PubMed Central

Duchene, Johan; Cayla, Cécile; Vessillier, Sandrine; Scotland, Ramona; Yamashiro, Kazuo; Lecomte, Florence; Syed, Irfan; Vo, Phuong; Marrelli, Alessandra; Pitzalis, Costantino; Cipollone, Francesco; Schanstra, Joost; Bascands, Jean-Loup; Hobbs, Adrian J; Perretti, Mauro; Ahluwalia, Amrita

2009-01-01

OBJECTIVE The pro-inflammatory phenotype induced by low laminar shear stress (LSS) is implicated in atherogenesis. The kinin B1 receptor (B1R), known to be induced by inflammatory stimuli, exerts many pro-inflammatory effects including vasodilatation and leukocyte recruitment. We investigated whether low LSS is a stimulus for endothelial B1R expression and function. METHODS AND RESULTS Human and mouse atherosclerotic plaques expressed high level of B1R mRNA and protein. In addition, B1R expression was upregulated in the aortic arch (low LSS region) of ApoE-/- mice fed a high fat diet compared to vascular regions of high LSS and animals fed normal chow. Of interest, a greater expression of B1R was noticed in endothelial cells from regions of low LSS in aortic arch of ApoE-/- mice. B1R was also upregulated in human umbilical vein endothelial cells (HUVEC) exposed to low LSS (0-2dyn/cm2) compared to physiological LSS (6-10dyn/cm2): an effect similarly evident in murine vascular tissue perfused ex vivo. Functionally, B1R activation increased prostaglandin and CXCL5 expression in cells exposed to low, but not physiological, LSS. IL-1β and ox-LDL induced B1R expression and function in HUVECs, a response substantially enhanced under low LSS conditions and inhibited by blockade of NFκB activation. CONCLUSION Herein, we show that LSS is a major determinant of functional B1R expression in endothelium. Furthermore, whilst physiological high LSS is a powerful repressor of this inflammatory receptor, low LSS at sites of atheroma are associated with substantial upregulation, identifying this receptor as a potential therapeutic target. CONDENSED ABSTRACT Low laminar shear stress (LSS) underlies the pro-inflammatory processes in atherogenesis. Herein, we demonstrate that whilst physiological LSS represses inflammatory kinin B1 receptor (B1R) expression/function, low atherogenic LSS is associated with profound upregulation of both in atherosclerosis in both humans and animal models, highlighting B1R as an exciting potential therapeutic target. PMID:19661485

Nuclear BK Channels Regulate Gene Expression via the Control of Nuclear Calcium Signaling

PubMed Central

Li, Boxing; Jie, Wei; Huang, Lianyan; Wei, Peng; Li, Shuji; Luo, Zhengyi; Friedman, Allyson K.; Meredith, Andrea L.; Han, Ming-Hu; Zhu, Xin-Hong; Gao, Tian-Ming

2014-01-01

Ion channels are essential for the regulation of neuronal functions. The significance of plasma membrane, mitochondrial, endoplasmic reticulum, and lysosomal ion channels in the regulation of Ca2+ is well established. In contrast, surprisingly less is known about the function of ion channels on the nuclear envelope (NE). Here we demonstrate the presence of functional large-conductance, calcium-activated potassium channels (BK channels) on the NE of rodent hippocampal neurons. Functionally blockade of nuclear BK channels (nBK channels) induces NE-derived Ca2+ release, nucleoplasmic Ca2+ elevation, and cAMP response element binding protein (CREB)-dependent transcription. More importantly, blockade of nBK channels regulates nuclear Ca2+-sensitive gene expression and promotes dendritic arborization in a nuclear Ca2+-dependent manner. These results suggest that nBK channel functions as a molecular linker between neuronal activity and nuclear Ca2+ to convey the signals from synapse to nucleus and is a new modulator for synaptic activity-dependent neuronal functions at the NE level. PMID:24952642

Expression of NK cell receptors on decidual T cells in human pregnancy.

PubMed

Tilburgs, Tamara; van der Mast, Barbara J; Nagtzaam, Nicole M A; Roelen, Dave L; Scherjon, Sicco A; Claas, Frans H J

2009-06-01

Specific receptors enable NK cells to discriminate between cells with normal expression of MHC class I and cells that have low or absent expression of MHC class I molecules. In addition to NK cells, these receptors can be expressed on T cell subsets, mainly on CD8+ T cells but also on gammadeltaTCR+ T cells and CD4+ T cells. Although the function of NK cell receptor expression on T cells is not completely understood, various studies have shown that they are involved in down regulation of T cell receptor (TCR)-mediated activation and influence effector functions, like cytotoxicity and cytokine production. The aim of this study was to analyze expression of NK cell receptors on peripheral blood and decidual T cells during human pregnancy using flow cytometry. We demonstrate that a proportion of decidual T cells express HLA-C specific killer immunoglobulin-like receptors (KIRs). Furthermore, a small proportion of decidual T cells express the HLA-E specific CD94-NKG2A inhibitory and CD94-NKG2C activating receptors. Decidual KIR+ and CD94-NKG2+ T cells mainly display a CD3+CD4-CD8- phenotype. However, decidual tissue also contains higher percentages of KIR and CD94-NKG2 expressing CD4+ and CD8+ T cells compared to peripheral blood. So far, the functional capacities of decidual T cells expressing the NK cell receptors are unknown but NK cell receptor expression on decidual T cells may provide an alternative means by which decidual T cells distinguish self (maternal) cells from allogeneic fetal cells, and act to modulate the decidual immune response.

Developmental expression and function analysis of protein tyrosine phosphatase receptor type D in oligodendrocyte myelination

PubMed Central

Zhu, Qiang; Tan, Zhou; Zhao, Shufang; Huang, Hao; Zhao, Xiaofeng; Hu, Xuemei; Zhang, Yiping; Shields, Christopher B; Uetani, Noriko; Qiu, Mengsheng

2015-01-01

Receptor protein tyrosine phosphatases (RPTPs) are extensively expressed in the central nervous system (CNS), and have distinct spatial and temporal patterns in different cell types during development. Previous studies have demonstrated possible roles for RPTPs in axon outgrowth, guidance, and synaptogenesis. In the present study, our results revealed that protein tyrosine phosphatase, receptor type D (PTPRD) was initially expressed in mature neurons in embryonic CNS, and later in oligodendroglial cells at postnatal stages when oligodendrocyte undergo active axonal myelination process. In PTPRD mutants, oligodendrocyte differentiation was normal and a transient myelination delay occurred at early postnatal stages, indicating the contribution of PTPRD to the initiation of axonal myelination. Our results also showed that the remyelination process was not affected in the absence of PTPRD function after a cuprizone-induced demyelination in adult animals. PMID:26341907

Cloning and function analysis of a drought-inducible gene associated with resistance to Curvularia leaf spot in maize.

PubMed

Zhu, Jiewei; Huang, Xiuli; Liu, Tong; Gao, Shigang; Chen, Jie

2012-08-01

ZmDIP was cloned and its function against Curvularia lunata was analyzed, according to a previous finding on a drought-inducible protein in resistant maize identified through MALDI-TOF-MS/MS. The ZmDIP expression varied in roots, leaf sheaths, and young, as well as old, leaves of different maize inbred lines. The ZmDIP transcript level changed in leaves over the course of time after inoculation with C. lunata. A prokaryotic expression analysis demonstrated that the gene can regulate the salt stress tolerance of Escherichia coli. The ZmDIP transient expression in the maize leaf showed that the gene was also linked to leaf resistance against the C. lunata infection. ZmDIP-mediated ROS and ABA signaling pathways were inferred to be closely associated with maize leaf resistance to the pathogen infection.

A CRISPR-Based Toolbox for Studying T Cell Signal Transduction

PubMed Central

Chi, Shen; Weiss, Arthur; Wang, Haopeng

2016-01-01

CRISPR/Cas9 system is a powerful technology to perform genome editing in a variety of cell types. To facilitate the application of Cas9 in mapping T cell signaling pathways, we generated a toolbox for large-scale genetic screens in human Jurkat T cells. The toolbox has three different Jurkat cell lines expressing distinct Cas9 variants, including wild-type Cas9, dCas9-KRAB, and sunCas9. We demonstrated that the toolbox allows us to rapidly disrupt endogenous gene expression at the DNA level and to efficiently repress or activate gene expression at the transcriptional level. The toolbox, in combination with multiple currently existing genome-wide sgRNA libraries, will be useful to systematically investigate T cell signal transduction using both loss-of-function and gain-of-function genetic screens. PMID:27057542

Sweet Vector for Gene Delivery: the Sugar Decoration of Polyplexes Reduces Cytotoxicity with a Balanced Effect on Gene Expression.

PubMed

Albuquerque, Lindomar J C; Alavarse, Alex C; Carlan da Silva, Maria C; Zilse, Morgana S; Barth, Maitê T; Bellettini, Ismael C; Giacomelli, Fernando C

2018-02-01

The use of sugar-functionalized polyplexes as a nonviral gene delivery vector with lower cytotoxicity than the well-known polymeric carrier branched polyethyleneimine (BPEI) is investigated. The substitution of primary amine groups in the BPEI chains with lactose residues leads to larger polyplexes, presumably due to the higher amount of polymer required to complete DNA condensation. Nevertheless, the sugar functionalization substantially reduces the cytotoxicity of the assemblies. The nanocomplexes are taken up by the cells to a greater extent, whereas the levels of gene expression are maintained compared to those obtained using BPEI, which is known for its excellent transfection efficiency. Accordingly, the preparation of lower-cytotoxicity polyplexes while maintaining gene expression, which is highly relevant to the field, is demonstrated. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Accelerated recruitment of new brain development genes into the human genome.

PubMed

Zhang, Yong E; Landback, Patrick; Vibranovski, Maria D; Long, Manyuan

2011-10-01

How the human brain evolved has attracted tremendous interests for decades. Motivated by case studies of primate-specific genes implicated in brain function, we examined whether or not the young genes, those emerging genome-wide in the lineages specific to the primates or rodents, showed distinct spatial and temporal patterns of transcription compared to old genes, which had existed before primate and rodent split. We found consistent patterns across different sources of expression data: there is a significantly larger proportion of young genes expressed in the fetal or infant brain of humans than in mouse, and more young genes in humans have expression biased toward early developing brains than old genes. Most of these young genes are expressed in the evolutionarily newest part of human brain, the neocortex. Remarkably, we also identified a number of human-specific genes which are expressed in the prefrontal cortex, which is implicated in complex cognitive behaviors. The young genes upregulated in the early developing human brain play diverse functional roles, with a significant enrichment of transcription factors. Genes originating from different mechanisms show a similar expression bias in the developing brain. Moreover, we found that the young genes upregulated in early brain development showed rapid protein evolution compared to old genes also expressed in the fetal brain. Strikingly, genes expressed in the neocortex arose soon after its morphological origin. These four lines of evidence suggest that positive selection for brain function may have contributed to the origination of young genes expressed in the developing brain. These data demonstrate a striking recruitment of new genes into the early development of the human brain.

Prostaglandin E2 modulates dendritic cell function via EP2 and EP4 receptor subtypes.

PubMed

Harizi, Hedi; Grosset, Christophe; Gualde, Norbert

2003-06-01

We have reported previously that PGE(2) inhibits dendritic cells (DC) functions. Because E prostanoid receptor (EPR) subtypes involved in this action are unknown, expression and functions of these receptors were examined in DC. Western blot and flow cytometry analyses showed that all EPRs were coexpressed in DC. In a dose-dependent manner, lipopolysaccharide (LPS) enhanced EP(2)R/EP(4)R but not EP(1)R/EP(3)R expressions. NS-398, a cyclooxygenase (COX)-2-selective inhibitor, suppressed LPS-enhanced EP(2)R/EP(4)R expression, suggesting that COX-2-issued prostaglandin E(2) (PGE(2)) modulates DC function through stimulation of specific EPR subtypes. Using selective agonists, we found that butaprost, an EP(2)R agonist, and PGE(1) alcohol, an EP(2)R and EP(2)R/EP(4)R agonist, inhibited major histocompatibility complex class II expression and enhanced interleukin-10 production from DC. However, no effect was observed with sulprostone and 17-phenyl-omega-trinor-PGE(2), selective agonists for EP(1)R and EP(1)R/EP(3)R, respectively. Treatment of DC with dibutyryl cyclic adenosine monophosphate (cAMP), an analog of cAMP, mimics PGE(2)-induced, inhibitory effects. Taken together, our data demonstrate that EP(2)R/EP(4)R are efficient for mediating PGE(2)-induced modulation of DC functions.

Impaired holistic coding of facial expression and facial identity in congenital prosopagnosia.

PubMed

Palermo, Romina; Willis, Megan L; Rivolta, Davide; McKone, Elinor; Wilson, C Ellie; Calder, Andrew J

2011-04-01

We test 12 individuals with congenital prosopagnosia (CP), who replicate a common pattern of showing severe difficulty in recognising facial identity in conjunction with normal recognition of facial expressions (both basic and 'social'). Strength of holistic processing was examined using standard expression composite and identity composite tasks. Compared to age- and sex-matched controls, group analyses demonstrated that CPs showed weaker holistic processing, for both expression and identity information. Implications are (a) normal expression recognition in CP can derive from compensatory strategies (e.g., over-reliance on non-holistic cues to expression); (b) the split between processing of expression and identity information may take place after a common stage of holistic processing; and (c) contrary to a recent claim, holistic processing of identity is functionally involved in face identification ability. Copyright © 2011 Elsevier Ltd. All rights reserved.

Impaired holistic coding of facial expression and facial identity in congenital prosopagnosia

PubMed Central

Palermo, Romina; Willis, Megan L.; Rivolta, Davide; McKone, Elinor; Wilson, C. Ellie; Calder, Andrew J.

2011-01-01

We test 12 individuals with congenital prosopagnosia (CP), who replicate a common pattern of showing severe difficulty in recognising facial identity in conjunction with normal recognition of facial expressions (both basic and ‘social’). Strength of holistic processing was examined using standard expression composite and identity composite tasks. Compared to age- and sex-matched controls, group analyses demonstrated that CPs showed weaker holistic processing, for both expression and identity information. Implications are (a) normal expression recognition in CP can derive from compensatory strategies (e.g., over-reliance on non-holistic cues to expression); (b) the split between processing of expression and identity information may take place after a common stage of holistic processing; and (c) contrary to a recent claim, holistic processing of identity is functionally involved in face identification ability. PMID:21333662

Conifer reproductive development involves B-type MADS-box genes with distinct and different activities in male organ primordia.

PubMed

Sundström, Jens; Engström, Peter

2002-07-01

The Norway spruce MADS-box genes DAL11, DAL12 and DAL13 are phylogenetically related to the angiosperm B-function MADS-box genes: genes that act together with A-function genes in specifying petal identity and with C-function genes in specifying stamen identity to floral organs. In this report we present evidence to suggest that the B-gene function in the specification of identity of the pollen-bearing organs has been conserved between conifers and angiosperms. Expression of DAL11 or DAL12 in transgenic Arabidopsis causes phenotypic changes which partly resemble those caused by ectopic expression of the endogenous B-genes. In similar experiments, flowers of Arabidopsis plants expressing DAL13 showed a different homeotic change in that they formed ectopic anthers in whorls one, two or four. We also demonstrate the capacity of the spruce gene products to form homodimers, and that DAL11 and DAL13 may form heterodimers with each other and with the Arabidopsis B-protein AP3, but not with PI, the second B-gene product in Arabidopsis. In situ hybridization experiments show that the conifer B-like genes are expressed specifically in developing pollen cones, but differ in both temporal and spatial distribution patterns. These results suggest that the B-function in conifers is dual and is separated into a meristem identity and an organ identity function, the latter function possibly being independent of an interaction with the C-function. Thus, even though an ancestral B-function may have acted in combination with C to specify micro- and megasporangia, the B-function has evolved differently in conifers and angiosperms.

Identification and functional analysis of long non-coding RNAs in human and mouse early embryos based on single-cell transcriptome data

PubMed Central

Qiu, Jia-jun; Ren, Zhao-rui; Yan, Jing-bin

2016-01-01

Epigenetics regulations have an important role in fertilization and proper embryonic development, and several human diseases are associated with epigenetic modification disorders, such as Rett syndrome, Beckwith-Wiedemann syndrome and Angelman syndrome. However, the dynamics and functions of long non-coding RNAs (lncRNAs), one type of epigenetic regulators, in human pre-implantation development have not yet been demonstrated. In this study, a comprehensive analysis of human and mouse early-stage embryonic lncRNAs was performed based on public single-cell RNA sequencing data. Expression profile analysis revealed that lncRNAs are expressed in a developmental stage–specific manner during human early-stage embryonic development, whereas a more temporal-specific expression pattern was identified in mouse embryos. Weighted gene co-expression network analysis suggested that lncRNAs involved in human early-stage embryonic development are associated with several important functions and processes, such as oocyte maturation, zygotic genome activation and mitochondrial functions. We also found that the network of lncRNAs involved in zygotic genome activation was highly preservative between human and mouse embryos, whereas in other stages no strong correlation between human and mouse embryo was observed. This study provides insight into the molecular mechanism underlying lncRNA involvement in human pre-implantation embryonic development. PMID:27542205

Direction of Amygdala-Neocortex Interaction During Dynamic Facial Expression Processing.

PubMed

Sato, Wataru; Kochiyama, Takanori; Uono, Shota; Yoshikawa, Sakiko; Toichi, Motomi

2017-03-01

Dynamic facial expressions of emotion strongly elicit multifaceted emotional, perceptual, cognitive, and motor responses. Neuroimaging studies revealed that some subcortical (e.g., amygdala) and neocortical (e.g., superior temporal sulcus and inferior frontal gyrus) brain regions and their functional interaction were involved in processing dynamic facial expressions. However, the direction of the functional interaction between the amygdala and the neocortex remains unknown. To investigate this issue, we re-analyzed functional magnetic resonance imaging (fMRI) data from 2 studies and magnetoencephalography (MEG) data from 1 study. First, a psychophysiological interaction analysis of the fMRI data confirmed the functional interaction between the amygdala and neocortical regions. Then, dynamic causal modeling analysis was used to compare models with forward, backward, or bidirectional effective connectivity between the amygdala and neocortical networks in the fMRI and MEG data. The results consistently supported the model of effective connectivity from the amygdala to the neocortex. Further increasing time-window analysis of the MEG demonstrated that this model was valid after 200 ms from the stimulus onset. These data suggest that emotional processing in the amygdala rapidly modulates some neocortical processing, such as perception, recognition, and motor mimicry, when observing dynamic facial expressions of emotion. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Elabela-apelin receptor signaling pathway is functional in mammalian systems.

PubMed

Wang, Zhi; Yu, Daozhan; Wang, Mengqiao; Wang, Qilong; Kouznetsova, Jennifer; Yang, Rongze; Qian, Kun; Wu, Wenjun; Shuldiner, Alan; Sztalryd, Carole; Zou, Minghui; Zheng, Wei; Gong, Da-Wei

2015-02-02

Elabela (ELA) or Toddler is a recently discovered hormone which is required for normal development of heart and vasculature through activation of apelin receptor (APJ), a G protein-coupled receptor (GPCR), in zebrafish. The present study explores whether the ELA-APJ signaling pathway is functional in the mammalian system. Using reverse-transcription PCR, we found that ELA is restrictedly expressed in human pluripotent stem cells and adult kidney whereas APJ is more widely expressed. We next studied ELA-APJ signaling pathway in reconstituted mammalian cell systems. Addition of ELA to HEK293 cells over-expressing GFP-AJP fusion protein resulted in rapid internalization of the fusion receptor. In Chinese hamster ovarian (CHO) cells over-expressing human APJ, ELA suppresses cAMP production with EC50 of 11.1 nM, stimulates ERK1/2 phosphorylation with EC50 of 14.3 nM and weakly induces intracellular calcium mobilization. Finally, we tested ELA biological function in human umbilical vascular endothelial cells and showed that ELA induces angiogenesis and relaxes mouse aortic blood vessel in a dose-dependent manner through a mechanism different from apelin. Collectively, we demonstrate that the ELA-AJP signaling pathways are functional in mammalian systems, indicating that ELA likely serves as a hormone regulating the circulation system in adulthood as well as in embryonic development.

Spontaneous and natural cytotoxicity receptor-mediated cytotoxicity are effector functions of distinct natural killer subsets in hepatitis C virus-infected chimpanzees.

PubMed

Verstrepen, B E; Nieuwenhuis, I G; Mooij, P; Bogers, W M; Boonstra, A; Koopman, G

2016-07-01

In humans, CD16 and CD56 are used to identify functionally distinct natural killer (NK) subsets. Due to ubiquitous CD56 expression, this marker cannot be used to distinguish between NK cell subsets in chimpanzees. Therefore, functional analysis of distinct NK subsets during hepatitis C virus (HCV) infection has never been performed in these animals. In the present study an alternative strategy was used to identify four distinct NK subsets on the basis of the expression of CD16 and CD94. The expression of activating and inhibiting surface receptors showed that these subsets resemble human NK subsets. CD107 expression was used to determine degranulation of the different subsets in naive and HCV-infected chimpanzees. In HCV-infected chimpanzees increased spontaneous cytotoxicity was observed in CD94(high/dim) CD16(pos) and CD94(low) CD16(pos) subsets. By contrast, increased natural cytotoxicity receptor (NCR)- mediated degranulation after NKp30 and NKp44 triggering was demonstrated in the CD94(dim) CD16(neg) subset. Our findings suggest that spontaneous and NCR-mediated cytotoxicity are effector functions of distinct NK subsets in HCV-infected chimpanzees. © 2016 British Society for Immunology.

Functional analysis of free fatty acid receptor GPR120 in human eosinophils: implications in metabolic homeostasis.

PubMed

Konno, Yasunori; Ueki, Shigeharu; Takeda, Masahide; Kobayashi, Yoshiki; Tamaki, Mami; Moritoki, Yuki; Oyamada, Hajime; Itoga, Masamichi; Kayaba, Hiroyuki; Omokawa, Ayumi; Hirokawa, Makoto

2015-01-01

Recent evidence has shown that eosinophils play an important role in metabolic homeostasis through Th2 cytokine production. GPR120 (FFA4) is a G protein-coupled receptor (GPCR) for long-chain fatty acids that functions as a regulator of physiological energy metabolism. In the present study, we aimed to investigate whether human eosinophils express GPR120 and, if present, whether it possesses a functional capacity on eosinophils. Eosinophils isolated from peripheral venous blood expressed GPR120 at both the mRNA and protein levels. Stimulation with a synthetic GPR120 agonist, GW9508, induced rapid down-regulation of cell surface expression of GPR120, suggesting ligand-dependent receptor internalization. Although GPR120 activation did not induce eosinophil chemotactic response and degranulation, we found that GW9508 inhibited eosinophil spontaneous apoptosis and Fas receptor expression. The anti-apoptotic effect was attenuated by phosphoinositide 3-kinase (PI3K) inhibitors and was associated with inhibition of caspase-3 activity. Eosinophil response investigated using ELISpot assay indicated that stimulation with a GPR120 agonist induced IL-4 secretion. These findings demonstrate the novel functional properties of fatty acid sensor GPR120 on human eosinophils and indicate the previously unrecognized link between nutrient metabolism and the immune system.

Antigenic and functional properties of the human red blood cell urea transporter hUT-B1.

PubMed

Lucien, Nicole; Sidoux-Walter, Frédéric; Roudier, Nathalie; Ripoche, Pierre; Huet, Martine; Trinh-Trang-Tan, Marie-Marcelle; Cartron, Jean-Pierre; Bailly, Pascal

2002-09-13

The Kidd (JK) blood group locus encodes the urea transporter hUT-B1, which is expressed on human red blood cells and other tissues. The common JK*A/JK*B blood group polymorphism is caused by a single nucleotide transition G838A changing Asp-280 to Asn-280 on the polypeptide, and transfection of erythroleukemic K562 cells with hUT-B1 cDNAs carrying either the G838 or the A838 nucleotide substitutions resulted in the isolation of stable clones that expressed the Jk(a) or Jk(b) antigens, respectively, thus providing the first direct demonstration that the hUT-B1 gene encodes the Kidd blood group antigens. In addition, immunochemical analysis of red blood cells demonstrated that hUT-B1 also exhibits ABO determinants attached to the single N-linked sugar chain at Asn-211. Moreover, immunoadsorption studies, using inside-out and right-side-out red cell membrane vesicles as competing antigen, demonstrated that the C- and N-terminal ends of hUT-B1 are oriented intracellularly. Mutagenesis and functional studies by expression in Xenopus oocytes revealed that both cysteines Cys-25 and Cys-30 (but not alone) are essential for plasma membrane addressing. Conversely, the transport function was not affected by the JK*A/JK*B polymorphism, C-terminal deletion (residues 360-389), or mutation of the extracellular N-glycosylation consensus site and remains poorly para-chloromercuribenzene sulfonate (pCMBS)-sensitive. However, transport studies by stopped flow light scattering using Jk-K562 transfectants demonstrated that the hUT-B1-mediated urea transport is pCMBS-sensitive in an erythroid context, as reported previously for the transporter of human red blood cells. Mutagenesis analysis also indicated that Cys-151 and Cys-236, at least alone, are not involved in pCMBS inhibition. Altogether, these antigenic, topologic, and functional properties might have implications into the physiology of hUT-B1 and other members of the urea transporter family.

Laboratory automation in a functional programming language.

PubMed

Runciman, Colin; Clare, Amanda; Harkness, Rob

2014-12-01

After some years of use in academic and research settings, functional languages are starting to enter the mainstream as an alternative to more conventional programming languages. This article explores one way to use Haskell, a functional programming language, in the development of control programs for laboratory automation systems. We give code for an example system, discuss some programming concepts that we need for this example, and demonstrate how the use of functional programming allows us to express and verify properties of the resulting code. © 2014 Society for Laboratory Automation and Screening.

The reprogramming factor nuclear receptor subfamily 5, group A, member 2 cannot replace octamer-binding transcription factor 4 function in the self-renewal of embryonic stem cells.

PubMed

Choi, Kyeng-Won; Oh, Hye-Rim; Lee, Jaeyoung; Lim, Bobae; Han, Yong-Mahn; Oh, Junseo; Kim, Jungho

2014-02-01

Although octamer-binding transcription factor 4 (Oct-4) is one of the most intensively studied factors in mammalian development, no cellular genes capable of replacing Oct-4 function in embryonic stem (ES) cells have been found. Recent data show that nuclear receptor subfamily 5, group A, member 2 (Nr5a2) is able to replace Oct-4 function in the reprogramming process; however, it is unclear whether Nr5a2 can replace Oct-4 function in ES cells. In this study, the ability of Nr5a2 to maintain self-renewal and pluripotency in ES cells was investigated. Nr5a2 localized to the nucleus in ES cells, similarly to Oct-4. However, expression of Nr5a2 failed to rescue the stem cell phenotype or to maintain the self-renewal ability of ES cells. Furthermore, as compared with Oct-4-expressing ES cells, Nr5a2-expressing ES cells showed a reduced number of cells in S-phase, did not expand normally, and did not remain in an undifferentiated state. Ectopic expression of Nr5a2 in ES cells was not able to activate transcription of ES cell-specific genes, and gene expression profiling demonstrated differences between Nr5a2-expressing and Oct-4-expressing ES cells. In addition, Nr5a2-expressing ES cells were not able to form teratomas in nude mice. Taken together, these results strongly suggest that the gene regulation properties of Nr5a2 and Oct-4 and their abilities to confer self-renewal and pluripotency of ES cells differ. The present study provides strong evidence that Nr5a2 cannot replace Oct-4 function in ES cells. © 2013 FEBS.

Lipopolysaccharide-Elicited TSLPR Expression Enriches a Functionally Discrete Subset of Human CD14+ CD1c+ Monocytes.

PubMed

Borriello, Francesco; Iannone, Raffaella; Di Somma, Sarah; Vastolo, Viviana; Petrosino, Giuseppe; Visconte, Feliciano; Raia, Maddalena; Scalia, Giulia; Loffredo, Stefania; Varricchi, Gilda; Galdiero, Maria Rosaria; Granata, Francescopaolo; Del Vecchio, Luigi; Portella, Giuseppe; Marone, Gianni

2017-05-01

Thymic stromal lymphopoietin (TSLP) is a cytokine produced mainly by epithelial cells in response to inflammatory or microbial stimuli and binds to the TSLP receptor (TSLPR) complex, a heterodimer composed of TSLPR and IL-7 receptor α (CD127). TSLP activates multiple immune cell subsets expressing the TSLPR complex and plays a role in several models of disease. Although human monocytes express TSLPR and CD127 mRNAs in response to the TLR4 agonist LPS, their responsiveness to TSLP is poorly defined. We demonstrate that TSLP enhances human CD14 + monocyte CCL17 production in response to LPS and IL-4. Surprisingly, only a subset of CD14 + CD16 - monocytes, TSLPR + monocytes (TSLPR + mono), expresses TSLPR complex upon LPS stimulation in an NF-κB- and p38-dependent manner. Phenotypic, functional, and transcriptomic analysis revealed specific features of TSLPR + mono, including higher CCL17 and IL-10 production and increased expression of genes with important immune functions (i.e., GAS6 , ALOX15B , FCGR2B , LAIR1 ). Strikingly, TSLPR + mono express higher levels of the dendritic cell marker CD1c. This evidence led us to identify a subset of peripheral blood CD14 + CD1c + cells that expresses the highest levels of TSLPR upon LPS stimulation. The translational relevance of these findings is highlighted by the higher expression of TSLPR and CD127 mRNAs in monocytes isolated from patients with Gram-negative sepsis compared with healthy control subjects. Our results emphasize a phenotypic and functional heterogeneity in an apparently homogeneous population of human CD14 + CD16 - monocytes and prompt further ontogenetic and functional analysis of CD14 + CD1c + and LPS-activated CD14 + CD1c + TSLPR + mono. Copyright © 2017 by The American Association of Immunologists, Inc.

Heterologous expression of an α-amylase inhibitor from common bean (Phaseolus vulgaris) in Kluyveromyces lactis and Saccharomyces cerevisiae.

PubMed

Brain-Isasi, Stephanie; Álvarez-Lueje, Alejandro; Higgins, Thomas Joseph V

2017-06-15

Phaseolamin or α-amylase inhibitor 1 (αAI) is a glycoprotein from common beans (Phaseolus vulgaris L.) that inhibits some insect and mammalian α-amylases. Several clinical studies support the beneficial use of bean αAI for control of diabetes and obesity. Commercial extracts of P. vulgaris are available but their efficacy is still under question, mainly because some of these extracts contain antinutritional impurities naturally present in bean seeds and also exhibit a lower specific activity αAI. The production of recombinant αAI allows to overcome these disadvantages and provides a platform for the large-scale production of pure and functional αAI protein for biotechnological and pharmaceutical applications. A synthetic gene encoding αAI from the common bean (Phaseolus vulgaris cv. Pinto) was codon-optimised for expression in yeasts (αAI-OPT) and cloned into the protein expression vectors pKLAC2 and pYES2. The yeasts Kluyveromyces lactis GG799 (and protease deficient derivatives such as YCT390) and Saccharomyces cerevisiae YPH499 were transformed with the optimised genes and transformants were screened for expression by antibody dot blot. Recombinant colonies of K. lactis YCT390 that expressed and secreted functional αAI into the culture supernatants were selected for further analyses. Recombinant αAI from K. lactis YCT390 was purified using anion-exchange and affinity resins leading to the recovery of a functional inhibitor. The identity of the purified αAI was confirmed by mass spectrometry. Recombinant clones of S. cerevisiae YPH499 expressed functional αAI intracellularly, but did not secrete the protein. This is the first report describing the heterologous expression of the α-amylase inhibitor 1 (αAI) from P. vulgaris in yeasts. We demonstrated that recombinant strains of K. lactis and S. cerevisiae expressed and processed the αAI precursor into mature and active protein and also showed that K. lactis secretes functional αAI.

The insulator protein Suppressor of Hairy-wing is an essential transcriptional repressor in the Drosophila ovary.

PubMed

Soshnev, Alexey A; Baxley, Ryan M; Manak, J Robert; Tan, Kai; Geyer, Pamela K

2013-09-01

Suppressor of Hairy-wing [Su(Hw)] is a DNA-binding factor required for gypsy insulator function and female germline development in Drosophila. The insulator function of the gypsy retrotransposon depends on Su(Hw) binding to clustered Su(Hw) binding sites (SBSs) and recruitment of the insulator proteins Centrosomal Protein 190 kD (CP190) and Modifier of mdg4 67.2 kD (Mod67.2). By contrast, the Su(Hw) germline function involves binding to non-clustered SBSs and does not require CP190 or Mod67.2. Here, we identify Su(Hw) target genes, using genome-wide analyses in the ovary to uncover genes with an ovary-bound SBS that are misregulated upon Su(Hw) loss. Most Su(Hw) target genes demonstrate enriched expression in the wild-type CNS. Loss of Su(Hw) leads to increased expression of these CNS-enriched target genes in the ovary and other tissues, suggesting that Su(Hw) is a repressor of neural genes in non-neural tissues. Among the Su(Hw) target genes is RNA-binding protein 9 (Rbp9), a member of the ELAV/Hu gene family. Su(Hw) regulation of Rbp9 appears to be insulator independent, as Rbp9 expression is unchanged in a genetic background that compromises the functions of the CP190 and Mod67.2 insulator proteins, even though both localize to Rbp9 SBSs. Rbp9 misregulation is central to su(Hw)(-/-) sterility, as Rbp9(+/-), su(Hw)(-/-) females are fertile. Eggs produced by Rbp9(+/-), su(Hw)(-/-) females show patterning defects, revealing a somatic requirement for Su(Hw) in the ovary. Our studies demonstrate that Su(Hw) is a versatile transcriptional regulatory protein with an essential developmental function involving transcriptional repression.

3D heterogeneous islet organoid generation from human embryonic stem cells using a novel engineered hydrogel platform.

PubMed

Candiello, Joseph; Grandhi, Taraka Sai Pavan; Goh, Saik Kia; Vaidya, Vimal; Lemmon-Kishi, Maya; Eliato, Kiarash Rahmani; Ros, Robert; Kumta, Prashant N; Rege, Kaushal; Banerjee, Ipsita

2018-05-25

Organoids, which exhibit spontaneous organ specific organization, function, and multi-cellular complexity, are in essence the in vitro reproduction of specific in vivo organ systems. Recent work has demonstrated human pluripotent stem cells (hPSCs) as a viable regenerative cell source for tissue-specific organoid engineering. This is especially relevant for engineering islet organoids, due to the recent advances in generating functional beta-like cells from human pluripotent stem cells. In this study, we report specific engineering of regenerative islet organoids of precise size and cellular heterogeneity, using a novel hydrogel system, Amikagel. Amikagel facilitated controlled and spontaneous aggregation of human embryonic stem cell derived pancreatic progenitor cells (hESC-PP) into robust homogeneous spheroids. This platform further allowed fine control over the integration of multiple cell populations to produce heterogeneous spheroids, which is a necessity for complex organoid engineering. Amikagel induced hESC-PP spheroid formation enhanced pancreatic islet-specific Pdx-1 and NKX6.1 gene and protein expression, while also increasing the percentage of committed population. hESC-PP spheroids were further induced towards mature beta-like cells which demonstrated increased Beta-cell specific INS1 gene and C-peptide protein expression along with functional insulin production in response to in vitro glucose challenge. Further integration of hESC-PP with biologically relevant supporting endothelial cells resulted in multicellular organoids which demonstrated spontaneous maturation towards islet-specific INS1 gene and C-peptide protein expression along with a significantly developed extracellular matrix support system. These findings establish Amikagel -facilitated platform ideal for islet organoid engineering. Copyright © 2018. Published by Elsevier Ltd.

GPER-targeted, 99mTc-labeled, nonsteroidal ligands demonstrate selective tumor imaging and in vivo estrogen binding.

PubMed

Nayak, Tapan K; Ramesh, Chinnasamy; Hathaway, Helen J; Norenberg, Jeffrey P; Arterburn, Jeffrey B; Prossnitz, Eric R

2014-11-01

Our understanding of estrogen (17β-estradiol, E2) receptor biology has evolved in recent years with the discovery and characterization of a 7-transmembrane-spanning G protein-coupled estrogen receptor (GPER/GPR30) and the development of GPER-selective functional chemical probes. GPER is highly expressed in certain breast, endometrial, and ovarian cancers, establishing the importance of noninvasive methods to evaluate GPER expression in vivo. Here, we developed (99m)Tc-labeled GPER ligands to demonstrate the in vivo status of GPER as an estrogen receptor (ER) and for GPER visualization in whole animals. A series of (99m)Tc(I)-labeled nonsteroidal tetrahydro-3H-cyclopenta[c]quinolone derivatives was synthesized utilizing pyridin-2-yl hydrazine and picolylamine chelates. Radioligand receptor binding studies revealed binding affinities in the 10 to 30 nmol/L range. Cell signaling assays previously demonstrated that derivatives retaining a ketone functionality displayed agonist properties, whereas those lacking such a hydrogen bond acceptor were antagonists. In vivo biodistribution and imaging studies performed on mice bearing human endometrial and breast cancer cell xenografts yielded significant tumor uptake (0.4-1.1%ID/g). Blocking studies revealed specific uptake in multiple organs (adrenals, uterus, and mammary tissue), as well as tumor uptake with similar levels of competition by E2 and G-1, a GPER-selective agonist. In conclusion, we synthesized and evaluated a series of first-generation (99m)Tc-labeled GPER-specific radioligands, demonstrating GPER as an estrogen-binding receptor for the first time in vivo using competitive binding principles, and establishing the utility of such ligands as tumor imaging agents. These results warrant further investigation into the role of GPER in estrogen-mediated carcinogenesis and as a target for diagnostic/therapeutic/image-guided drug delivery. These studies provide a molecular basis to evaluate GPER expression and function as an ER through in vivo imaging. ©2014 American Association for Cancer Research.

High Aldehyde Dehydrogenase Activity Identifies a Subset of Human Mesenchymal Stromal Cells with Vascular Regenerative Potential.

PubMed

Sherman, Stephen E; Kuljanin, Miljan; Cooper, Tyler T; Putman, David M; Lajoie, Gilles A; Hess, David A

2017-06-01

During culture expansion, multipotent mesenchymal stromal cells (MSCs) differentially express aldehyde dehydrogenase (ALDH), an intracellular detoxification enzyme that protects long-lived cells against oxidative stress. Thus, MSC selection based on ALDH-activity may be used to reduce heterogeneity and distinguish MSC subsets with improved regenerative potency. After expansion of human bone marrow-derived MSCs, cell progeny was purified based on low versus high ALDH-activity (ALDH hi ) by fluorescence-activated cell sorting, and each subset was compared for multipotent stromal and provascular regenerative functions. Both ALDH l ° and ALDH hi MSC subsets demonstrated similar expression of stromal cell (>95% CD73 + , CD90 + , CD105 + ) and pericyte (>95% CD146 + ) surface markers and showed multipotent differentiation into bone, cartilage, and adipose cells in vitro. Conditioned media (CDM) generated by ALDH hi MSCs demonstrated a potent proliferative and prosurvival effect on human microvascular endothelial cells (HMVECs) under serum-free conditions and augmented HMVEC tube-forming capacity in growth factor-reduced matrices. After subcutaneous transplantation within directed in vivo angiogenesis assay implants into immunodeficient mice, ALDH hi MSC or CDM produced by ALDH hi MSC significantly augmented murine vascular cell recruitment and perfused vessel infiltration compared with ALDH l ° MSC. Although both subsets demonstrated strikingly similar mRNA expression patterns, quantitative proteomic analyses performed on subset-specific CDM revealed the ALDH hi MSC subset uniquely secreted multiple proangiogenic cytokines (vascular endothelial growth factor beta, platelet derived growth factor alpha, and angiogenin) and actively produced multiple factors with chemoattractant (transforming growth factor-β, C-X-C motif chemokine ligand 1, 2, and 3 (GRO), C-C motif chemokine ligand 5 (RANTES), monocyte chemotactic protein 1 (MCP-1), interleukin [IL]-6, IL-8) and matrix-modifying functions (tissue inhibitor of metalloprotinase 1 & 2 (TIMP1/2)). Collectively, MSCs selected for ALDH hi demonstrated enhanced proangiogenic secretory functions and represent a purified MSC subset amenable for vascular regenerative applications. Stem Cells 2017;35:1542-1553. © 2017 AlphaMed Press.

Rationally engineered Troponin C modulates in vivo cardiac function and performance in health and disease.

PubMed

Shettigar, Vikram; Zhang, Bo; Little, Sean C; Salhi, Hussam E; Hansen, Brian J; Li, Ning; Zhang, Jianchao; Roof, Steve R; Ho, Hsiang-Ting; Brunello, Lucia; Lerch, Jessica K; Weisleder, Noah; Fedorov, Vadim V; Accornero, Federica; Rafael-Fortney, Jill A; Gyorke, Sandor; Janssen, Paul M L; Biesiadecki, Brandon J; Ziolo, Mark T; Davis, Jonathan P

2016-02-24

Treatment for heart disease, the leading cause of death in the world, has progressed little for several decades. Here we develop a protein engineering approach to directly tune in vivo cardiac contractility by tailoring the ability of the heart to respond to the Ca(2+) signal. Promisingly, our smartly formulated Ca(2+)-sensitizing TnC (L48Q) enhances heart function without any adverse effects that are commonly observed with positive inotropes. In a myocardial infarction (MI) model of heart failure, expression of TnC L48Q before the MI preserves cardiac function and performance. Moreover, expression of TnC L48Q after the MI therapeutically enhances cardiac function and performance, without compromising survival. We demonstrate engineering TnC can specifically and precisely modulate cardiac contractility that when combined with gene therapy can be employed as a therapeutic strategy for heart disease.

Rationally engineered Troponin C modulates in vivo cardiac function and performance in health and disease

PubMed Central

Shettigar, Vikram; Zhang, Bo; Little, Sean C.; Salhi, Hussam E.; Hansen, Brian J.; Li, Ning; Zhang, Jianchao; Roof, Steve R.; Ho, Hsiang-Ting; Brunello, Lucia; Lerch, Jessica K.; Weisleder, Noah; Fedorov, Vadim V.; Accornero, Federica; Rafael-Fortney, Jill A.; Gyorke, Sandor; Janssen, Paul M. L.; Biesiadecki, Brandon J.; Ziolo, Mark T.; Davis, Jonathan P.

2016-01-01

Treatment for heart disease, the leading cause of death in the world, has progressed little for several decades. Here we develop a protein engineering approach to directly tune in vivo cardiac contractility by tailoring the ability of the heart to respond to the Ca2+ signal. Promisingly, our smartly formulated Ca2+-sensitizing TnC (L48Q) enhances heart function without any adverse effects that are commonly observed with positive inotropes. In a myocardial infarction (MI) model of heart failure, expression of TnC L48Q before the MI preserves cardiac function and performance. Moreover, expression of TnC L48Q after the MI therapeutically enhances cardiac function and performance, without compromising survival. We demonstrate engineering TnC can specifically and precisely modulate cardiac contractility that when combined with gene therapy can be employed as a therapeutic strategy for heart disease. PMID:26908229

Surfactant protein-A nanobody-conjugated liposomes loaded with methylprednisolone increase lung-targeting specificity and therapeutic effect for acute lung injury.

PubMed

Li, Nan; Weng, Dong; Wang, Shan-Mei; Zhang, Yuan; Chen, Shan-Shan; Yin, Zhao-Fang; Zhai, Jiali; Scoble, Judy; Williams, Charlotte C; Chen, Tao; Qiu, Hui; Wu, Qin; Zhao, Meng-Meng; Lu, Li-Qin; Mulet, Xavier; Li, Hui-Ping

2017-11-01

The advent of nanomedicine requires novel delivery vehicles to actively target their site of action. Here, we demonstrate the development of lung-targeting drug-loaded liposomes and their efficacy, specificity and safety. Our study focuses on glucocorticoids methylprednisolone (MPS), a commonly used drug to treat lung injuries. The steroidal molecule was loaded into functionalized nano-sterically stabilized unilamellar liposomes (NSSLs). Targeting functionality was performed through conjugation of surfactant protein A (SPANb) nanobodies to form MPS-NSSLs-SPANb. MPS-NSSLs-SPANb exhibited good size distribution, morphology, and encapsulation efficiency. Animal experiments demonstrated the high specificity of MPS-NSSLs-SPANb to the lung. Treatment with MPS-NSSLs-SPANb reduced the levels of TNF-α, IL-8, and TGF-β1 in rat bronchoalveolar lavage fluid and the expression of NK-κB in the lung tissues, thereby alleviating lung injuries and increasing rat survival. The nanobody functionalized nanoparticles demonstrate superior performance to treat lung injury when compared to that of antibody functionalized systems.

Identification of a µ opiate receptor signaling mechanism in human placenta.

PubMed

Mantione, Kirk J; Angert, Robert M; Cadet, Patrick; Kream, Richard M; Stefano, George B

2010-11-01

Previous studies report that genes in the morphine biosynthetic pathway have been found in placental tissue. Prior researchers have shown that kappa opioid receptors are present in human placenta. We determined if a µ opiate receptor was present and which subtype was expressed in human placenta. We also sought to demonstrate a functional µ opiate receptor in human placenta. Polymerase chain reactions as well as DNA sequencing were performed to identify the µ opiate receptor subtypes present in human placenta. The functionality of the receptor was demonstrated by real time amperometric measurements of morphine induced NO release. The µ4 opiate receptor sequence was present as well as the µ1 opioid receptor transcript. The addition of morphine to placental tissue resulted in immediate nitric oxide release and this effect was blocked by naloxone. In the present study, an intact morphine signaling system has been demonstrated in human placenta. Morphine signaling in human placenta probably functions to regulate the immune, vascular, and endocrine functions of this organ via NO.

Cloning and functional characterization of the guinea pig apoptosis inhibitor protein Survivin.

PubMed

Habtemichael, Negusse; Wünsch, Desiree; Bier, Carolin; Tillmann, Sarah; Unruhe, Britta; Frauenknecht, Katrin; Heinrich, Ulf-Rüdiger; Mann, Wolf J; Stauber, Roland H; Knauer, Shirley K

2010-12-01

The guinea pig is widely used as a model to study (patho)physiological processes, such as neurodegenerative disorders. Survivin's dual function as an apoptosis inhibitor and a mitotic regulator is crucial not only for ordered development but its modulation seems crucial also under disease conditions. However, data on the expression and function of the guinea pig Survivin protein (Survivin(Gp)) are currently lacking. Here, we here report the cloning and functional characterization of Survivin(Gp). The respective cDNA was cloned from spleen mRNA, containing a 426 bp open reading frame encoding for a protein of 142aa. Survivin(Gp) displays a high homology to the human and murine orthologue, especially in domains critical for function, such as binding sites for chromosomal passenger complex (CPC) proteins and the nuclear export signal (NES). Notably, phylogenetic analyses revealed that Survivin(Gp) is more related to humans than to rodents. Ectopic expression studies of a Survivin(Gp)-GFP fusion confirmed its dynamic intracellular localization, analogous to the human and murine counterparts. In interphase cells, Survivin(Gp)-GFP was predominantly cytoplasmic and accumulated in the nucleus following export inhibition with leptomycin B (LMB). A typical CPC protein localization during mitosis was observed for Survivin(Gp)-GFP. Microinjection experiments together with genetic knockout demonstrated that the NES is essential for the anti-apoptotic and regulatory role of Survivin(Gp) during cell division. In vivo protein interaction assays further demonstrated its dimerization with human Survivin and its interaction with human CPC proteins. Importantly, RNAi-depletion studies show that Survivin(Gp) can fully substitute for human Survivin as an apoptosis inhibitor and a mitotic effector. Immunohistochemistry, immunofluorescence, and western blotting were employed to detect Survivin expression in guinea pig tissues. Besides its expression in proliferating tissues, such as spleen and liver, we also found Survivin in terminally differentiated cell types. Importantly, Survivin was detectable also in the cochlea, suggesting a potential role for Survivin in the auditory system. We provide the first experimental evidence for the expression of Survivin in the guinea pig. As Survivin(Gp) can substitute for known functions of human Survivin, the guinea pig model will now also allow investigating Survivin's (patho)physiological role and to test Survivin-directed potential therapeutic strategies. Copyright © 2010 Elsevier B.V. All rights reserved.

Three-dimensional biomimetic vascular model reveals a RhoA, Rac1, and N-cadherin balance in mural cell-endothelial cell-regulated barrier function.

PubMed

Alimperti, Stella; Mirabella, Teodelinda; Bajaj, Varnica; Polacheck, William; Pirone, Dana M; Duffield, Jeremy; Eyckmans, Jeroen; Assoian, Richard K; Chen, Christopher S

2017-08-15

The integrity of the endothelial barrier between circulating blood and tissue is important for blood vessel function and, ultimately, for organ homeostasis. Here, we developed a vessel-on-a-chip with perfused endothelialized channels lined with human bone marrow stromal cells, which adopt a mural cell-like phenotype that recapitulates barrier function of the vasculature. In this model, barrier function is compromised upon exposure to inflammatory factors such as LPS, thrombin, and TNFα, as has been observed in vivo. Interestingly, we observed a rapid physical withdrawal of mural cells from the endothelium that was accompanied by an inhibition of endogenous Rac1 activity and increase in RhoA activity in the mural cells themselves upon inflammation. Using a system to chemically induce activity in exogenously expressed Rac1 or RhoA within minutes of stimulation, we demonstrated RhoA activation induced loss of mural cell coverage on the endothelium and reduced endothelial barrier function, and this effect was abrogated when Rac1 was simultaneously activated. We further showed that N -cadherin expression in mural cells plays a key role in barrier function, as CRISPR-mediated knockout of N -cadherin in the mural cells led to loss of barrier function, and overexpression of N -cadherin in CHO cells promoted barrier function. In summary, this bicellular model demonstrates the continuous and rapid modulation of adhesive interactions between endothelial and mural cells and its impact on vascular barrier function and highlights an in vitro platform to study the biology of perivascular-endothelial interactions.

Expression of a non-coding RNA in ectromelia virus is required for normal plaque formation.

PubMed

Esteban, David J; Upton, Chris; Bartow-McKenney, Casey; Buller, R Mark L; Chen, Nanhai G; Schriewer, Jill; Lefkowitz, Elliot J; Wang, Chunlin

2014-02-01

Poxviruses are dsDNA viruses with large genomes. Many genes in the genome remain uncharacterized, and recent studies have demonstrated that the poxvirus transcriptome includes numerous so-called anomalous transcripts not associated with open reading frames. Here, we characterize the expression and role of an apparently non-coding RNA in orthopoxviruses, which we call viral hairpin RNA (vhRNA). Using a bioinformatics approach, we predicted expression of a transcript not associated with an open reading frame that is likely to form a stem-loop structure due to the presence of a 21 nt palindromic sequence. Expression of the transcript as early as 2 h post-infection was confirmed by northern blot and analysis of publicly available vaccinia virus infected cell transcriptomes. The transcription start site was determined by RACE PCE and transcriptome analysis, and early and late promoter sequences were identified. Finally, to test the function of the transcript we generated an ectromelia virus knockout, which failed to form plaques in cell culture. The important role of the transcript in viral replication was further demonstrated using siRNA. Although the function of the transcript remains unknown, our work contributes to evidence of an increasingly complex poxvirus transcriptome, suggesting that transcripts such as vhRNA not associated with an annotated open reading frame can play an important role in viral replication.

MicroRNA-490-5p inhibits proliferation of bladder cancer by targeting c-Fos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Shiqi; Xu, Xianglai; Xu, Xin

2013-11-29

Highlights: •We examined the level of miR-490-5p in bladder cancer tissues and three cancer cell lines. •We are the first to show the function of miR-490-5p in bladder cancer. •We demonstrate c-Fos may be a target of miR-490-5p. -- Abstract: MicroRNAs (miRNAs) are non-protein-coding sequences that play a crucial role in tumorigenesis by negatively regulating gene expression. Here, we found that miR-490-5p is down-regulated in human bladder cancer tissue and cell lines compared to normal adjacent tissue and a non-malignant cell line. To better characterize the function of miR-490-5p in bladder cancer, we over-expressed miR-490-5p in bladder cancer cell linesmore » with chemically synthesized mimics. Enforced expression of miR-490-5p in bladder cancer cells significantly inhibited the cell proliferation via G1-phase arrest. Further studies found the decreased c-Fos expression at both mRNA and protein levels and Luciferase reporter assays demonstrated that c-Fos is a direct target of miR-490-5p in bladder cancer. These findings indicate miR-490-5p to be a novel tumor suppressor of bladder cancer cell proliferation through targeting c-Fos.« less

Modeled Microgravity Disrupts Collagen I/Integrin Signaling During Osteoblastic Differentiation of Human Mesenchymal Stem Cells

NASA Technical Reports Server (NTRS)

Meyers, Valerie E.; Zayzafoon, Majd; Gonda, Steven R.; Gathings, William E.; McDonald, Jay M.

2004-01-01

Spaceflight leads to reduced bone mineral density in weight bearing bones that is primarily attributed to a reduction in bone formation. We have previously demonstrated severely reduced osteoblastogenesis of human mesenchymal stem cells (hMSC) following seven days culture in modeled microgravity. One potential mechanism for reduced osteoblastic differentiation is disruption of type I collagen-integrin interactions and reduced integrin signaling. Integrins are heterodimeric transmembrane receptors that bind extracellular matrix proteins and produce signals essential for proper cellular function, survival, and differentiation. Therefore, we investigated the effects of modeled microgravity on integrin expression and function in hMSC. We demonstrate that seven days of culture in modeled microgravity leads to reduced expression of the extracellular matrix protein, type I collagen (Col I). Conversely, modeled microgravity consistently increases Col I-specific alpha2 and beta1 integrin protein expression. Despite this increase in integrin sub-unit expression, autophosphorylation of adhesion-dependent kinases, focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2), is significantly reduced. Activation of Akt is unaffected by the reduction in FAK activation. However, reduced downstream signaling via the Ras-MAPK pathway is evidenced by a reduction in Ras and ERK activation. Taken together, our findings indicate that modeled microgravity decreases integrin/MAPK signaling, which likely contributes to the observed reduction in osteoblastogenesis.

An Evolutionary Conserved Epigenetic Mark of Polycomb Response Elements Implemented by Trx/MLL/COMPASS.

PubMed

Rickels, Ryan; Hu, Deqing; Collings, Clayton K; Woodfin, Ashley R; Piunti, Andrea; Mohan, Man; Herz, Hans-Martin; Kvon, Evgeny; Shilatifard, Ali

2016-07-21

Polycomb response elements (PREs) are specific DNA sequences that stably maintain the developmental pattern of gene expression. Drosophila PREs are well characterized, whereas the existence of PREs in mammals remains debated. Accumulating evidence supports a model in which CpG islands recruit Polycomb group (PcG) complexes; however, which subset of CGIs is selected to serve as PREs is unclear. Trithorax (Trx) positively regulates gene expression in Drosophila and co-occupies PREs to antagonize Polycomb-dependent silencing. Here we demonstrate that Trx-dependent H3K4 dimethylation (H3K4me2) marks Drosophila PREs and maintains the developmental expression pattern of nearby genes. Similarly, the mammalian Trx homolog, MLL1, deposits H3K4me2 at CpG-dense regions that could serve as PREs. In the absence of MLL1 and H3K4me2, H3K27me3 levels, a mark of Polycomb repressive complex 2 (PRC2), increase at these loci. By inhibiting PRC2-dependent H3K27me3 in the absence of MLL1, we can rescue expression of these loci, demonstrating a functional balance between MLL1 and PRC2 activities at these sites. Thus, our study provides rules for identifying cell-type-specific functional mammalian PREs within the human genome. Copyright © 2016 Elsevier Inc. All rights reserved.

Ectopic Expression of a Glycine soja myo-Inositol Oxygenase Gene (GsMIOX1a) in Arabidopsis Enhances Tolerance to Alkaline Stress

PubMed Central

Chen, Chen; Sun, Xiaoli; Duanmu, Huizi; Yu, Yang; Liu, Ailin; Xiao, Jialei; Zhu, Yanming

2015-01-01

Myo-inositol participates in various aspects of plant physiology, and myo-inositol oxygenase is the key enzyme of the myo-inositol oxygenation pathway. Previous studies indicated that myo-inositol oxygenase may play a role in plant responses to abiotic stresses. In this study, we focused on the functional characterization of GsMIOX1a, a remarkable alkaline stress-responsive gene of Glycine soja 07256, based on RNA-seq data. Using quantitative real-time PCR, we demonstrated that GsMIOX1a is rapidly induced by alkaline stress and expressed predominantly in flowers. We also elucidated the positive function of GsMIOX1a in the alkaline response in the wild type, atmiox1 mutant as well as GsMIOX1a-overexpressing Arabidopsis. We determined that atmiox1 mutant decreased Arabidopsis tolerance to alkaline stress, whereas GsMIOX1a overexpression increased tolerance. Moreover, the expression levels of some alkaline stress-responsive and inducible marker genes, including H+-Ppase, NADP-ME, KIN1 and RD29B, were also up-regulated in GsMIOX1a overexpression lines compared with the wild type and atmiox1 mutant. Together, these results suggest that the GsMIOX1a gene positively regulates plant tolerance to alkaline stress. This is the first report to demonstrate that ectopic expression of myo-inositol oxygenase improves alkaline tolerance in plants. PMID:26091094

Cajanus cajan Linn. (Leguminosae) prevents alcohol-induced rat liver damage and augments cytoprotective function.

PubMed

Kundu, Rakesh; Dasgupta, Suman; Biswas, Anindita; Bhattacharya, Anirban; Pal, Bikas C; Bandyopadhyay, Debashis; Bhattacharya, Shelley; Bhattacharya, Samir

2008-08-13

Cajanus cajan Linn. (Leguminosae) is a nontoxic edible herb, widely used in Indian folk medicine for the prevention of various liver disorders. In the present study we have demonstrated that methanol-aqueous fraction (MAF2) of Cajanus cajan leaf extract could prevent the chronically treated alcohol induced rat liver damage. Chronic doses of alcohol (3.7 g/ kg) orally administered to rats for 28 days and liver function marker enzymes such as GPT, GOT, ALP and anti-oxidant enzyme activities were determined. Effect of MAF2 at a dose of 50mg/kg body weight on alcohol treated rats was noted. Alcohol effected significant increase in liver marker enzyme activities and reduced the activities of anti-oxidant enzymes. Co-administration of MAF2 reversed the liver damage due to alcohol; it decreased the activities of liver marker enzymes and augmented antioxidant enzyme activities. We also demonstrate significant decrease of the phase II detoxifying enzyme, UDP-glucuronosyl transferase (UGT) activity along with a three- and two-fold decrease of UGT2B gene and protein expression respectively. MAF2 co-administration normalized UGT activity and revived the expression of UGT2B with a concomitant expression and nuclear translocation of Nrf2, a transcription factor that regulates the expression of many cytoprotective genes. Cajanus cajan extract therefore shows a promise in therapeutic use in alcohol induced liver dysfunction.

FAS/FASL are dysregulated in chordoma and their loss-of-function impairs zebrafish notochord formation

PubMed Central

Libera, Laura; Boari, Nicola; Mortini, Pietro; Bellipanni, Gianfranco; Giordano, Antonio; Cotelli, Franco; Riva, Paola

2014-01-01

Chordoma is a rare malignant tumor that recapitulates the notochord phenotype and is thought to derive from notochord remnants not correctly regressed during development. Apoptosis is necessary for the proper notochord development in vertebrates, and the apoptotic pathway mediated by Fas and Fasl has been demonstrated to be involved in notochord cells regression. This study was conducted to investigate the expression of FAS/FASL pathway in a cohort of skull base chordomas and to analyze the role of fas/fasl homologs in zebrafish notochord formation. FAS/FASL expression was found to be dysregulated in chordoma leading to inactivation of the downstream Caspases in the samples analyzed. Both fas and fasl were specifically expressed in zebrafish notochord sorted cells. fas and fasl loss-of-function mainly resulted in larvae with notochord multi-cell-layer jumps organization, larger vacuolated notochord cells, defects in the peri-notochordal sheath structure and in vertebral mineralization. Interestingly, we observed the persistent expression of ntla and col2a1a, the zebrafish homologs of the human T gene and COL2A1 respectively, which are specifically up-regulated in chordoma. These results demonstrate for the first time the dysregulation of FAS/FASL in chordoma and their role in notochord formation in the zebrafish model, suggesting their possible implication in chordoma onset. PMID:25071022

Sumoylation of the Basic Helix-Loop-Helix Transcription Factor Sharp-1 Regulates Recruitment of the Histone Methyltransferase G9a and Function in Myogenesis*

PubMed Central

Wang, Yaju; Shankar, Shilpa Rani; Kher, Devaki; Ling, Belinda Mei Tze; Taneja, Reshma

2013-01-01

Sumoylation is an important post-translational modification that alters the activity of many transcription factors. However, the mechanisms that link sumoylation to alterations in chromatin structure, which culminate in tissue specific gene expression, are not fully understood. In this study, we demonstrate that SUMO modification of the transcription factor Sharp-1 is required for its full transcriptional repression activity and function as an inhibitor of skeletal muscle differentiation. Sharp-1 is modified by sumoylation at two conserved lysine residues 240 and 255. Mutation of these SUMO acceptor sites in Sharp-1 does not impact its subcellular localization but attenuates its ability to act as a transcriptional repressor and inhibit myogenic differentiation. Consistently, co-expression of the SUMO protease SENP1 with wild type Sharp-1 abrogates Sharp-1-dependent inhibition of myogenesis. Interestingly, sumoylation acts as a signal for recruitment of the co-repressor G9a. Thus, enrichment of G9a, and histone H3 lysine 9 dimethylation (H3K9me2), a signature of G9a activity, is dramatically reduced at muscle promoters in cells expressing sumoylation-defective Sharp-1. Our findings demonstrate how sumoylation of Sharp-1 exerts an impact on chromatin structure and transcriptional repression of muscle gene expression through recruitment of G9a. PMID:23637228

Chickpea transcription factor CaTLP1 interacts with protein kinases, modulates ROS accumulation and promotes ABA-mediated stomatal closure

PubMed Central

Wardhan, Vijay; Pandey, Aarti; Chakraborty, Subhra; Chakraborty, Niranjan

2016-01-01

Tubby and Tubby-like proteins (TLPs), in mammals, play critical roles in neural development, while its function in plants is largely unknown. We previously demonstrated that the chickpea TLP, CaTLP1, participates in osmotic stress response and might be associated with ABA-dependent network. However, how CaTLP1 is connected to ABA signaling remains unclear. The CaTLP1 was found to be engaged in ABA-mediated gene expression and stomatal closure. Complementation of the yeast yap1 mutant with CaTLP1 revealed its role in ROS scavenging. Furthermore, complementation of Arabidopsis attlp2 mutant displayed enhanced stress tolerance, indicating the functional conservation of TLPs across the species. The presence of ABA-responsive element along with other motifs in the proximal promoter regions of TLPs firmly established their involvement in stress signalling pathways. The CaTLP1 promoter driven GUS expression was restricted to the vegetative organs, especially stem and rosette leaves. Global protein expression profiling of wild-type, attlp2 and complemented Arabidopsis plants revealed 95 differentially expressed proteins, presumably involved in maintaining physiological and biological processes under dehydration. Immunoprecipitation assay revealed that protein kinases are most likely to interact with CaTLP1. This study provides the first demonstration that the TLPs act as module for ABA-mediated stomatal closure possibly via interaction with protein kinase. PMID:27934866

Expression of resolvin D1 biosynthetic pathways in salivary epithelium.

PubMed

Leigh, N J; Nelson, J W; Mellas, R E; Aguirre, A; Baker, O J

2014-03-01

Resolvins are potent anti-inflammatory mediators derived from ω-3 fatty acids. Results from our previous studies indicated that resolvin D1 (RvD1) blocks pro-inflammatory responses in salivary glands. Furthermore, RvD1 enhances salivary epithelial integrity, demonstrating its potential use for the restoration of salivary gland function in Sjögren's syndrome (SS). We investigated whether the RvD1 biosynthetic machinery (e.g., cytosolic phospholipase A2, calcium-independent phospholipase A2, 12/15 and 5-lipoxygenase) is expressed in mouse submandibular glands (mSMG), using qPCR and Western blot analyses. Additionally, we determined the localization of RvD1 biosynthetic machinery in mSMG and human minor salivary glands (hMSG), with and without SS, using confocal microscopy. Finally, we measured RvD1 levels in cell supernatants from mSMG cell cultures and freshly isolated mSMG cells, with and without SS, using ELISA. Our results indicate that: (1) RvD1 machinery is expressed in mouse and human salivary glands; (2) polar distribution of RvD1 biosynthetic machinery is lost in hMSG with SS; (3) RvD1 levels in mSMG cell culture supernatants increased with time; and (4) RvD1 levels in mSMG cell supernatants, with and without SS, were similar. These studies demonstrate that the RvD1 biosynthesis machinery is expressed and functional in salivary glands with and without SS.

Cell-specific vacuolar calcium storage mediated by CAX1 regulates apoplastic calcium concentration, gas exchange, and plant productivity in Arabidopsis.

PubMed

Conn, Simon J; Gilliham, Matthew; Athman, Asmini; Schreiber, Andreas W; Baumann, Ute; Moller, Isabel; Cheng, Ning-Hui; Stancombe, Matthew A; Hirschi, Kendal D; Webb, Alex A R; Burton, Rachel; Kaiser, Brent N; Tyerman, Stephen D; Leigh, Roger A

2011-01-01

The physiological role and mechanism of nutrient storage within vacuoles of specific cell types is poorly understood. Transcript profiles from Arabidopsis thaliana leaf cells differing in calcium concentration ([Ca], epidermis <10 mM versus mesophyll >60 mM) were compared using a microarray screen and single-cell quantitative PCR. Three tonoplast-localized Ca(2+) transporters, CAX1 (Ca(2+)/H(+)-antiporter), ACA4, and ACA11 (Ca(2+)-ATPases), were identified as preferentially expressed in Ca-rich mesophyll. Analysis of respective loss-of-function mutants demonstrated that only a mutant that lacked expression of both CAX1 and CAX3, a gene ectopically expressed in leaves upon knockout of CAX1, had reduced mesophyll [Ca]. Reduced capacity for mesophyll Ca accumulation resulted in reduced cell wall extensibility, stomatal aperture, transpiration, CO(2) assimilation, and leaf growth rate; increased transcript abundance of other Ca(2+) transporter genes; altered expression of cell wall-modifying proteins, including members of the pectinmethylesterase, expansin, cellulose synthase, and polygalacturonase families; and higher pectin concentrations and thicker cell walls. We demonstrate that these phenotypes result from altered apoplastic free [Ca(2+)], which is threefold greater in cax1/cax3 than in wild-type plants. We establish CAX1 as a key regulator of apoplastic [Ca(2+)] through compartmentation into mesophyll vacuoles, a mechanism essential for optimal plant function and productivity.

Levels of Lycopene β-Cyclase 1 Modulate Carotenoid Gene Expression and Accumulation in Daucus carota

PubMed Central

Moreno, Juan Camilo; Pizarro, Lorena; Fuentes, Paulina; Handford, Michael; Cifuentes, Victor; Stange, Claudia

2013-01-01

Plant carotenoids are synthesized and accumulated in plastids through a highly regulated pathway. Lycopene β-cyclase (LCYB) is a key enzyme involved directly in the synthesis of α-carotene and β-carotene through the cyclization of lycopene. Carotenoids are produced in both carrot (Daucus carota) leaves and reserve roots, and high amounts of α-carotene and β-carotene accumulate in the latter. In some plant models, the presence of different isoforms of carotenogenic genes is associated with an organ-specific function. D. carota harbors two Lcyb genes, of which DcLcyb1 is expressed in leaves and storage roots during carrot development, correlating with an increase in carotenoid levels. In this work, we show that DcLCYB1 is localized in the plastid and that it is a functional enzyme, as demonstrated by heterologous complementation in Escherichia coli and over expression and post transcriptional gene silencing in carrot. Transgenic plants with higher or reduced levels of DcLcyb1 had incremented or reduced levels of chlorophyll, total carotenoids and β-carotene in leaves and in the storage roots, respectively. In addition, changes in the expression of DcLcyb1 are accompanied by a modulation in the expression of key endogenous carotenogenic genes. Our results indicate that DcLcyb1 does not possess an organ specific function and modulate carotenoid gene expression and accumulation in carrot leaves and storage roots. PMID:23555569

[Construction and functional identification of eukaryotic expression vector carrying Sprague-Dawley rat MSX-2 gene].

PubMed

Yang, Xian-Xian; Zhang, Mei; Yan, Zhao-Wen; Zhang, Ru-Hong; Mu, Xiong-Zheng

2008-01-01

To construct a high effective eukaryotic expressing plasmid PcDNA 3.1-MSX-2 encoding Sprague-Dawley rat MSX-2 gene for the further study of MSX-2 gene function. The full length SD rat MSX-2 gene was amplified by PCR, and the full length DNA was inserted in the PMD1 8-T vector. It was isolated by restriction enzyme digest with BamHI and Xhol, then ligated into the cloning site of the PcDNA3.1 expression plasmid. The positive recombinant was identified by PCR analysis, restriction endonudease analysis and sequence analysis. Expression of RNA and protein was detected by RT-PCR and Western blot analysis in PcDNA3.1-MSX-2 transfected HEK293 cells. Sequence analysis and restriction endonudease analysis of PcDNA3.1-MSX-2 demonstrated that the position and size of MSX-2 cDNA insertion were consistent with the design. RT-PCR and Western blot analysis showed specific expression of mRNA and protein of MSX-2 in the transfected HEK293 cells. The high effective eukaryotic expression plasmid PcDNA3.1-MSX-2 encoding Sprague-Dawley Rat MSX-2 gene which is related to craniofacial development can be successfully reconstructed. It may serve as the basis for the further study of MSX-2 gene function.

A systematic expression analysis implicates Plexin-B2 and its ligand Sema4C in the regulation of the vascular and endocrine system.

PubMed

Zielonka, Matthias; Xia, Jingjing; Friedel, Roland H; Offermanns, Stefan; Worzfeld, Thomas

2010-09-10

Plexins serve as receptors for semaphorins and play important roles in the developing nervous system. Plexin-B2 controls decisive developmental programs in the neural tube and cerebellum. However, whether Plexin-B2 also regulates biological functions in adult nonneuronal tissues is unknown. Here we show by two methodologically independent approaches that Plexin-B2 is expressed in discrete cell types of several nonneuronal tissues in the adult mouse. In the vasculature, Plexin-B2 is selectively expressed in functionally specialized endothelial cells. In endocrine organs, Plexin-B2 localizes to the pancreatic islets of Langerhans and to both cortex and medulla of the adrenal gland. Plexin-B2 expression is also detected in certain types of immune and epithelial cells. In addition, we report on a systematic comparison of the expression patterns of Plexin-B2 and its ligand Sema4C, which show complementarity or overlap in some but not all tissues. Furthermore, we demonstrate that Plexin-B2 and its family member Plexin-B1 display largely nonredundant expression patterns. This work establishes Plexin-B2 and Sema4C as potential regulators of the vascular and endocrine system and provides an anatomical basis to understand the biological functions of this ligand-receptor pair. Copyright 2010 Elsevier Inc. All rights reserved.

Kcnh1 Voltage-gated Potassium Channels Are Essential for Early Zebrafish Development*

PubMed Central

Stengel, Rayk; Rivera-Milla, Eric; Sahoo, Nirakar; Ebert, Christina; Bollig, Frank; Heinemann, Stefan H.; Schönherr, Roland; Englert, Christoph

2012-01-01

The Kcnh1 gene encodes a voltage-gated potassium channel highly expressed in neurons and involved in tumor cell proliferation, yet its physiological roles remain unclear. We have used the zebrafish as a model to analyze Kcnh1 function in vitro and in vivo. We found that the kcnh1 gene is duplicated in teleost fish (i.e. kcnh1a and kcnh1b) and that both genes are maternally expressed during early development. In adult zebrafish, kcnh1a and kcnh1b have distinct expression patterns but share expression in brain and testis. Heterologous expression of both genes in Xenopus oocytes revealed a strong conservation of characteristic functional properties between human and fish channels, including a unique sensitivity to intracellular Ca2+/calmodulin and modulation of voltage-dependent gating by extracellular Mg2+. Using a morpholino antisense approach, we demonstrate a strong kcnh1 loss-of-function phenotype in developing zebrafish, characterized by growth retardation, delayed hindbrain formation, and embryonic lethality. This late phenotype was preceded by transcriptional up-regulation of known cell-cycle inhibitors (p21, p27, cdh2) and down-regulation of pro-proliferative factors, including cyclin D1, at 70% epiboly. These results reveal an unanticipated basic activity of kcnh1 that is crucial for early embryonic development and patterning. PMID:22927438

Analytical solution for boundary heat fluxes from a radiating rectangular medium

NASA Technical Reports Server (NTRS)

Siegel, R.

1991-01-01

Reference is made to the work of Shah (1979) which demonstrated the possibility of partially integrating the radiative equations analytically to obtain an 'exact' solution. Shah's solution was given as a double integration of the modified Bessel function of order zero. Here, it is shown that the 'exact' solution for a rectangular region radiating to cold black walls can be conveniently derived, and expressed in simple form, by using an integral function, Sn, analogous to the exponential integral function appearing in plane-layer solutions.

Characterization of a stearoyl-acyl carrier protein desaturase gene family from chocolate tree, Theobroma cacao L.

PubMed

Zhang, Yufan; Maximova, Siela N; Guiltinan, Mark J

2015-01-01

In plants, the conversion of stearoyl-ACP to oleoyol-ACP is catalyzed by a plastid-localized soluble stearoyl-acyl carrier protein (ACP) desaturase (SAD). The activity of SAD significantly impacts the ratio of saturated and unsaturated fatty acids, and is thus a major determinant of fatty acid composition. The cacao genome contains eight putative SAD isoforms with high amino acid sequence similarities and functional domain conservation with SAD genes from other species. Sequence variation in known functional domains between different SAD family members suggested that these eight SAD isoforms might have distinct functions in plant development, a hypothesis supported by their diverse expression patterns in various cacao tissues. Notably, TcSAD1 is universally expressed across all the tissues, and its expression pattern in seeds is highly correlated with the dramatic change in fatty acid composition during seed maturation. Interestingly, TcSAD3 and TcSAD4 appear to be exclusively and highly expressed in flowers, functions of which remain unknown. To test the function of TcSAD1 in vivo, transgenic complementation of the Arabidopsis ssi2 mutant was performed, demonstrating that TcSAD1 successfully rescued all AtSSI2 related phenotypes further supporting the functional orthology between these two genes. The identification of the major SAD gene responsible for cocoa butter biosynthesis provides new strategies for screening for novel genotypes with desirable fatty acid compositions, and for use in breeding programs to help pyramid genes for quality and other traits such as disease resistance.

Ectopic Expression of α6 and δ GABAA Receptor Subunits in Hilar Somatostatin Neurons Increases Tonic Inhibition and Alters Network Activity in the Dentate Gyrus

PubMed Central

Tong, Xiaoping; Peng, Zechun; Zhang, Nianhui; Cetina, Yliana; Huang, Christine S.; Wallner, Martin; Otis, Thomas S.

2015-01-01

The role of GABAA receptor (GABAAR)-mediated tonic inhibition in interneurons remains unclear and may vary among subgroups. Somatostatin (SOM) interneurons in the hilus of the dentate gyrus show negligible expression of nonsynaptic GABAAR subunits and very low tonic inhibition. To determine the effects of ectopic expression of tonic GABAAR subtypes in these neurons, Cre-dependent viral vectors were used to express GFP-tagged GABAAR subunits (α6 and δ) selectively in hilar SOM neurons in SOM-Cre mice. In single-transfected animals, immunohistochemistry demonstrated strong expression of either the α6 or δ subunit; in cotransfected animals, both subunits were consistently expressed in the same neurons. Electrophysiology revealed a robust increase of tonic current, with progressively larger increases following transfection of δ, α6, and α6/δ subunits, respectively, indicating formation of functional receptors in all conditions and likely coassembly of the subunits in the same receptor following cotransfection. An in vitro model of repetitive bursting was used to determine the effects of increased tonic inhibition in hilar SOM interneurons on circuit activity in the dentate gyrus. Upon cotransfection, the frequency of GABAAR-mediated bursting in granule cells was reduced, consistent with a reduction in synchronous firing among hilar SOM interneurons. Moreover, in vivo studies of Fos expression demonstrated reduced activation of α6/δ-cotransfected neurons following acute seizure induction by pentylenetetrazole. The findings demonstrate that increasing tonic inhibition in hilar SOM interneurons can alter dentate gyrus circuit activity during strong stimulation and suggest that tonic inhibition of interneurons could play a role in regulating excessive synchrony within the network. SIGNIFICANCE STATEMENT In contrast to many hippocampal interneurons, somatostatin (SOM) neurons in the hilus of the dentate gyrus have very low levels of nonsynaptic GABAARs and exhibit very little tonic inhibition. In an effort to increase tonic inhibition selectively in these interneurons, we used Cre-dependent viral vectors in SOM-Cre mice to achieve interneuron-specific expression of the nonsynaptic GABAAR subunits (α6 and δ) in vivo. We show, for the first time, that such recombinant GFP-tagged GABAAR subunits are expressed robustly, assemble to form functional receptors, substantially increase tonic inhibition in SOM interneurons, and alter circuit activity within the dentate gyrus. PMID:26658866

Dendritic cells provide a potential link between smoking and inflammation in rheumatoid arthritis

PubMed Central

2012-01-01

Introduction Smoking increases the risk of developing rheumatoid arthritis (RA) and affects the severity of established RA. Smoking can impact on Th17 lymphocyte differentiation and function through activation of the aryl hydrocarbon receptor (AHR), a process with implications for the pathogenic mechanisms in RA that involve the cytokine, interleukin (IL)-17A. The objective of this study was to establish any effect of smoking on the inflammatory tissue lesions of rheumatoid arthritis via the AHR and IL-17A. Methods Twenty synovial and eighteen subcutaneous nodule tissue samples from 31 patients with RA were studied. Patient smoking status at the time of tissue collection was established. Expression of AHR, CYP1A1, AHRR, IL6, IL17A, IL17F, IL22, IL23, IL23R, IFNG, TBX21, IDO1 and FOXP3 genes were assessed in tissues and cultured cells using real-time PCR. Two-colour immunofluorescence was used to co-localise AHR and CYP1A1 protein in synovial tissues. The response of monocytes and monocyte-derived dendritic cells (mo-DCs) to the AHR agonist, benzo(a)pyrene (BaP) was compared in vitro. Results AHR gene expression was demonstrated in rheumatoid synovial tissues and nodules with significantly greater expression in synovia. Expression was not influenced by smoking in either tissue. Evidence of AHR activation, indicated by CYP1A1 and AHRR gene expression, was found only in synovia from patients who smoked. However, IL17A gene expression was lower in synovia from smokers. TBX21 and FOXP3 expression was not affected by smoking. Within the synovial tissues of smokers the principal cell type with evidence of AHR activation was a subset of synovial DCs. This observation was consistent with the sensitivity of human mo-DCs to BaP stimulation demonstrated in vitro. Exposure to BaP affected mo-DC function as demonstrated by decreased IL6 expression induced by PolyI:C, without affecting indoleamine 2,3 dioxygenase (IDO)1 expression. Conclusion Our findings show that one effect of smoking on inflamed rheumatoid synovial tissue involves activation of the AHR pathway. A subset of synovial DCs is important in the response to cigarette smoke. The potential for smoking to affect DC behaviour in joint tissues has relevance to both early and late phases of RA pathogenesis and warrants further investigation. PMID:23036591

CXCR5+CD8+ T cells infiltrate the colorectal tumors and nearby lymph nodes, and are associated with enhanced IgG response in B cells.

PubMed

Xing, Junjie; Zhang, Chenxin; Yang, Xiaohong; Wang, Shaoxuan; Wang, Zhongchuan; Li, Xu; Yu, Enda

2017-07-01

Colorectal cancer is the third most prevalent cancer type worldwide and contributes to a significant percentage of cancer-related mortality. Recent studies have shown that the CXCR5 + CD8 + T cells present more potent proinflammatory function than CXCR5 - CD8 + T cells in chronic virus infections and in follicular lymphoma, but the role of CXCR5 + CD8 + T cells in colorectal cancer is yet unclear. In this study, we demonstrated that CXCR5 + CD8 + T cells were very rare in peripheral blood mononuclear cells from healthy and colorectal cancer individuals, but were significantly enriched in resected tumors and tumor-associated lymph nodes. Compared to CXCR5 - CD8 + T cells, the CXCR5 + CD8 + T cells demonstrated significantly higher Bcl-6 expression and lower Blimp1 expression, suggesting that CXCR5 + CD8 + T cells might represent a memory CD8 + T cell subset. CXCR5 + CD8 + T cells also enhanced the IgG expression by autologous B cells. Under ex vivo condition, the CXCR5 + CD8 + T cells demonstrated lower degranulation, TNFα expression and IFNγ expression than CXCR5 - CD8 + T cells. However, after PMA + ionomycin stimulation, the degranulation and TNFα expression by CXCR5 + CD8 + T cells were significantly elevated to a level comparable with CXCR5 - CD8 + T cells, whereas the IFNγ expression by PMA + ionomycin-stimulated CXCR5 + CD8 + T cells were significantly higher than that by CXCR5 - CD8 + T cells. Following long-term TCR-stimulation, CXCR5 + CD8 + T cells demonstrated significantly more potent proliferation capacity and higher IFNγ expression than CXCR5 - CD8 + T cells. TCR-stimulated CXCR5 + CD8 + T cells also showed a gradual downregulation in CXCR5 expression. We further found that TCR-stimulated CXCR5 + CD8 + T cells demonstrated higher granzyme B production and induced more specific lysis of autologous tumor cells than CXCR5 - CD8 + T cells. Together, these data demonstrate that CXCR5 + CD8 + T cells represent a significant CD8 + T cell subset in colorectal tumors and have the potential to contribute to antitumor immunity, but their specific roles require further studies in vivo. Copyright © 2017. Published by Elsevier Inc.

Facial expressions of emotion and the course of conjugal bereavement.

PubMed

Bonanno, G A; Keltner, D

1997-02-01

The common assumption that emotional expression mediates the course of bereavement is tested. Competing hypotheses about the direction of mediation were formulated from the grief work and social-functional accounts of emotional expression. Facial expressions of emotion in conjugally bereaved adults were coded at 6 months post-loss as they described their relationship with the deceased; grief and perceived health were measured at 6, 14, and 25 months. Facial expressions of negative emotion, in particular anger, predicted increased grief at 14 months and poorer perceived health through 25 months. Facial expressions of positive emotion predicted decreased grief through 25 months and a positive but nonsignificant relation to perceived health. Predictive relations between negative and positive emotional expression persisted when initial levels of self-reported emotion, grief, and health were statistically controlled, demonstrating the mediating role of facial expressions of emotion in adjustment to conjugal loss. Theoretical and clinical implications are discussed.

Intestinal inflammation reduces expression of DRA, a transporter responsible for congenital chloride diarrhea.

PubMed

Yang, H; Jiang, W; Furth, E E; Wen, X; Katz, J P; Sellon, R K; Silberg, D G; Antalis, T M; Schweinfest, C W; Wu, G D

1998-12-01

The pathogenesis of diarrhea in intestinal inflammatory states is a multifactorial process involving the effects of inflammatory mediators on epithelial transport function. The effect of colonic inflammation on the gene expression of DRA (downregulated in adenoma), a chloride-sulfate anion transporter that is mutated in patients with congenital chloridorrhea, was examined in vivo as well as in an intestinal epithelial cell line. DRA mRNA expression was diminished five- to sevenfold in the HLA-B27/beta2m transgenic rat compared with control. In situ hybridization showed that DRA, which is normally expressed in the upper crypt and surface epithelium of the colon, was dramatically reduced in the surface epithelium of the HLA-B27/beta2m transgenic rat, the interleukin-10 (IL-10) knockout mouse with spontaneous colitis, and in patients with ulcerative colitis. Immunohistochemistry demonstrated that mRNA expression of DRA reflected that of protein expression in vivo. IL-1beta reduced DRA mRNA expression in vitro by inhibiting gene transcription. The loss of transport function in the surface epithelium of the colon by attenuation of transporter gene expression, perhaps inhibited at the level of gene transcription by proinflammatory cytokines, may play a role in the pathogenesis of diarrhea in colitis.

Rrp1b, a New Candidate Susceptibility Gene for Breast Cancer Progression and Metastasis

PubMed Central

Crawford, Nigel P. S; Qian, Xiaolan; Ziogas, Argyrios; Papageorge, Alex G; Boersma, Brenda J; Walker, Renard C; Lukes, Luanne; Rowe, William L; Zhang, Jinghui; Ambs, Stefan; Lowy, Douglas R; Anton-Culver, Hoda; Hunter, Kent W

2007-01-01

A novel candidate metastasis modifier, ribosomal RNA processing 1 homolog B (Rrp1b), was identified through two independent approaches. First, yeast two-hybrid, immunoprecipitation, and functional assays demonstrated a physical and functional interaction between Rrp1b and the previous identified metastasis modifier Sipa1. In parallel, using mouse and human metastasis gene expression data it was observed that extracellular matrix (ECM) genes are common components of metastasis predictive signatures, suggesting that ECM genes are either important markers or causal factors in metastasis. To investigate the relationship between ECM genes and poor prognosis in breast cancer, expression quantitative trait locus analysis of polyoma middle-T transgene-induced mammary tumor was performed. ECM gene expression was found to be consistently associated with Rrp1b expression. In vitro expression of Rrp1b significantly altered ECM gene expression, tumor growth, and dissemination in metastasis assays. Furthermore, a gene signature induced by ectopic expression of Rrp1b in tumor cells predicted survival in a human breast cancer gene expression dataset. Finally, constitutional polymorphism within RRP1B was found to be significantly associated with tumor progression in two independent breast cancer cohorts. These data suggest that RRP1B may be a novel susceptibility gene for breast cancer progression and metastasis. PMID:18081427